Author response: Evidence of human infection by a new mammarenavirus endemic to Southeastern Asia (2016)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Southeastern Asia is a recognised hotspot for emerging infectious diseases, many of which have an animal origin. Mammarenavirus infections contribute significantly to the human disease burden in both Africa and the Americas, but little data exists for Asia. To date only two mammarenaviruses, the widely spread lymphocytic choriomeningitis virus and the recently described Wēnzhōu virus have been identified in this region, but the zoonotic impact in Asia remains unknown. Here we report the presence of a novel mammarenavirus and of a genetic variant of the Wēnzhōu virus and provide evidence of mammarenavirus-associated human infection in Asia. The association of these viruses with widely distributed mammals of diverse species, commonly found in human dwellings and in peridomestic habitats, illustrates the potential for widespread zoonotic transmission and adds to the known aetiologies of infectious diseases for this region. https://doi.org/10.7554/eLife.13135.001 eLife digest Rodents have long been notorious for spreading disease among humans. Often the animals can carry viruses and transmit them to humans without becoming ill. Certain species thrive in cities and agricultural areas where they come in close contact with humans; this creates many opportunities to spread infection. As humans urbanize and farm larger swaths of previously wild lands, the risk of rodent-transmitted infections increases. As a result, some scientists are working to identify viruses carried by rodents in human settlements and hopefully prevent them from spreading to humans. The mammarenavirsuses are a group of rodent-transmitted viruses that commonly cause illness in people in Africa and Latin America. Each year, one such virus – the Lassa virus –sickens as many as 300,000 people in Africa and kills 5,000. So far, only two mammarenaviruses have been found in Asia: one called the Wēnzhōu virus and another called LCMV. However only LCMV is known to cause human illness and many cases of illness caused by mammarenaviruses in Asia may go undetected because they often cause mild symptoms similar to the common cold. Blasdell et al. have now tested lung samples from 20 species of rodents collected at 7 sites in Cambodia, Thailand, and Laos to look for molecules produced by mammarenaviruses. The tests revealed a strain of Wēnzhōu virus circulating in Cambodian rats that often live in urban areas. A new mammarenavirus was also detected in rodents that live in Thai rice fields. However, infecting wild and domestic rodents with the viruses in the laboratory did not cause many noticeable signs of illness. Blasdell et al. then tested samples from Cambodian patients who either had influenza-like symptoms or more serious symptoms that are associated with a condition called Dengue fever (which is common in the area). Some patients with respiratory symptoms tested positive for the Wēnzhōu virus. Because the symptoms are mild and similar to those of other common diseases it is likely that the Wēnzhōu virus may be spreading more widely among humans in Asia. The next challenges are to provide a better estimate of the frequency of this disease in the human population in Asia and to describe the full spectrum of disease that might be associated with this newly discovered infectious disease. https://doi.org/10.7554/eLife.13135.002 Introduction Rodents of several species are known hosts of numerous zoonotic pathogens (Luis et al., 2013), and are also amongst the peridomestic ensemble that benefit from how humans are modifying the landscape (Shochat et al., 2006). Their increased presence will amplify human-rodent encounter rates and opportunities for zoonotic transmission, likely creating an increased risk for human health (Young et al., 2014). A concerted effort to establish which human pathogens are present in the environments heavily utilized by humans would therefore be beneficial for the prevention, control and where possible, treatment of such zoonoses. Mammarenaviruses are predominantly rodent-borne viruses, several of which have