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[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. 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Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. 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Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4.", "Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge UK : Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE.", "Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A H1N1 viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG.", "Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene NS segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.", "12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK.", "J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18.", "Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927.", "Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds.", "Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol.", "Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl N-acetyllactosamine . Virology. 1997;232: 345–50.", "Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31.", "A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33.", "J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD.", "J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger\" and David M. Morens1- The “Spanish\" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis.", "But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population or z500 million persons were infected and had clinical- ly apparent illnesses 1,2 during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics 3,4 . Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion ..", "Total deaths were estimated at z50 million . and were arguably as high as 100 mil- lion .. The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide except- ing human infections from avian Viruses such as H5N1 and H7N7 , have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown.", "The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses now known to be H1N1 Viruses were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs.", "Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic .. Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 “Asian flu” , the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically.", "But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer .. They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage so-called classic swine flu , and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present .", "Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains prevalent from 1968 until the present . H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates.", "No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus .. These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies .", "These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies . and a number of reviews covering different aspects of the 1918 pandemic have recently been published 8720 and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic.", "The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus . and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 .", "Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America the first wave was best described in the United States in March 1918 . Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus ., and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context 9 . Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 .. Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively Figure 1 . Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected.", "Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps ., but a counter argument is that if a strain with a new hemagglutinin HA was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous or nearly simultaneous infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 .", "The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 12,20 . Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase NA surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before 12,15, 24 . More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 .. Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling.", "Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date .. In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants.", "Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 .. In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal.", "In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months Figure 1 . Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 .. Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases.", "The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited.", "The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures bene- ficial to thermolabile Viruses such as influenza , optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread.", "However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter of the Northern Hemisphere , respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later?", "Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported.", "And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 more than a year after the first pandemic appearance and the third in early 1892 21 . As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months.", "As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients 6 , nothing can yet be said about whether the first spring wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed.", "Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes.", "Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source 17,19 , as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods 24,25 . For example, the 1918 nucleoprotein NP gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains 13,19 . One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement.", "One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far.", "Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from?", "One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses . ; both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity?", "Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans.", "Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 . linked sugars .. Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 2% link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it ..", "The switch from this avian receptor configuration requires of the Virus only 1 amino acid change ., and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it .. This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced.", "Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice .. Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage .. These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice.", "These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA and possibly the NA contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned.", "The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age.", "The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped Figure 2 , exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third middle distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years .. Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates .", "The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic .. A sharper perspective emerges when 1918 age-specific influenza morbidity rates . are used to adj ust the W- shaped mortality curve Figure 3, panels, A, B, and C 35,37 . Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence Figure 3, panel A . But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups.", "But even after adjusting age-specific deaths by age-specif— ic clinical attack rates Figure 3, panel B , a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 Figure 3, panel C . Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology e.g., antibody-dependent infection enhancement associated with prior Virus exposures and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough >35 years to have been infected during that prior era .. But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases.", "But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 .. Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3.", "Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 dashed line and for the pandemic year 1918 solid line 33,34 . Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia P&l combined age-specific incidence rates per 1,000 persons per age group panel A , death rates per 1,000 persons, ill and well combined panel B , and case-fatality rates panel C, solid line , US Public Health Service house-to-house surveys, 8 states, 1918 .. A more typical curve of age-specific influenza case-fatality panel C, dotted line is taken from US Public Health Service surveys during 1928—1929 .. the 5 to 15-year-old group jumped to 25% of influenza cases compatible with exposure to an antigenically novel Virus strain , while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics.", "In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum Figure 2 , a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today.", "In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 >95% in most locales in industrialized nations were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group.", "Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time Figure 1 . Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic.", "Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus ., though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus.", "There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza HPAI Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation e.g., receptor binding are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths.", "Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the alleged pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second fall and third winter waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses.", "Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history.", "His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. 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Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9.", "Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: taubenberger@afip.osd.mil The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus 2019-nCoV was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses.", "The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet s containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.", "Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses CoVs are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus αCoV , Betacoronavirus βCoV , Deltacoronavirus δCoV , and Gammacoronavirus γCoV . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens.", "Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV SARS-CoV which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV MERS-CoV which has caused a persistent epidemic in the Arabian Peninsula since 2012 . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host the Himalayan palm civet Paguma larvata for SARS-CoV and the dromedary camel Camelus dromedarius for MERS-CoV before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively .", "Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV HCoV-229E and HKU-NL63 and 4 βCoV HCoV-OC43 HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly . In contrast, SARS-CoV lineage B βCoV and MERS-CoV lineage C βCoV may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively . On 31 December 2019, the World Health Organization WHO was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 .", "As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan . The earliest date of symptom onset was 1 December 2019 . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 32% required intensive care and 6 15% died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan .", "All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank accession no. MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis.", "MN975262 Table 1 . The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.", "The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test 1000 replicates was shown next to the branches . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site . All ambiguous positions were removed for each sequence pair pairwise deletion option . Evolutionary analyses were conducted in MEGA X . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED .", "Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction PSIPRED . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics.", "Prediction of transmembrane domains was performed using the TMHMM 2.0 server Secondary structure prediction in the 5 ′ -untranslated region UTR and 3 ′ -UTR was performed using the RNAfold WebServer RNAWebSuite/RNAfold.cgi with minimum free energy MFE and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%.", "The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other Table 2 . There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV Table 3 . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits.", "The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain NTD and receptor-binding domain RBD , while the S2 subunit contains conserved fusion peptide FP , heptad repeat HR 1 and 2, transmembrane domain TM , and cytoplasmic domain CP . We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs SL-CoV ZXC21 and ZC45 and human SARS-CoV Figure 2 . Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B .", "Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV Figure 3 A , the core domain of RBD excluding the external subdomain are highly conserved Figure 3 B . Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor.", "Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain RBD when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 .", "The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b Figure 4 . It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV .", "Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses.", "The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients . In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences.", "In addition, we have previously identified two bat SARS-related-CoV Bat-CoV YNLF_31C and YNLF_34C and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 human SARS-CoV GZ02 , the mid-and late-phase SARS-CoV that includes the split orf8b human SARS-CoV Tor2 , civet SARS-CoV paguma SARS-CoV , two bat SARS-related-CoV containing full-length orf8 bat-CoV YNLF_31C and YNLF_34C , 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45 , and bat SARS-related-CoV HKU3-1 Figure 5 A . As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C .", "Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5 B which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet s containing six strands Figure 5 C . The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 MG772934.1 , and 82% with human SARS-CoV BJ01 2003 AY278488 and human SARS-CoV Tor2 AY274119 . The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes S, E, M, and N were shown Figure 6 A-E . For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8.", "For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 A-C , the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b.", "In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b marked in red , which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8.", "All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs .", "The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR Figure 7 D-F . The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "2019 Novel Coronavirus 2019-nCoV is an emerging infectious disease closely related to MERS-CoV and SARS-CoV that was first reported in Wuhan City, Hubei Province, China in December 2019. As of January 2020, cases of 2019-nCoV are continuing to be reported in other Eastern Asian countries as well as in the United States, Europe, Australia, and numerous other countries. An unusually high volume of domestic and international travel corresponding to the beginning of the 2020 Chinese New Year complicated initial identification and containment of infected persons. Due to the rapidly rising number of cases and reported deaths, all countries should be considered at risk of imported 2019-nCoV. Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease.", "Therefore, it is essential for prehospital, clinic, and emergency department personnel to be able to rapidly assess 2019-nCoV risk and take immediate actions if indicated. The Identify-Isolate-Inform 3I Tool, originally conceived for the initial detection and management of Ebola virus and later adjusted for other infectious agents, can be adapted for any emerging infectious disease. This paper reports a modification of the 3I Tool for use in the initial detection and management of patients under investigation for 2019-nCoV. After initial assessment for symptoms and epidemiological risk factors, including travel to affected areas and exposure to confirmed 2019-nCoV patients within 14 days, patients are classified in a risk-stratified system. Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV.", "Upon confirmation of a suspected 2019-nCoV case, affected persons must immediately be placed in airborne infection isolation and the appropriate public health agencies notified. This modified 3I Tool will assist emergency and primary care clinicians, as well as out-of-hospital providers, in effectively managing persons with suspected or confirmed 2019-nCoV. Text: 2019 Novel Coronavirus 2019-nCoV is a novel respiratory disease first reported in Wuhan, Hubei Province, China in December 2019. 1 Chinese health officials were originally investigating a sudden increase in cases of pneumonia which were later determined to be linked to 2019-nCoV. While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available.", "While most cases originated within mainland China, the disease spread to neighboring countries including Taiwan, Thailand, South Korea, and Japan, and later to the United States, Europe, and Australia. A near real-time updated tracking website for cases and locations worldwide, along with reported deaths is available. 2 Chinese health authorities have sequenced 2019-nCoV and freely shared its genetic profile online. 