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Identifying Influenza Viruses with Resequencing Microarrays
research-article
Author(s):
Zheng Wang
*
,
† ,
Luke T. Daum
‡ ,
Gary J. Vora
* ,
David Metzgar
§ ,
Elizabeth A. Walter
¶ ,
Linda C. Canas
‡ ,
Anthony P. Malanoski
* ,
Baochuan Lin
* ,
David A. Stenger
*
,
Publication date (Print):
April 2006
Journal:
Emerging Infectious Diseases
Publisher:
Centers for Disease Control and Prevention
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Abstract
Resequencing microarrays rapidly identify influenza viruses.
Abstract
Identification of genetic variations of influenza viruses is essential for epidemic and pandemic outbreak surveillance and determination of vaccine strain selection. In this study, we combined a random amplification strategy with high-density resequencing microarray technology to demonstrate simultaneous detection and sequence-based typing of 25 geographically distributed human influenza virus strains collected in 2004 and 2005. In addition to identification, this method provided primary sequence information, which suggested that distinct lineages of influenza viruses co-circulated during the 2004–2005 season, and simultaneously identified and typed all component strains of the trivalent FluMist intranasal vaccine. The results demonstrate a novel, timely, and unbiased method for the molecular epidemiologic surveillance of influenza viruses.
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Most cited references22
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Influenza: lessons from past pandemics, warnings from current incidents.
Taisuke Horimoto, Yoshihiro Kawaoka (2005)
Recent outbreaks of highly pathogenic avian influenza A virus infections (H5 and H7 subtypes) in poultry and in humans (through direct contact with infected birds) have had important economic repercussions and have raised concerns that a new influenza pandemic will occur in the near future. The eradication of pathogenic avian influenza viruses seems to be the most effective way to prevent influenza pandemics, although this strategy has not proven successful so far. Here, we review the molecular factors that contribute to the emergence of pandemic strains.
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Whole-Genome Analysis of Human Influenza A Virus Reveals Multiple Persistent Lineages and Reassortment among Recent H3N2 Viruses
Edward C Holmes, Elodie Ghedin, Naomi Miller … (2005)
Introduction Influenza A viruses are negative-strand RNA viruses of the Family Orthomyxoviridae that infect a wide variety of warm-blooded animals, including domestic and wild birds and mammals (e.g., humans, pigs, and horses). The natural reservoir for influenza virus is thought to be wild waterfowl, and genetic material from avian strains episodically emerges in strains infectious to humans. These human viruses continually circulate in yearly epidemics (mainly during the winter months in temperate climates), and antigenically novel strains emerge sporadically as pandemic viruses [1,2]. In the United States, influenza is estimated to kill 30,000 people in an average year [3,4]. Every few years, influenza epidemics boost the annual mortality level above this average, causing 10,000–15,000 additional deaths. Occasionally, and unpredictably, global pandemics of influenza occur, infecting 20% to 40% of the population in a single year and raising death rates dramatically above normal levels. Pandemic influenza A viruses emerged three times during the last century: in 1918 (H1N1 subtype), in 1957 (H2N2), and in 1968 (H3N2) [2,5]. The recent circulation of highly pathogenic avian H5N1 viruses in Asia from 2003–2005 has caused at least 52 human deaths [6,7] and has raised concern about the development of a new pandemic [5]. How and when novel influenza viruses emerge as pandemic strains and their precise mechanisms of pathogenesis are still not understood. While the risk of pandemic influenza poses a significant public health concern, inter-pandemic or epidemic influenza remains a major cause of morbidity and mortality. The influenza A surface glycoprotein hemagglutinin (HA) protein is under selective pressure for change in order to evade the host's immune system [8]. Antibodies against the HA protein inhibit receptor binding and are very effective at preventing reinfection with the same strain. However, HA can change to evade previously acquired immunity either by antigenic drift, whereby mutations of the currently circulating HA gene disrupt antibody binding, or by antigenic shift, in which the virus acquires an HA of a new subtype by reassortment of one or more gene segments. While it is generally accepted that drift is responsible for inter-pandemic influenza outbreaks and shift for pandemics, there are exceptions to this rule. For example, in 1977, an H1N1 virus re-emerged but failed either to cause a pandemic or to replace the prevailing H3N2 subtype [9]. The importance of predicting the emergence of new circulating influenza strains for subsequent annual vaccine development cannot be underestimated [10]. To this end, the global influenza surveillance network coordinated by the World Health Organization was established to select the candidate strains of influenza A and B for the yearly production of influenza vaccine in both the northern and southern hemispheres. The network characterizes the antigenic properties of influenza viruses using HA inhibition assays and sequencing of the HA1 domain (globular head) of HA of a select number of strains [11,12]. Antigenic, genetic, and epidemiologic data are then examined to make recommendations of candidate vaccine strains. A number of retrospective studies have been performed using partial HA gene sequences to understand, and sometimes predict, the evolution of human H3N2 strains [13–17]. Accumulation of amino acid replacements in HA is clustered in five variable antigenic sites [18] around the receptor binding site. Phylogenetic analysis has revealed that 18 codons in the HA1 domain exhibit significantly more nonsynonymous nucleotide substitutions than synonymous ones, constituting a signature of strong, selectively driven, antigenic drift [15]. More recently the antigenic and genetic evolution of HA was compared through the construction of an antigenic map of H3N2 viruses [17]. Although antigenic evolution was found to be more punctuated than genetic evolution over the same time period, the two measures of HA drift were generally correlated. Despite the wealth of data on the molecular evolution of influenza viruses, how the entire genome of influenza A virus evolves