been associated with human disease. In Western Africa, Lassa virus (LASV) infection causes an estimated 100,000 to 300,000 infections and approximately 5000 deaths annually (McCormick et al., 1987), whilst Junín virus (JUNV) causes regular seasonal outbreaks of viral haemorrhagic fever in Argentina (Gómez et al., 2011). So far only Wēnzhōu virus and lymphocytic choriomeningitis virus (LCMV) have been conclusively demonstrated to be present in Asia (Li et al., 2014; Morita et al., 1996; 1991). Wēnzhōu virus was identified in China in rodents belonging to four Rattus species, Niviventer niviventer and in Asian house shrews (Suncus murinus), but has not yet been associated with disease. However, LCMV is zoonotic, is primarily hosted by the widespread house mouse (Mus musculus), and consequently has the widest known distribution of any mammarenavirus (Buchmeier et al., 2007). Human infection with LCMV predominantly results in a relatively mild influenza-like illness (Bonthius, 2012; Macneil et al., 2012; Centers for Disease Control and Prevention (CDC), 2012). This propensity of some mammarenaviruses to cause mild disease with symptoms common to those found for many other viral infections, illustrates the potential for these infections to go undiagnosed or misdiagnosed. That human infections caused by arenaviruses other than LCMV have not previously been detected in Southeastern Asia (Charrel and de Lamballerie, 2003) should therefore not be considered as evidence of absence. As each mammarenavirus is primarily adapted to rodents of distinct species or to closely related rodents of diverse species (Gonzalez et al., 2007), the apparent absence of these viruses from a region with high rodent biodiversity (Pagès et al., 2010) is surprising. A large proportion of infectious diseases remain undiagnosed in resource poor countries where people rarely visit healthcare professionals and where clinicians often do not have the training or laboratory support for accurate diagnosis (Mueller et al., 2014). Here, in the course of a survey for rodent-borne mammarenaviruses, we identified a genetic variant of the recently identified Wēnzhōu virus in Cambodia - provisionally named Cardamones - in rodents belonging to two widespread species, brown rats (Rattus norvegicus) and Pacific rats (R. exulans), which are commonly found in proximity to humans. We also identified a novel mammarenavirus species in Thailand in rice-field associated greater bandicoot rats (Bandicota indica), Savile’s bandicoot rats (B. savilei) and an Indomalayan niviventer (Niviventer fulvescens). In addition, we detected Wēnzhōu virus in several Cambodian patients presenting with fever and respiratory symptoms, indicating that this virus may be causally related with human disease. Results Identification of two mammarenaviruses in Southeastern Asia To assess whether mammarenaviruses are present in Southeastern Asian small mammals, we used a previously described RT-PCR (Vieth et al., 2007) (Supplementary file 1A) to screen 627 homogenised lung samples from small mammals of twenty species (Supplementary file 1B) collected from seven sites (two in Cambodia, three in Thailand and two in Laos). Based on the hypothesis of rodent-mammarenavirus interrelation (Gonzalez et al., 2007) and the presence of only Old World rodents in the region (Marshall et al., 1988), primers targeting Old World mammarenaviruses were used. We detected evidence of mammarenavirus RNA in twenty seven animals from two locations: Veal Renh in Cambodia and Loei in Thailand. Seventeen individuals from two peridomestic species, brown rats (four individuals) and Pacific rats (thirteen individuals), were identified positive in Veal Renh. In Loei, ten individuals from three agricultural-associated species were found positive, namely six Savile’s bandicoot rats, three greater bandicoot rats and one Indomalayan niviventer (Supplementary file 1B). All but one of the Cambodian positive rodents were collected in settlements, whilst five of the Thai rodents were collected in plantations (Supplementary file 1C). Sanger sequencing of the amplicons indicated the presence of a novel mammarenavirus in the Thai samples. Full genome sequencing