3, 4 Additionally, on January 28, 2020, an Australian laboratory reported growing the virus from a patient sample. As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin.", "As of January 30, 2020, there have been at least 9,776 persons infected and 213 verified deaths. 2 These numbers are likely underestimates due to the limited information available regarding incubation time, transmissibility, and virus origin. The What was the research question? Investigators adapted the \"Identify, Isolate, Inform\" 3I Tool for use in suspected cases of 2019-nCoV. What was the major finding of the study? A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients.", "A novel 2019-nCoV 3I Tool is designed for frontline clinicians in the management of suspected patients. This 2019-nCoV 3I adaptation will aid healthcare providers most likely to encounter the disease in the containment and effective treatment of patients. age distribution of these verified deaths is currently not available. One preliminary, small-scale study of 41 patients in Wuhan China, reported 6 deaths 15% mortality with a median age of 49.0 years. 5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported.", "5 Additionally, transmission of the virus has reportedly occurred in healthcare facilities in Wuhan City, raising concerns of spread to healthcare workers, as was seen during prior outbreaks of the novel coronaviruses, Middle Eastern Respiratory Syndrome MERS and Severe Acute Respiratory Syndrome SARS . Due to the dynamic nature of the outbreak, exposure criteria may change depending on where new cases of 2019-nCoV are detected, the degree of transmissibility, and when additional information regarding the origin of the virus is discovered and reported. On January 15, 2020, the Centers for Disease Control and Prevention CDC confirmed the first known imported case of 2019-nCoV in the US state of Washington. The patient had recently returned from Wuhan City, where he likely contracted the disease. Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020.", "Chicago health authorities reported a second US case on January 24, 2020. This was quickly followed by additional imported cases reported in Orange and Los Angeles Counties, California on January 26, 2020. Additional suspected cases continue to be evaluated. On January 30, 2020, the CDC reported the first local transmission in the US between members in a household. On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency.", "On the same day, the World Health Organization declared 2019-nCoV to be a Public Health Emergency of International Concern PHEIC . 6 On January 31, 2020, the US Department of Health and Human Services declared coronavirus a public health emergency. 7 Healthy individuals and those with mild illness may be asymptomatic, while others may have more pronounced symptoms of fever or lower respiratory illness. Upon identification of a suspected patient, that individual should immediately be isolated with airborne precautions. Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies.", "Further workup and laboratory confirmation can then proceed. Emergency physicians EPs , emergency medical services EMS personnel, and other healthcare workers who encounter patients with suspected 2019-nCoV infection must inform the appropriate authorities, including but not limited to hospital infection control and local or state public health agencies. Healthcare workers must follow on-going developments related to the outbreak, especially new information concerning detection and management. 8, 9 The 3I Tool outlined in this paper is consistent with current US CDC guidelines and can be applied in a variety of settings such as those in emergency departments, urgent-care clinics, physicians' offices, and prehospital settings. This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever.", "This paper will first briefly review 2019-nCoV and then present the novel 2019-nCoV 3I Tool as modified from its initial conception for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 Coronavirus 2019-nCoV infection commonly presents with signs and symptoms of pneumonia or as a nonspecific lower respiratory illness, with coughing or difficulty breathing accompanied by fever. 5, 19, 20 Fever and cough constitute the most common presentations. However, patients may have other respiratory symptoms, sore throat, nasal congestion, malaise, myalgia, and headache. Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock.", "Bilateral infiltrates may be seen on chest X-ray. Severe cases may present with sepsis and even shock. Conversely, some patients may present as only mildly ill or asymptomatic altogether. 21 To date, patients with underlying medical conditions and the elderly are more likely to become severely ill, require hospitalization, and ultimately die. 22 Early predictions for incubation time are between 2 and 14 days, based on data from similar coronaviruses. The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition.", "The 14-day criterion for epidemiological risk assumes the longest estimated incubation time. 23 In addition, the World Health Organization WHO has created its own interim case definition. 24 By definition, the main features of a novel virus, for example, how it is transmitted, will not be immediately known. However, as with the development of any 3I Tool, it is essential to understand specific characteristics of the disease. In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al.", "In the case of a novel virus such as 2019-CoV, this is challenging since information is rapidly evolving and the science is not yet fully understood. It is possible that the virus will undergo mutations over time that could substantially change its The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus Koenig et al. features. Nevertheless, an appreciation of the key concepts that drive evidence-based management is beneficial Table 1 . Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick.", "Management guidance will likely change over time. With the initial discovery of a new potential public health threat, it will likely be unclear how patients become sick. For example, rather than a contagion, there could be a contaminant or a toxin responsible for signs and symptoms. In this case, the possibility of an environmental toxin in the Wuhan Market was a consideration early on when limited to no human-tohuman transmission was reported. The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins.", "The mode of transmission has implications for the types of personal protective equipment PPE needed to protect healthcare providers in the prehospital, clinic, and hospital settings. 25 In addition, patients may need decontamination after exposure to certain toxins. 26 Another important consideration for application of the 3I Tool is whether the disease is contagious prior to symptom onset like measles or only after symptoms develop like Ebola . A January 30, 2020 letter to the New England Journal of Medicine describes a purported confirmed instance of transmission from an asymptomatic individual. Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question.", "Researchers state that, before symptom onset, the primary case infected two individuals, one of which infected two additional colleagues. 27 Subsequent investigation suggested that the source patient did have mild symptoms and had taken an antipyretic, calling this reported asymptomatic transmission into question. While quarantine may not be feasible and can have unintended consequences, 28, 29, 30 it is a public health tool that can be considered in cases when disease is transmissible before symptom onset. 30 Conversely, if a disease is known not to be transmissible prior to symptom onset, asymptomatic exposed patients must be monitored, but do not require quarantine or isolation unless they develop symptoms. Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur.", "Initially, it may be unclear whether an infectious agent occurred naturally or was deliberately or accidentally released. In this case, a BSL-4 laboratory studying coronaviruses was located approximately 32 kilometers away from the market where initial exposures were felt to occur. 31 Recall that in 2001, the anthrax letter attacks were initially thought to be naturally occurring. Once determined to be bioterrorism, management of the event was similar to that for a chemical exposure with a sudden impact, defined scene, and need for a rapid response and decontamination on site. This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread.", "This differed from the WHO's modeling predicting an aerosolized release that would result in an incubation period with 100,000 or more persons exposed rather than the 22 people who contracted anthrax in 2001. 32 By understanding the key features of a novel disease, healthcare workers can take evidence-based measures to protect themselves, optimize individual patient management, and prevent further disease spread. It is currently unclear how 2019-nCoV is spread, but it is suspected to be transmitted through contact with infected respiratory secretions, like other known coronaviruses. There are instances of sustained human-to-human transmission across generations of cases, especially near the epicenter in Wuhan City. 21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE.", "21 Current evidence suggests that close contact with an infected person is a major factor in disease transmission. CDC defines \"close contact\" 33 as being in or within two meters of an area with a confirmed patient or being directly exposed to infectious secretions without appropriate PPE. Healthcare facilities in China have reported spread from person to person. In addition, some mildly ill or potentially even asymptomatic patients may have a higher chance of spreading the disease to others as they may be less likely to seek medical care. 34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries.", "34 The possibility that patients may be infectious prior to symptom onset further compounds the difficulty of containing the virus and effectively preventing transmission. The current majority of 2019-nCoV cases have been within China and its bordering countries. 2 Persons with recent travel within 14 days to Wuhan City or another region with widespread disease, or exposure to a patient under investigation, are considered to have an epidemiologic risk factor and should be assessed for signs and symptoms of a viral illness such as fever and respiratory symptoms. Coronavirus is a zoonotic virus The Identify-Isolate-Inform 3I Tool Applied to a Novel Emerging Coronavirus that is transmitted to humans via contact with infected animals. Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory.", "Preliminary reports suggest the disease may have originated in a seafood and live animal market in Wuhan City, but it is still unknown how or whether such transmission occurred. Clinicians working with local public health departments must arrange to have specimens from patients under investigation PUIs sent to the CDC laboratory. At this time, the CDC has the only laboratory that can definitively test for 2019-nCoV, though laboratory testing capacity is being rapidly expanded. Polymerase chain reaction PCR assays conducted on samples from a patient's upper and lower respiratory tracts will be used to confirm potential cases. In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website.", "In addition, serum antibody titers can be analyzed for confirmation of infection or evidence of immunity. Up-to-date information about the needed specimens and handling requirements to test for 2019-nCoV are available on the CDC website. 