during epidemic years is unclear, particularly as past sample sizes have been inadequate. While antigenic drift of HA is clearly of vital importance in the survival of an influenza strain, other factors, including HA receptor binding specificity [19], antigenic drift of neuraminidase (NA) [20], matched activity between HA and NA [21–23], and the interaction of the other influenza proteins with each other and their host cells, are all likely to affect viral fitness in a polygenic manner. Similarly, it is unclear how many lineages of influenza A viruses persist between seasonal epidemics, particularly in genes other than that encoding HA. To this end the National Institute of Allergy and Infectious Diseases of the National Institutes of Health has funded the Influenza Genome Sequencing Project with several partners [24,25] including the Institute for Genomic Research. To date, 156 genomes of human H3N2 viruses collected between 1999 and 2004 from New York State have been completely sequenced and deposited in GenBank. We have performed an initial analysis of these viruses and have found evidence for both the existence of multiple clades of viruses co-circulating at the same time point and for multiple reassortment events among these clades. One of these reassortment events was the likely progenitor of the A/Fujian/411/2002-like drift epidemic of the 2003–2004 influenza season, in which there was a poor match between the vaccine strain and the predominant circulating viruses of that year [26,27]. This report extends recent observations of reassortant H3N2 influenza A viruses from the southern hemisphere during this same time period [28]. Results Analysis of the Concatenated Complete Genome of the New York State Influenza Isolates Three major clusters of sequences were apparent in phylogenetic trees of the 156 complete genomes of H3N2 influenza A viruses sampled from New York State. These corresponded to particular influenza seasons (winter months): (a) 1999–2000, (b) 2001–2002 and 2002–2003 together (although only five members of latter season are present in these data), and (c) 2003–2004 (Figure 1). Such temporal structure is commonly observed in trees of influenza A virus, and this is thought to be largely driven by positive selection acting on the HA gene [17]. However, a number of isolates did not fall into these three groups. First, three isolates from the 2002–2003 and 2003–2004 seasons—A/New York/32/2003, A/New York/198/2003, and A/New York/199/2003—formed a distinct phylogenetic group whose closest relationship was with those viruses sampled in the 1999–2000 season, rather than with those sampled during later seasons. We have denoted the two groups of viruses circulating after the 2001–2002 season as clade A (the major cluster after the 2001–2002 season) and clade B (the minor cluster comprising isolates 32, 198, and 199). Second, isolates A/New York/52/2004 and A/New York/59/2003 (designated as clade C in Figure 1) occupied a position intermediate between clade A and the 2001–2002 group of viruses, as did isolate A/New York/11/2003. Finally, isolates A/New York/137/1999 and A/New York/138/1999 formed a group that was divergent from the main group of viruses sampled in the 1999–2000 season. Analysis of Individual Gene Segments To investigate the evolutionary history of the outlier viruses in more detail we inferred phylogenetic trees for each of the eight individual gene segments (Figure 2). Strikingly, although the distinction between clades A, B, and C was apparent in seven of the eight genes, no such separation was seen in the HA phylogeny. In this case, clades B and C clearly clustered within clade A and with strong bootstrap support. The close phylogenetic relationship of these three groups of viruses in HA set against a background of genetic divergence in all other segments strongly suggests that these data contain evidence for at least two independent reassortment events, one involving clades A and B and another involving clade C and either clade A or clade B. In the case of the clade C viruses, the separate gene phylogenies also reveal that these isolates share a common ancestry with viruses first sampled during the 2001–2002 season, while the clade B viruses share a closer relationship with those viruses of the 1999–2000 season. Two more major phylogenetic displacements suggestive of reassortment involving other segments were similarly identified. First, isolate A/New York/11/2003, which fell within clade A in seven of the gene trees (including HA), clustered with clade B viruses in PB2. Consequently, isolate A/New York/11/2003 represents a reassortment of two segments between clades A and B. Second, isolate A/New York/182/2000, which clustered with the main set of viruses sampled during the 1999–2000 season in most of the gene trees, was very closely related to the divergent A/New York/137/1999 and A/New York/138/1999 isolates in PA and M1, although the high degree of genetic similarity among all viruses in M1 precludes a further analysis of reassortment in this case. To determine the direction of the reassortment events in HA, we inferred phylogenetic trees of larger datasets comprising the New York State isolates and representatives of the other human and swine H3N2 viruses sampled during the same time period. Because sequences from the core genes have only been sporadically collected, this analysis necessarily focused on HA and NA. As expected from the phylogenetic analysis of the New York State viruses, the distinction between clades A, B, and C was apparent in the NA gene tree (Figure 3) as well as the core genes (trees shown in Figure S1). Moreover, in the NA gene tree, viruses sampled from a variety of locations during 2000–2005 fell into clade B, including Europe (Denmark and Norway), Asia (China and Singapore), and the Americas (Argentina, Brazil, and the United States). Hence, although clade B was at low frequency in the New York State dataset, it represents a distinct lineage of H3N2 viruses globally circulating from at least 2000 to 2005. Similarly, a third North American virus, A/Charlottesville/03/2004, was designated as clade C. A very different evolutionary history was revealed in HA. In this case, clade A of the New York State viruses expanded to contain the majority of viruses sampled after 2002 and from a variety of locations (Asia, Australasia, Europe, and North America), as well as a number of Asian viruses from 2002 (Figure 4). This large group of viruses then clustered within clade B, such that most clade B viruses sampled from 2000 to 2002 formed a clear, but closely related, outgroup to the later clade A