was attempted on a single sample from each site: C0649, originally obtained from a wild-caught Pacific rat from Veal Renh, and R5074, originally obtained from a wild-caught Savile’s bandicoot rat from Loei. Coding-complete genomes of isolates were acquired by deep sequencing (Illumina HiSeq 2000) of random amplified RNAs from the lungs of Wistar laboratory rats inoculated with either the C0649 or R5074 lung homogenate (C0649: S segment accession KC669696, length 3331 nucleotides (nt); L segment accession KC669690, length 7181nt. R5074: S segment accession KC669698, length 3345nt; L segment accession KC669693, length 7186nt). Primers were designed based on this sequence and in conjunction with a previously published protocol (Bowen et al., 2000) (Supplementary file 1A), were used to obtain the coding-complete genome of two further isolates: C0617, obtained from a wild-caught brown rat, (S segment accession KC669694, length 3344nt; L segment accession KC669691, length 7171nt) and R4937, obtained from a wild-caught greater bandicoot rat (S segment accession KC669697, length 3379nt; L segment accession KC669692, length 7185nt). Each segment encoded two open reading frames (ORFs) in an ambisense organization with an intergenic region containing a predicted hairpin between the ORFs. Deduced amino acid (aa) and nt sequences from the four isolates were compared to those of other representative mammarenaviruses. An aa sequence divergence of >25% for the nucleoprotein (NP) was found between the Thai isolates and all other known mammarenavirus species, whilst a 3.5–12.7% aa sequence divergence was found between the Cambodian isolates and Wēnzhōu virus (Table 1). PAirwise Sequence Comparison (PASC) was performed on both segments for each of the four sequenced viruses. All samples were found most closely related to Wēnzhōu virus, with the Cambodian virus demonstrating 88.5–88.8% identity for the S segment and 86.0–86.3% identity for the L segment, whilst the Thai virus showed 70.3–70.6% identity for the S segment and 62.7–63.1% identity for the L segment (Table 2; Table 2—source data 1). The International Committee on Taxonomy of Viruses (ICTV) arenavirus species demarcation criteria includes, among others, the association with a main host species or group of sympatric hosts, presence in a defined geographical area, and significant protein amino acid sequence differences, including a difference of at least 12% in the amino acid (aa) sequence of the nucleoprotein to other species in the genus (King et al., 2012; Radoshitzky et al., 2015). A recent update from the ICTV Arenaviridae study group has also recommended the use of the PASC tool for the assessment of novel arenaviruses. Cut-off values chosen for classifying arenaviruses belonging to the same species using this tool are >80% and >76% nucleotide sequence identity in the S and L segments respectively (Radoshitzky et al., 2015). The Thai virus is the first mammarenavirus to be detected in Bandicota species and, alongside the Cambodian virus is the first to be detected in this geographic region. As this virus also meets nucleoprotein amino acid sequence identities and PASC requirements, we propose that this Thai virus represents a member of a novel species. We suggest to call this novel virus Loei River mammarenavirus (after a river close to the site where it was detected) with the abbreviation LORV. Although the Cambodian virus is the first mammarenavirus to be associated with Pacific rats (R. exulans) and Cambodia is geographically distant to the Chinese region in which Wēnzhōu virus was originally detected, the sequence homology to Wēnzhōu virus and the association with rodents of a common host species (R. norvegicus), indicates that the Cambodian virus represents a genetic variant of Wēnzhōu mammarenavirus (Li et al., 2014). We propose to provisionally name this variant Cardamones (after the chain of mountains near Veal Renh). Table 1 Nucleotide and amino acid sequence identities (%) between Cambodian and Thai isolates and selected other arenaviruses. https://doi.org/10.7554/eLife.13135.003 IsolatesSegment or ORFnt/ aaCambodian isolatesThai isolatesWēnzhōuLassaIppyMopeiaLCMVJunínLunaMorogoroCambodian isolatesL segmentnt98.969.2-69.487.5-88.655.8-56.457.7-57.860.856.950.2-50.461.1-61.460.9-61.0L ORFnt99.367.3-67.588.0-89.059.7-60.660.4-60.559.2-59.455.650.6-50.959.6-60.959.6aa99.669.2-69.492.2-94.855.5-56-457.7-57.856.6-57.148.537.9-38.055.8-55.955.6Z ORFnt99.573.4-74.5837.