35 Like other related coronaviruses, patients with 2019-nCoV frequently present with non-specific symptoms resembling that of influenza. Physicians may consider differential diagnoses related to a wide variety of respiratory infections. In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV.", "In order to relate these symptoms to 2019-nCoV, it is imperative that the identification of a potential exposure event epidemiologic risk factor within 14 days of symptom onset is made so that a more focused work-up for 2019-nCoV can be completed. Although the likelihood of coinfection of 2019-nCoV and another respiratory virus is thought to be low, a positive finding of another respiratory pathogen does not exclude the diagnosis of 2019-nCoV. Many commercially available respiratory panels include \"coronavirus\" in the results, but neither a positive nor a negative finding on these panels should be used to include or exclude a diagnosis of 2019-nCoV. Supportive care with appropriate infection control is the mainstay of current CDC treatment guidelines for 2019-nCoV. There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments.", "There are not yet any approved antiviral treatments for 2019-nCoV. Emergency Use Authorizations EUA for compassionate use cases may be forthcoming from the US federal government for normally unapproved treatments. Supportive treatment predominantly includes respiratory support, hydration, and antipyretics. General treatment for severe cases should focus on the preservation of vital organ function. In the future, antiviral medications may be available. If a secondary bacterial infection such as pneumonia develops, targeted antibiotics are indicated. Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis.", "Prevention of 2019-nCoV transmission, like any other infectious agent, involves minimizing risk of exposure. Vaccines are under accelerated development and may be useful in the future for post-exposure prophylaxis. Healthcare personnel are at increased risk and should practice standard, droplet, and airborne precautions when encountering an infected person, a PUI, or any symptomatic close contacts. Healthcare workers handling specimens should also adhere to CDC guidelines and should not attempt to perform any virus isolation or characterization. Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset.", "Fever screening has been implemented at numerous airports, including major international hubs within Asia and the US. The efficacy of this intervention is not well documented, however, as some infected persons may be afebrile and disease transmission might occur prior to symptom onset. 27 In addition, people can artificially lower their temperature readings, e.g., by applying ice to their foreheads. As outlined above, admission criteria for 2019-nCoV are similar to that of other patients. If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted.", "If patients do not meet medical criteria for hospitalization, they may be discharged home with isolation precautions and continued observation. EPs must notify local public health authorities so appropriate monitoring and community protective measures can be instituted. The Identify-Isolate-Inform 3I Tool was initially developed for Ebola virus disease 10,11 and later adapted for measles, 12 MERS, 13 mumps, 14 Zika virus disease, 15 hepatitis A, 16 pertussis, 17 and scabies. 18 This novel tool for suspected 2019-nCoV patients Figure 1 provides frontline clinicians with a simple algorithm to manage an emerging disease. Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk.", "Identification of exposed patients with an epidemiologic risk factor within 14 days of symptom onset is a crucial first step. An automatic prompt in the electronic health record can be useful in assisting clinicians with early identification of patients at risk. Case definitions promulgated by the WHO 24 and CDC 33 provide useful comprehensive definitions that have been incorporated into the 3I Tool. The 2019-nCoV Tool provides an accurate, summarized algorithm to immediately, and effectively manage suspected patients until additional resources can be consulted. Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept.", "Patients who do not have an exposure risk or any symptoms may be triaged normally. However, before making patient contact, providers must first apply the Vital Sign Zero concept. 36 Vital Sign Zero is a preliminary, non-contact assessment i.e., performed prior to touching a patient to take traditional vital signs to first determine whether specific PPE is indicated before the examination commences. By taking the additional time to complete this assessment, risk of exposure and further transmission can be minimized. while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities.", "while in the treatment facility should be started and maintained to assist with the possibility of contact tracing. Following isolation, physicians should immediately inform the appropriate authorities. Patients who do not meet medical criteria for admission can be isolated at home during the evaluation phase. 37 Health department officials can help prevent transmission in isolated patients by providing in-home monitoring and implementing appropriate exposure-control measures. Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever.", "Providers in the prehospital setting who have a high likelihood of encountering 2019-nCoV patients, such as those near international ports of entry, should adhere to established exposure control guidelines. 38 Along with appropriate PPE, providers should also carry thermometers to quantify any fever. In the US, providers should contact the appropriate CDC quarantine station upon isolation of infected or suspected patients, especially those from Wuhan, China or other regions with widespread disease, who report symptoms in the last 14 days. As for other infectious diseases, assessing travel history is essential. Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV.", "Dispatch protocols have been instituted to facilitate identification of callers to 911 or the country-equivalent emergency number prior to prehospital personnel arrival. 39 In addition, CDC has promulgated EMS guidelines for prehospital PPE, transportation of PUIs, vehicle decontamination, and 911 Public Safety Answering Points PSAPs for 2019-nCoV. 40 2019-nCoV is an emerging infectious disease with rapidly evolving features, the full scope of which will be defined over time. Prior outbreaks of coronaviruses can help inform needed actions in the short term to assist with both treatment of individual patients and prevention of global disease spread. This adaptation of the Identify-Isolate-Inform Tool serves as a resource for healthcare workers who need to make clear, rapid assessments when confronted with potential patients. The concise nature of the 2019-nCoV 3I Tool allows for the rapid and effective management of a novel disease by healthcare providers." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]