viruses, with the remaining clade B viruses falling within clade A (Figure 4). The three clade C viruses also fell within this expanded clade A. Such a phylogenetic pattern strongly suggests that the HA from both the clade A and clade C viruses was acquired from that present in clade B through reassortment. In this context it is of interest to note that the HA of the A/Fujian/411/2002 virus and related isolates are present in this reassorted clade. Further, the fact that the clade A and B isolates closest to the phylogenetic location of the reassortment event were both sampled in 2002 suggests that the reassortment occurred in this year, although pinpointing the exact phylogenetic location of the recombinant event is difficult given the relatively small number of samples available from this critical time period. Similarly, the fact that these viruses had Asian origins is also compatible with the reassortment event occurring in this region, a hypothesis also supported by a recent analysis of comparable partial genomic analysis of H3N2 isolates from the southern hemisphere [28]. Analysis of the Coding Differences between Clades A, B, and C Because both clade A and clade B contain viruses sampled on a near global basis, it is important to determine possible phenotypic differences between them. Table 1 shows the amino acid replacements that consistently distinguish the clade A and B viruses. These changes are not uniformly distributed among the seven segments (not including HA). Of the 48 amino acid differences between clades A and B, 14 fall in NA and nine in NP. PB1, PB2, and PA have five differences each, while M2 and NS1 have three differences, and M1 and NS2 have two. In contrast, there are only nine amino acid differences between the clades A and C: PB2—T9N; PA—A20T, L226I, and N272D; NP—G450S; NA—H40Y and V263I; and NS1—A56T and N143T. Discussion Our analysis of whole genomes of H3N2 influenza A viruses sampled during 1999–2004 has identified two key evolutionary patterns. First, although the majority of viruses isolated after 2002 fall into a single phylogenetic group (clade A), multiple, co-circulating viral lineages are present at particular time points. The genetic diversity of influenza A virus is therefore not as restricted as previously suggested, particularly when genes other than that encoding HA are analyzed. This co-circulation of lineages is most apparent with the identification of three clades of H3N2 viruses that appear to infect the same populations until 2002, after which they acquired a common HA gene through reassortment. Second, and more dramatically, these multiple, co-circulating lineages may have complex genealogical histories and interact through reassortment. Indeed, we have documented two reassortment events involving the HA gene of clade B: one in which it was acquired by the clade A viruses and another in which it was independently acquired by those isolates assigned to clade C. Two further reassortment events involving the PB2 and PA genes were also evident from our phylogenetic analysis. Given that we are only able to reliably detect reassortment when it is associated with major changes in tree topology, it is likely that reassortment among closely related lineages is also commonplace in influenza A viruses. Reassortment between influenza A viruses has been described in both human and animal viruses [1,29]. Notably, antigenic shift by reassortment between human and avian influenza A viruses has been documented in the formation of the 1957 H2N2 and 1968 H3N2 pandemics [30–32]. Other recent examples of reassortment between human and animal influenza A viruses have resulted in the emergence of novel H3N2 and H1N2 swine viruses in North America and Europe [33,34] and the evolution of H5N1 viruses in Asia from 1997 to the present [35]. Reassortment between co-circulating lineages of human influenza A and more recently influenza B viruses following mixed infection has also been described [36–41]. For example, human H2N2 viruses formed two distinct clades in the 1960s prior to the emergence of the 1968 H3N2 pandemic virus, with one virus a reassortant containing genes of both clades [42]. Similarly, the early H3N2 viruses (1968–1972) acquired the H3 HA and the PB1 gene via reassortment with an avian virus [30,31]. Reassortment between H3N2 and H2N2 viruses may therefore have assisted successful cross-species transmission [42]. Reassortant viruses were also described following the re-emergence of the H1N1 subtype in 1977 that did not replace the previously circulating H3N2 viruses. In this case, co-circulation of influenza viruses of both subtypes continued, and co-infection with both subtypes was reported [43]. While reassortant H3N1 strains were not isolated, H1N1 strains containing reassorted internal protein-encoding gene segments from H3N2 viruses were observed [44,45]. Occasional isolates of H1N2 viruses were also detected after the re-emergence of H1N1 [46,47]. More recently, the widespread circulation of viruses with the H1N2 subtype has been documented [41]. These viruses contained the HA segment of contemporary H1N1 viruses reassorted onto an H3N2 background, a 7:1 reassortment pattern similar to that observed with the sporadically circulating H1N2 viruses of the 1980s and early 1990s [47] and to the dominant reassortments described in this analysis. Since the H1 and N2 subtype proteins were antigenically and genetically similar to co-circulating H1N1 and H3N2 subtype viruses, the emergence of this new subtype did not result in an epidemiologically significant event [41]. Reassortment among co-circulating clades of H3N2 viruses like that observed in the current study has also been previously described, including reassortment of the NA gene segment [48] and the core protein-encoding segments [49]. Most prior phylogenetic studies of human influenza A have suggested that inter-pandemic evolution may be essentially described as a series of successions by variants of the previous season's dominant strain. These successions are largely determined by strong positive selection acting on the abundance of mutational diversity in the HA of the dominant strain. However, we found that at least four reassortment events occurred among human viruses during the period 1999–2004 and that two of these involved a major change in HA. Recently, Barr et al. independently provided phylogenetic evidence of the clade A–clade B reassortment described here in an analysis of predominantly southern hemispheric influenza A H3N2 isolates collected during the same period [28]. To our knowledge, these analyses are the first demonstrations of