-87.966.8-67.463.662.557.6-58.254.9-55.469.0-70.164.7-65.2Aa98.879.289.4-93.970.1-75.370.164.959.440.363.6-64.961.0S segmentnt99.571.7-72.187.5-89.861.7-68.166.6-66.866.6-67.061.6-61.854.6-54.867.6-68.266.7-66.9NP ORFnt99.373.1-74.486.6-90.067.1-68.268.3-68.467.6-67.962.9-63.255.1-55.669.0-70.368.7aa99.882.9-84.287.3-96.572.2-73.874.4-74.673.5-73.864.051.9-52.573.3-74.074.0-74.6GPCnt99.769.1-70.088.6-89.767.3-68.664.7-65.765.3-65.761.2-61.453.4-53.666.3-66.649.9-65.2aa99.879.5-81.195.5-96.474.2-76.269.5-71.371.5-72.857.2-57.548.2-43.072.6-74.473.1-74.4Thai virusL segmentnt68-68.194.666.6-67.560.7-61.861.3-61.561.1-61.457.150.2-50.560.7-61.461.4-61.5L ORFnt67.3-67.595.167.9-68.859.6-59.959.9-60.461.6-62.155.7-56.249.9-50.860.4-61.659.9-60.2aa69.2-69.496.769.6-70.755.7-56.456.556.6-57.149.0-49.437.1-37.555.9-56.356.5-56.7Z ORFnt73.4-74.595.469.4-75.065.2-66.864.1-65.869.0-69.658.7-59.254.3-56.065.8-72.866.3-66.8aa79.298.573.1-74.667.5-71.468.870.158.442.9-44.270.1-71.466.2S segmentnt71.7-72.194.471.0-72.265.8-67.366.1-66.866.2-6761.7-62.653.9-55.065.2-68.166.1-67.9NP ORFnt73.1-74.494.672.2-74.165.2-67.866.6-67.366.9-67.762.4-63.254.1-54.466.1-68.067.3-68.9aa82.9-84.298.178.3-87.273.3-74.672.0-72.973.8-74.464.2-64.649.9-51.072.0-73.574-75.5GPCnt69.1-7094.169.2-69.966.0-67.665.0-66.166.4-6761.5-62.653.9-56.065.8-69.266.1-67.5aa79.5-81.197.580.1-81.173.5-75.771.5-72.471.0-74.259.7-61.043.0-43.974.2-74.873.1-75.9 Note where (L & S gene): Cambodian = KC669690, KC669691& KC669694, KC669696; Thai = KC669692, KC669693 & KC669697, KC669698; Wenhzhou = KM386661, KM051421, KJ909795, KM051420 and KM386660, KM051423, KJ909794, and KM051422; Lassa = GU481076 & GU481077; Ippy = DQ328877 & DQ328878; Mopeia = DQ328874 & DQ328875; LCMV = AY847350 & AY847351; Junín = D10072 & AY216507; ORF = Open reading frame; NP = Nucleoprotein; GPC = Glycoprotein; nt = Nucleotide; aa = Amino acid; LCMV =: Lymphocytic Choriomeningitis Virus Table 2 Summary of PASC analysis. https://doi.org/10.7554/eLife.13135.004 Sample IDCountry of originPASC: S segmentPASC: L segmentSequence identity (%)Closest virusSequence identity (%)Closest virusC0617Cambodia88.80Wēnzhōu virus86.27Wēnzhōu virusC0649Cambodia88.51Wēnzhōu virus85.98Wēnzhōu virusR4937Thailand70.63Wēnzhōu virus62.71Wēnzhōu virusR5074Thailand70.29Wēnzhōu virus63.08Wēnzhōu virus Table 2—source data 1 PASC analysis. https://doi.org/10.7554/eLife.13135.005 Download elife-13135-table2-data1-v2.docx In maximum likelihood phylogenetic analyses, the Southeastern Asian viruses, along with the Chinese isolates of Wēnzhōu virus, formed an independent clade within the Old World mammarenaviruses. The Cambodian virus sequences clustered with the Wēnzhōu virus sequences, whilst LORV sequences clustered independently. This Asian mammarenavirus clade formed a sister clade to Ippy virus (IPPYV) in both full RNA-dependent RNA polymerase (L) and NP gene analysis, and to the African Lassa-related viruses as a whole in glycoprotein precursor (GPC) gene analysis (Figure 1a and Figure 2). Figure 1 Download asset Open asset Maximum likelihood phylogenetic tree of novel arenavirus isolates and other representative arenaviruses for a, the complete ORF of the L gene with sequences from rodents only, b, partial L sequences including sequences from rodents and patients. Cambodian strains detected in rodent (triangle), human (square) and Thai strains detected in rodent (circle) are in bold. Clade A, B and C are three evolutionary lineages of New World arenaviruses within the Tacaribe complex. A/Rec denotes the recombinant clade including the three Northern American viruses. The virus names are in abbreviation according to Radoshitzky et al. (2015). https://doi.org/10.7554/eLife.13135.006 Figure 2 Download asset Open asset Maximum likelihood phylogenetic tree of novel arenavirus isolates and other representative arenaviruses for a, the complete ORF of GPC gene and b, complete