[ "nan Text: Over 168 million people across 50 countries are estimated to need humanitarian assistance in 2020 . Response to epidemics in complex humanitarian crisessuch as the recent cholera epidemic in Yemen and the Ebola epidemic in the Democratic Republic of Congois a global health challenge of increasing scale . The thousands of Yemeni and Congolese who have died in these years-long epidemics demonstrate the difficulty of combatting even well-known pathogens in humanitarian settings. The novel severe acute respiratory syndrome coronavirus-2 SARS-CoV-2 may represent a still greater threat to those in complex humanitarian crises, which lack the infrastructure, support, and health systems to mount a comprehensive response. Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization.", "Poor governance, public distrust, and political violence may further undermine interventions in these settings. Populations affected by humanitarian crises are expected to be particularly susceptible to COVID-19, the disease caused by SARS-CoV-2, due to displacement, crowded housing, malnutrition, inadequate water, sanitation, and hygiene WASH tools, and stigmatization. Disease outbreaks further reduce access to limited healthcare, which is increasingly disrupted by attacks on health facilities and the persistent overburdening of health systems. These situations escalate both the necessity and the difficulty of delivering accurate and actionable information to potentially affected populations . As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings.", "As the international community responds to SARS-CoV-2, public health authorities in humanitarian crises begin at a disadvantage to enact appropriate infection control to prevent transmission in healthcare settings, identify infectious cases, administer supportive care and novel treatments for the seriously ill, and trace contacts. These standard public health measures are particularly difficult to perform in humanitarian settings. For example, limited public health, laboratory, and primary care services represent a barrier to testing. Providing the limited healthcare worker cadre with appropriate training and personal protective equipment, and ensuring a continuous supply chain for such, is a challenge in all settings, exacerbated in complex humanitarian crises. Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill.", "Frequent displacement and limited contact information may prevent effective contact tracing. Finally, intractable structural challenges such as overcrowding limit the implementation of both quarantine of those exposed and isolation of those who are ill. Given these increased vulnerabilities, humanitarian crises should be viewed as a priority for national and international bodies that seek to combat this unfolding pandemic. Resources must be identified to protect healthcare workers, develop and deploy rapid testing, improve surveillance, and enact quarantine and isolation of contacts and cases. To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing .", "To mitigate the impact of COVID-19 on crisesaffected populations, governments and agencies will implement the familiar, global evidence-based approaches for combatting respiratory viruses. Respiratory hygiene is a highly effective public health intervention, supported by evidence demonstrating that the spread of respiratory viruses, such as SARS-CoV-2, can be prevented by hand hygiene, safe cough practice, and social distancing . Hand hygiene is a readily implemented behavior: the distribution of soap to households in humanitarian settings has been shown to increase handwashing by over 30% . Furthermore, hand hygiene is an avenue of agency for protecting one's own health, consistent with the rights to dignity and to fully participate in decisions related to assistance in humanitarian crises. Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance .", "Widespread introduction of alcohol-based hand rubs is also possible in many resource-limited settings, with published protocols for local production . The Sphere Handbook, a collection of rights-based guidelines for humanitarian response, is the foremost authority on minimum standards for humanitarian assistance . However, despite the indisputable evidence for the efficacy of hand hygiene for reducing both bacterial and viral pathogen transmission, humanitarian WASH standards are based on evidence pertaining to the prevention of illnesses transmitted by the faecal-oral route, with the focus on hand hygiene proximate to latrines . And yet, latrines in crisis settings are often shared and distant from residential shelters, conferring a high risk of gender-based violence . Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic.", "Gender-based violence around latrines is an important deterrent for accessing latrine-adjacent handwashing stations, particularly for hand hygiene to prevent respiratory pathogen transmission. Evidence-based guidelines alone in complex humanitarian crises may not suffice during the emergence of the current SARS-CoV-2 pandemic. Without the adaptation of existing standards, mitigation plans will fall short of health and human rights obligations in outbreak response. Crisis-affected community engagement is integral in pandemic planning, in order to maximize the real-world effectiveness of efficacious interventions. Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings .", "Transparent and credible information-sharing mechanisms are increasingly essential when pandemics threaten vulnerable populations . Diplomacy bridging long-standing mistrust of public health and biomedical interventions and facilitating engagement with contentious actors is a necessary component of effective health governance in complex crisis settings . Interventions tailored to the needs of crisis-affected populations, delivered with transparent information, in the context of inclusive governance practices, are urgently needed in the global response to the COVID-19 pandemic." ]