the emergence of a major antigenically variant virus derived by reassortment between two distinct clades of co-circulating H3N2 viruses rather than by antigenic drift. These findings suggest that the ongoing evolution of human influenza A virus is likely to be more complex than depicted in standard models of antigenic drift; multiple lineages of antigenically distinct viruses can persist within populations and, through their reassortment, produce major changes in antigen space. Similarly, the persistence of multiple lineages of H3N2 within a single population indicates that human populations represent a larger reservoir of genetically distinct viruses than previously anticipated. Indeed, it is possible that key changes in antigen type, depicted as jumps between cluster types [17], could be strongly influenced by reassortment among co-circulating human strains. Crucially, the real importance of both lineage persistence and reassortment in influenza A virus evolution could not be determined until a representative sample of full-genome sequences was collected from a single population. In the 2003–2004 influenza season, a major drift variant emerged in both the northern and southern hemispheres [50–52]—the A/Fujian/411/2002-like variant. In the United States, the 2001–2002 influenza was an H3N2-predominant season, and all antigenically characterized isolates matched the A/Moscow/10/1999 vaccine strain [53]. In the 2002–2003 season, which was an H1- and influenza B–dominant season in the United States, a minority of antigenically characterized H3N2 isolates were different from the Moscow/10/1999-like vaccine strain [54], probably coinciding with the emergence of the Fujian strain. Indeed, in our phylogenetic analysis two of five H3N2 viruses from the 2002–2003 season fall into clade B, along with two newly sequenced isolates that were not included in our analysis (A/New York/201/2003 and A/New York/203/2003). Consequently, clade B viruses made up a significant fraction of the few H3N2 isolates in that season. In contrast, only a small minority of influenza H3N2 viruses sampled in Europe at this time were antigenically characterized as Fujian-like [50], while in the southern hemisphere's 2003 influenza season the Fujian strain was predominant [28,51]. The 2003–2004 influenza season in the northern hemisphere was also dominated by the Fujian strain [55], although the vaccine contained the H3N2 (A/Panama/2007/1999) from the previous year. This strain was a poor antigenic match to the Fujian strain [26,27], which in turn led to reduced vaccine effectiveness. Thirteen amino acid changes distributed across the five antigenic sites of the HA1 domain distinguish the A/Panama/2007/1999-like and A/Fujian/411/2002-like strains. While these replacement changes appear to have phenotypic consequences (e.g., replication efficiency in eggs), only two residues, 155 and 156, are responsible for the major antigenic differences between the strains [27]. Although data are insufficient for precise determination of the timing of these two critical mutations, the available data are most consistent with these changes occurring in a relatively short time period before the reassortment event. The histidine to threonine change at site 155 and the glutamine to histidine change at site 156 are present in all the clade A reassortant isolates as well as in clade B isolates from 2003–2004, thus suggesting that they occurred prior to the reassortment event. No available clade B isolates prior to 2002–2003 have either of these mutations, and we were able to identify only three “intermediate” isolates from 2002–2003 (A/Kwangju/219/2002, A/Kwangju/243/2002, and A/Cheonnam/340/2002) with the replacement at site 155 but not at site 156. Overall, the data presented here, coupled with those recently reported [28], reveal that the HA segment of the H3N2 clade B viruses, present in low frequencies at least since 1999, was reassorted into clade A of H3N2, probably in 2002 soon after the appearance of these two critical mutations, and that this reassortment was central to the production of antigenically variant strains that were poorly matched to the vaccine strain in the 2003–2004 season [27]. In addition, presumably because of the high rate of reassortment and the fitness advantage conferred by these two mutations, this clade B HA segment also appeared to replace the HA segment of the clade C strains. Finally, though previously present only in low frequencies, recent sequencing of HA and NA by investigators in Denmark [56] and published phylogenetic trees from Australia [28,51] show the existence of clade B virus, suggesting that it continued to have a global distribution and sometimes at appreciable frequency. Several questions remain unanswered by our study. Since the HA donated by clade B led to a major expansion of the reassorted clade A, it is uncertain why clade B did not initially out-compete clade A without reassortment. One possibility is that the HA of clade B had an intrinsically higher fitness than other HAs circulating at the same time but was unable to reach a high frequency in the New York population owing to linkage to mutations located in other segments that reduced the overall fitness of this genotype. According to this hypothesis, it was not until it was placed by reassortment into a more favorable genetic background, in this case the clade A viruses, that its fitness advantage was realized. Since clade B itself appeared to proliferate in other regions, it will be useful to analyze whole-genome sequence from these isolates when they are available. More generally, it is clear that the genotypic basis to viral fitness has not been entirely elucidated. In particular, it is likely that interactions among viral proteins and between viral proteins and host factors play a key role. In this respect it is notable that of the 48 amino acid differences that distinguish the clade B viruses, nine fall in NP and 14 fall in NA (see Table 1). In NP, the replacement at residue 425 falls within an HLA-B35-restricted cytotoxic T lymphocyte (CTL) epitope and is not commonly seen in human populations [57]. Two other changes, at residues 27 and 197, have also been identified as being contained in two HLA-A11-restricted CTL epitopes [58]. Another unusual change in NP is M136I: a change to methionine at this site was proposed as one of six human adaptive changes distinguishing the 1918 NP from avian NPs [59]. Indeed, all 45 available NP sequences from human H2N2 viruses, and all but four of approximately 250 human H3N2 NP sequences, maintain this methionine. Only the three New York clade B