ORF of NP gene. Cambodian strains (triangle) and Thai strains (circle) detected in rodents are in bold. Clade A, B and C are three evolutionary lineages of New World arenaviruses within the Tacaribe complex. A/Rec denotes the recombinant clade including Northern American viruses. The virus names are in abbreviation according to Radoshitzky et al. (2015). https://doi.org/10.7554/eLife.13135.007 Experimental infections in rodents To establish if infection and transmission of the Cardamones variant of Wēnzhōu virus could be replicated in rodents of two of the suspect host species, experimental infections were performed. Despite several attempts to obtain a culture isolates in tissue culture, including multiple passages, we were unable to propagate the Cambodian isolates in the cell lines available in our laboratory (Vero E6, MDCK, BHK), or in primary cell lines from lungs obtained from a Wistar laboratory rat. Experimental infections were therefore conducted using homogenised lung tissue from wild-caught RNA-positive animals to infect both Wistar laboratory rats and wild-caught or first generation Pacific rats captured in the Mondulkiri site. Male and female adult animals were used. Due to logistical limitations, parallel experiments were not conducted for LORV. Of 85 animals inoculated intraperitoneally with the Cardamones variant of Wēnzhōu virus-positive homogenate, 77 (64/68 Wistar laboratory rats; 13/17 Pacific rats) were successfully infected as measured by detection of viral RNA by RT-PCR in organs and/or seroconversion by IFA and/or ELISA (Supplementary file 1D). Organs positive for Wēnzhōu virus (i.e. brain, heart, lung, liver, spleen, kidney, bladder, peritoneum) varied between individuals (data not shown), with individuals tested usually demonstrating a generalised infection. Organs from randomly selected adult individuals collected between 3dpi and 22dpi or 28dpi for Wistar laboratory rats and Pacific rats respectively, were subjected to quantitative real-time RT-PCR (qRT-PCR) and copy number was determined. Copy numbers increased over time with high copy numbers (108–1011 cDNA copy/mg) detected in several organs from 7dpi in Pacific rats and 11dpi in Wistar laboratory rats. Specifically, high copy numbers were detected in the liver, spleen and lung of most individuals, with the latter suggesting the potential for respiratory transmission. Mammarenavirus RNA was detected in most inoculated Wistar laboratory rats killed between 3-56dpi (52/68 individuals) and Pacific rats killed between 3-37dpi (11/17 individuals), although no animals were tested beyond these time points. Mammarenaviruses are known to cause both acute and chronic infections in their rodent hosts 17 and the duration of infection observed here is comparative to that seen in experimental infections of the natural hosts of other mammarenaviruses (Fulhorst et al., 1999; Walker et al., 1975). However, as lung homogenates were used instead of titrated virus, the variability of the dose received by each animal may have impacted on the infection course, likely resulting in the variations and inconsistencies observed (Childs et al., 1993). In both species, we observed horizontal transmission, but only between pups and their dams (Supplementary file 1D). Vertical transmission could not be confirmed for either species. Mammarenavirus RNA was detected in a single pup from each of the two Pacific rat litters but all other pups from these litters were negative, and antibodies detected in additional pups may have been of maternal origin. LCMV infection of house mice must occur shortly before or after mating in order for dams to transmit the virus to a high proportion of their pups (Skinner and Knight, 1974). Therefore the absence of vertical transmission observed here could have been due to the late stage of gestation at which most dams were inoculated (Supplementary file 1D), although vertical transmission has not been confirmed in all mammarenaviruses (Mills et al., 2007). Clinical signs are rarely seen in mammarenavirus rodent reservoirs, although more subtle signs including reduced weight and body mass index have been observed (Borremans et al., 2011). To establish if