viruses, as well as A/Taiwan/1/71, have a change to an isoleucine at this residue. Of the changes in NA, six of them lie in five regions previously identified as being phylogenetically important regions, residues 40, 42, 143, 199, 307, and 385 [60], and therefore play a role in virus–host interaction. Three further changes, at residues 172, 199, and 307, map to antigenic sites [20], while a change at residue 18 has been mapped as an HLA-A2-restricted CTL epitope [58]. Similarly, two changes in M2, at residues 51 and 56, map to an HLA-A11-restricted CTL epitope, while residue 82 of NS1 maps to an HLA-A2-restricted CTL epitope [58]. Another change between clade A and B viruses, at residue 226 of PA, also maps to an HLA-A2-restricted CTL epitope [58]. It is possible that some of the mutations that fall in CTL epitopes assist the persistence of clade B by elongating the viral infectious period [61]. However, any combination of the constellation of amino acid changes may have altered the fitness of the clade B viruses in a way that we do not have the ability to understand from sequence analysis alone. Interestingly, Gulati et al. [62] have recently shown that Fujian-like strains have a mismatch in their HA and NA activities that is probably the result of the reassortment event described in the current study. The significance of this for the pathophysiology of the virus is currently unknown. In summary, our study clearly demonstrates the utility of whole-genome analyses of influenza A viruses, and further makes clear that additional whole-genome analyses are required to understand fully the evolutionary mechanisms and epidemiological dynamics of this virus. While antigenic variance of HA is still the dominant selective pressure on human influenza A virus evolution, the finding that antigenically novel clades emerge by reassortment among persistent viral lineages rather than via antigenic drift is of major significance for vaccine strain selection. Materials and Methods Influenza viruses used in this study The influenza virus isolates were collected as part of the diagnostic service provided by the Virus Reference and Surveillance Laboratory at the Wadsworth Center, New York State Department of Health. Viruses were received as part of outbreak investigations, through the reference function of the laboratory, and, since 2001, as part of a sentinel physician influenza surveillance program. Viruses were passaged minimally in primary rhesus monkey kidney cell culture and the RNA extracted from the clarified supernatant. Whole-genome sequence information was derived at the Institute for Genomic Research using methods described elsewhere (E. Ghedin, N. A. Miller, M. Shumway, J. Zaborsky, T. Feldblyum, et al., unpublished data). Use of the diagnostic samples in this study was approved by the New York State Department of Health Institutional Review Board. Sequences used in the analysis Sequence data for 156 complete genomes of influenza A virus (H3N2) sampled from New York State during the period 1999–2004 were downloaded from GenBank (1,248 separate accessions, representing the eight gene segments of 156 individual influenza isolates; GenBank accession numbers available from http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Separate sequence alignments were then manually compiled for the major coding regions of each segment: PB2, 2,277 bp; PB1, 2,271 bp (PB1 protein); PA, 2,148 bp; HA, 1,698 bp; NP, 1,494 bp; NA, 1,407 bp; MP, 756 bp (M1 protein); and NS, 690 bp (NS1 protein). An alignment of the concatenated major coding regions was also constructed for all 156 isolates (12,741 bp). To place the New York State viruses in the context of global H3N2 evolution, larger datasets were complied comprising the New York State isolates plus all other mammalian influenza A viruses sampled from 1999–2004 available on GenBank and the Los Alamos Influenza Sequence Database (http://www.flu.lanl.gov/). For the six core genes, few such background sequences were available, particularly from clades B and C (see below). However, far larger numbers of background sequences were available for the HA1 domain portion of HA and for sequence of NA genes. To facilitate the computational analysis of these background datasets, those New York State isolates with identical sequences were removed from the analysis. This resulted in alignments of the following sizes: PB2, 134 sequences, 2,277 bp; PB1, 135 sequences, 2,271 bp; PA, 140 sequences, 2,148 bp; HA, 256 sequences, 987 bp; NP, 117 sequences, 1,494 bp; NA, 197 sequences, 1,407 bp; M1, 73 sequences, 756 bp; and NS1, 75 sequences, 690 bp. A full list of the isolates used in this study is provided in Table S1. Phylogenetic analysis Phylogenetic trees were inferred for all of the datasets described above using the maximum likelihood method available in the PAUP* package [63] (see Table S2). In each case the general time-reversible model of nucleotide substitution was used also incorporating a proportion of invariable sites and a gamma distribution of rate variation among sites with four rate categories. All parameter values were estimated from the empirical data and are given in Table S2. Tree bisection–reconnection branch-swapping was used in all cases apart from the expansive (“background”) HA and NA datasets, which contained so many sequences that the analysis was restricted to subtree pruning–regrafting branch-swapping. To assess the reliability of key nodes on the phylogenetic trees, a bootstrap resampling analysis was also undertaken in each case. This involved the inference of 1,000 replicate neighbor-joining trees using the maximum likelihood substitution model inferred for each dataset. Supporting Information Figure S1 Phylogenetic Trees of New York State and Background H3N2 Strains Inferred from the Core Genes Maximum likelihood phylogenetic trees depicting the evolutionary relationships of the remaining six segments (major coding regions only) from the H3N2 influenza A viruses sampled in New York State during the period 1999–2004 (unique sequences only) and the “background” viruses taken from GenBank or the Los Alamos Influenza Sequence Database. All trees are mid-point rooted for purposes of clarity only, and all horizontal branch lengths are drawn to scale. Bootstrap values are shown for clades A, B, and C. Colors are as in Figure 1. (715 KB EPS). Click here for additional data file. Table S1 Isolates of Influenza A Virus H3N2 Used in This Study (164 KB DOC). Click here for additional data file. Table S2 Parameter Values for Maximum Likelihood Phylogenetic Analysis (50 KB DOC). Click here for additional data file.
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Evolution of H5N1 Avian Influenza Viruses in Asia
Yulia Desheva (2005)
Highly pathogenic avian influenza viruses of the H5N1 subtype are circulating in eastern Asia with unprecedented epizootic and epidemic effects ( 1 ). Nine Asian countries reported H5N1 outbreaks in poultry in 2004: Cambodia, China, Indonesia, Japan, Laos, Malaysia, South Korea, Thailand, and Vietnam ( 1 ). Between 2004 and the first 3 months of 2005, a total of 89 laboratory-confirmed human infections, 52 of which were fatal, were reported to the World Health Organization (WHO) by public health authorities in Vietnam, Thailand, and Cambodia. These records indicate that this outbreak of human H5N1 infections is the largest documented since its emergence in humans in 1997 ( 2 ). Efficient viral transmission among poultry caused the virus to spread regionally, leading to the loss of >100 million birds from disease and culling. In contrast, human-to-human transmission of the virus is exceptional but has been described, most recently in a family cluster in Thailand ( 3 ). The 3 viral envelope proteins of influenza A virus are most medically relevant. The hemagglutinin (HA), neuraminidase (NA), and M2 are essential viral proteins targeted by host antibodies or antiviral drugs such as oseltamivir and rimantadine ( 4 – 6 ). The HA glycoprotein forms spikes at the surface of virions, mediating attachment to host cell sialoside receptors and subsequent entry by membrane fusion. The NA forms knoblike structures on the surface of virus particles and catalyzes their release from infected cells, allowing virus spread. The M2 is a transmembrane protein that forms an ion channel required for the uncoating process that precedes viral gene expression. We report on phylogenetic, phenotypic, and antigenic analysis of H5N1 viruses from the 2004–2005 outbreak, focusing on these 3 genes, to address questions relevant to the public health response to the outbreak: 1) What is the genetic diversity of H5N1 viruses involved in human infections? 2) Can the relationship between human and avian H5N1 isolates help explain the source of infection? 3) Do genetic changes correlate with enhanced viral transmissibility in humans? 4) How sensitive are H5N1 isolates to antiviral drugs? 5) What is the antigenic similarity between human H5N1 viruses and current candidate vaccines? and 6) Can candidate vaccine reference stocks be developed in time for an effective public health response? Methods All work involving infectious H5N1 influenza was performed in government-approved biosafety level 3–enhanced containment facilities with experimental protocols in compliance with applicable federal statutes and institutional guidelines. Influenza A (H5N1) viruses isolated in Asia and A/Puerto Rico/8/34 (PR8) (H1N1) were propagated in embryonated chicken eggs or in Madin-Darby canine kidney (MDCK) cells. The African green monkey kidney Vero cell line was from a cell bank certified for human vaccine production. Viral RNA was extracted by using a commercial lysis solution and resin kit and amplified by reverse transcriptase–polymerase chain reaction with specific oligonucleotide primers. Nucleotide sequencing reactions were performed with a cycle sequencing kit and resolved on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). DNA sequence analysis was performed by using version 10 of the GCG sequence analysis package ( 7 ), and phylogeny was inferred by using a neighbor-joining tree reconstruction method implemented in the Phylip package ( 8 ). Postinfection ferret antisera were prepared as previously described ( 9 ). Hemagglutination inhibition (HI) testing was performed as previously described with turkey erythrocytes ( 10 ). Median inhibitory concentration (IC50) values for oseltamivir and zanamivir were determined by using NA-Star substrate and Light Emission Accelerator IITM (Applied Biosystems, Bedford, MA, USA) as previously described ( 11 ). Biological susceptibility to rimantadine was determined by recording the yield of viral progeny in MDCK cells infected with the H5N1 strains of interest at a multiplicity of >10 median egg infectious doses in the absence or presence of 2 μg/mL rimantadine. Plasmids with full-length cDNA from the 6 internal genes (PB1, PB2, PA, NP, M, NS) of influenza virus PR8 strain ( 12 ), flanked by human RNA polymerase I (PolI) promoter and polyadenylation site at the 3´ end and a PolI terminator as well as a PolII promoter at the 5´ end, were generated as described previously ( 12 – 14 ). The cDNA of N1 NA or H5 HA genes of VN/1203/2004 or VN/1194/2004 (VN/04-like) were inserted into plasmids as described above. The 4 basic amino acid codons from the cleavage site of HA were deleted by overlap extension PCR, as described previously (sequences available upon request) ( 13 , 15 – 17 ). PR8 reassortant viruses with HA and NA from VN/04-like viruses were generated by plasmid DNA-based reverse genetics in Vero cell under good laboratory practice conditions appropriate for future human use. Candidate vaccine reference reagent reassortant viruses were generated at the National Institute of Biological Standards and Control (NIBSC), South Mimms, United Kingdom; Saint Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA; and Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. For