Wēnzhōu virus infection of rodent hosts results in lesions, histopathological analysis and chromogenic immunohistochemistry were performed on tissues from two Pacific rats inoculated intra-peritoneally with lung tissue homogenates obtained from a wild rat that tested positive for Cardamones variant of Wēnzhōu virus. Electron microscopy clearly identified virus particles with a specific mammarenavirus-type morphology (Figures 3a and b) in the lung tissues. Histopathological examination of the lungs revealed only severe diffuse pneumonia (Figures 3c–h). Other organs were not affected except for vascular congestion, probably due to pre mortem conditions. This relatively limited pathology is in congruence with the suspected reservoir status of this species. Figure 3 Download asset Open asset Electron microscope images of the Cardamones variant of Wēnzhōu virus in the rat lung tissues, a (scale: 200 nm) & b (scale: 2 µm). Histopathological examination of the lungs revealed only severe diffuse pneumonia, with lesions associated with acute exudative inflammation characterised by foci of consolidation surrounded by extremely rare aerated parenchyma, c, vascular congestion, acute bronchiolitis and diffuse leukocytic infiltration with lymphocytes, polymorphonuclear and macrophages in the and and demonstrated the severe of lung parenchyma, immunohistochemistry identified foci with numerous positive primarily including but in the more and aerated some and were also clearly Human mammarenavirus infections in Cambodia The peridomestic of the hosts of Wēnzhōu virus et al., indicates that this virus would have opportunities to transmit to humans. We therefore to establish if zoonotic transmission occur and if it is associated with disease by human samples from (Table In group a of patients presenting with and influenza-like but who tested for and infection tested positive for The of positive patients was to which was not from patients 7 to The was detected in the to group was no difference in the between and patients and The was high in some of (Figure were observed in 7 patients with fever or influenza-like illness (Table patients had a of with a of which were not significantly to mammarenavirus patients with 7 to between the of the acute and the samples for these patients between 2 and with a of (Supplementary file data were available for two of these patients. The first was a 3 who with fever and The was a presenting with mild fever and and were in both fever to was in the five cases Of with is extremely common in Cambodia and therefore the measured at the time of examination is often In to samples from individuals from who in a study were also for antibodies and were found positive (Table The of positive individuals was with a The and of positive cases in individuals compared to patients presenting with and influenza-like symptoms were not significantly and A limited number of human samples = file were tested by both mammarenavirus IFA and demonstrating in results of of with a The those two is only as the tests were used for due to IFA was chosen for because of the of the to antibodies a of arenaviruses the ELISA was with the nucleoprotein of the Cardamones variant of Wēnzhōu virus is order to the of the the Wēnzhōu virus ELISA was tested LCMV positive human the only other zoonotic mammarenavirus to be present in Southeastern but no was The ELISA to be more than the mammarenavirus additional positive samples (Supplementary file The mammarenavirus IFA identified 2 samples found by the ELISA as However these infections may have been caused by more related viruses, such as not of detection by the more specific Despite as mammarenaviruses are by ELISA et al., it is that this is specific to Wēnzhōu virus. it is likely that this would be of other closely related including novel species. However to other arenaviruses were not available for As for most the in these patients can therefore not be identified Table 3 of samples of in of influenza-like and including and to - - - with evidence of seroconversion in 7 individuals collected stage to illness for four common respiratory to illness for four common respiratory respiratory to individuals – to – Cambodia to