brevity, the reverse genetics derivation method described represents a consensus of the institutions; minor unpublished protocol details unique to each laboratory were not described and are available upon request. The VN/04x/PR8 reassortant virus was recovered in embryonated eggs and identified in the allantoic fluid by HA assay. The genetic and antigenic properties of the resulting reassortant virus were determined as described previously ( 15 , 18 – 20 ). Candidate vaccine stocks were subjected to virulence studies in avian, murine, and ferret models to establish their safety ( 19 ). Results Analysis of HA, NA, and M2 Genes from H5N1 Viruses Phylogenetic analyses of the H5 HA genes from the 2004 and 2005 outbreak showed 2 different lineages of HA genes, termed clades 1 and 2. Viruses in each of these clades are distributed in nonoverlapping geographic regions of Asia (Figure 1). The H5N1 viruses from the Indochina peninsula are tightly clustered within clade 1, whereas H5N1 isolates from several surrounding countries are distinct from clade 1 isolates and belong in the more divergent clade 2. Clade 1 H5N1 viruses were isolated from humans and birds in Vietnam, Thailand, and Cambodia but only from birds in Laos and Malaysia. The clade 2 viruses were found in viruses isolated exclusively from birds in China, Indonesia, Japan, and South Korea. Viruses isolated from birds and humans in Hong Kong in 2003 and 1997 made up clades 1´ and 3, respectively. Figure 1 Phylogenetic relationships among H5 hemagglutinin (HA) genes from H5N1 avian influenza viruses and their geographic distribution. Viral isolates collected before and during the 2004–2005 outbreak in Asia and selected ancestors were included in the analysis (Table A1). HA clades 1, 1′, and 2, discussed in the text, are colored in blue, red, and green fonts, respectively. Virus names in boldface denote isolates from human infections. Phylogenetic trees were inferred from nucleotide sequences by the neighbor-joining method with A/chicken/Scotland/56 genes as outgroup (not shown, denoted by arrowhead). Bootstrap analysis values >90% are shown. A) HA gene tree phylogeny was based on the coding region of the segment. Presence of a motif for glycosylation in HA is indicated as A156T by an arrow at the root of clade 1 and a diamond for other clades (Table 1). Stars denote absence of 1 arginine residue at the polybasic cleavage site, which starts at position 325 of HA1. Isolates to which ferret antisera were made for antigenic analyses are boxed (Table 2). B) Geographic distribution of H5N1 in east Asia: blue denotes countries reporting infections with clade 1 H5N1 in humans and birds (solid) or in birds only (hatched). Green denotes countries reporting bird infections with clade 2 H5N1 viruses. The HA genes from H5N1 viruses isolated from human specimens were closely related to HA genes from H5N1 viruses of avian origin; human HA gene sequences differ from the nearest gene from avian isolates from the same year in 2–14 nucleotides (<1% divergence). These findings are consistent with the epidemiologic data that suggest that humans acquired their infections by direct or indirect contact with poultry or poultry products ( 21 ). Analysis of the amino acid sequences showed that both clades of H5 HAs from the 2004–2005 outbreak have a multiple basic amino acid motif at the cleavage site, a defining feature of highly pathogenic avian influenza viruses. Among all H5N1 isolates collected in east Asia since 1997, only those in clades 1, 1´, and 3 appear to be associated with fatal human infections ( 22 , 23 ). We compared amino acid sequences of HA from contemporary isolates (clades 1 and 2) with those of the fatal H5N1 infections in Hong Kong in 1997 and 2003 to identify changes that may correlate with patterns of human infection (Table 1). Thirteen polymorphic sites were identified when the HA1 from the 4 consensus sequences were compared. One change in the 2004–2005 viruses is serine 129 to leucine (S129L). This change affects receptor binding because S129 makes atomic contact with cellular sialoside receptors ( 24 ). A second structural change in HA was the A156T substitution, which resulted in glycosylation of asparagine 154 and is predicted to reduce its affinity for sialosides. This change is commonly associated with viral adaptation to terrestrial poultry and increased virulence for these birds ( 25 – 27 ). Table 1 Amino acid differences among H5 hemagglutinins (HA1) Clade 3 Clade 2 Clade 1´ Clade 1 H3 No. Functional significance N45* D D D 54† Antigenic site C S84 N N N 92 Antigenic site E A86 A A V 93 Antigenic site E N94 D D D (1) 101 Near Y91; receptor binding? N124 D S S 129 Antigenic site B S129 S L L 133a Receptor binding L138 Q Q Q 142 Antigenic site A S155 – N155 – 159 Antigenic site B T156‡ A A T 160 N154 glycosylation motif L175 L L L (2) 179 Near H179; receptor binding? T188 T T T (3) 192 Near L190; receptor binding? K189 R R K 193 Adjacent to receptor binding, antigenic site B E212 K K R 216 Antigenic site D S223 – N223§ – (4) 227 Receptor binding T263 A A T 266 Antigenic site E 325R¶ Absent – – Absent HA cleavage efficiency *Amino acid residue in single-letter code and position in the mature H5 HA1.
†Equivalent residue number in the mature H3 HA1 aligned with H5 amino acid sequence; –, no change from HK97 clade HA consensus.
‡A156 or S156 were found in certain clade 3 HAs; A156 was present in some HAs from clade 2. HA genes from »50% of isolates collected in 2005 had these substitutions present in only one isolate: 1) to N or V; 2) to M or I; 3) to A, V, or I; or 4) to N.
§Change present exclusively in isolates from humans.
¶Arginine at the start of the polybasic cleavage site, position 325. Because of the heightened alert due to H5N1 infections in Vietnam during the first months of 2005, we examined the HA sequences for evidence of shared amino acid changes. The HA of viruses isolated in the first 3 months of 2005 showed several amino acid changes relative to 2004 viruses (Table 1). None of the changes in the HA were common to all the 2005 viruses, which suggests that these variant viruses are cocirculating independently in poultry. The most commonly observed changes are located within short distances of the receptor-binding site. For example, positions D94, L175, and T188 may modulate the interaction of Y91, H179, and L190 with sialosides. One of the isolates from a fatal infection in 2005 showed a substitution of serine 223 to asparagine, which is predicted to facilitate binding of sialosides commonly found in mammalian species (Table 1). The phylogenetic tree of the NA genes resembled that of the HA genes, which indicates coevolution of these 2 envelope genes (Figure 2). NA genes of isolates from Thailand seem to have diverged to form a group distinct from that of genes from Vietnam viruses. As reported previously, the NA of HK/213/03 did not co-evolve with the HA genes ( 28 ). NA genes from human and related avian H5N1 isolates from 2003–2005 as well as clade 3 isolates were characterized by deletions in the stalk region of the protein (positions 49–68 for clades 1–2 and 54–72 for clade 3) ( 29 ). Deletions in the stalk of the NA are thought to increase retention of virions at the plasma membrane ( 30 ) to balance weaker binding of sialic acid receptors by the HA with newly acquired N154 glycosylation. Figure 2 Phylogenetic relationships among N1 neuraminidase (NA) genes of H5N1 influenza viruses. The clade of the hemagglutinin of each of these viruses is indicated by font coloring as in Figure 1A. Brackets denote genes encoding NA protein with deletions in the stalk region; residues 49–68 for clades 1–2 and 57–75 in clade 3. Neuraminidase inhibitors are effective antiviral drugs against human influenza viruses, and preclinical studies suggest a similar effectiveness against avian influenza in humans ( 5 , 31 ). The IC50 of oseltamivir for the clade 1 and 2 NA of 2004–2005 isolates was <10 nmol/L, as compared to IC50 values of 85 and 1,600 nmol/L for resistant H1N1 or H3N2 mutants used as controls (Table 3). Thus, NA of H5N1 isolates is sensitive to this class of antiviral agents. Table 3 Sensitivity of H5N1 influenza isolates to oseltamivir Virus Oseltamivir IC50* H1N1 (H274)† 0.69 H1N1 (Y274)† 85.92 H3N2 (R292)‡ 1.99 H3N2 (K292)‡ 1,600.00 Hong Kong/483/97 4.86 Hong Kong/213/03 5.07 Vietnam /1194/04 2.49 Vietnam/1203/04 7.68 Chicken/VN/NCVD1/04 5.87 Chicken/VN/NCVD8/03 9.90 *Median inhibitory concentration (IC50) of oseltamivir (nmol/L) for H5N1 influenza isolates and control H1N1 or H3N2 isolates (results for viruses shown are representatives of 31 isolates tested).
†Wildtype (H274) and resistant mutant (Y274) influenza virus A/Texas/36/91 (H1N1).
‡Wildtype (R292) and resistant mutant (K292) influenza virus A/Victoria/3/75 (H3N2). The phylogenetic tree of the M genes resembled that of the HA genes, indicating coevolution of these genes (results not shown). The amino acid sequence of the M2 protein of clade 1 viruses as well as of HK/213/03 indicated a serine-to-asparagine substitution at residue 31 (S31N), known to confer resistance to adamantanes (including amantadine and rimantadine) ( 6 ). Clade 1 isolates from 2004 and 2005 cultured in the presence of 2 μg/mL rimantadine replicated as efficiently as in untreated cultures, whereas the replication of HK/483/97 was reduced to 1% of control values, indicating that all the currently circulating clade 1 isolates are resistant to adamantanes (data not shown). Origin of Internal Genes of H5N1 Viruses from Asia A complete genetic characterization of circulating H5N1 viruses is critical to identify the possible incorporation of human influenza virus genes by reassortment. To this end, we analyzed the phylogeny of the internal protein coding genes. The PB2, PB1, and PA polymerase genes from 2003–2005 H5N1 isolates from humans constitute a single clade (data not shown) and have coevolved with the respective HA genes (Figure 1). No evidence of reassortment with polymerase genes from circulating H1N1 or H3N2 human influenza virus was found. The phylogenies of the NP and NS genes also supported the avian origin of these genes, indicating that all the genes from the human H5N1 isolates analyzed are of avian origin, which confirms the absence of reassortment with human influenza genes. Taken together, the phylogenies of the 8 genomic segments show that the H5N1 viruses from human infections and the closely related avian viruses isolated in 2004 and 2005 belong to a single genotype, often referred to as genotype Z ( 1 ). Antigenic Analysis of H5N1 Viruses from Asia Influenza vaccines whose HA are antigenically similar to circulating strains provide the highest level of protection from infection ( 32 ). H5N1 isolates collected in 2004 and 2005 analyzed by the HI test showed reactivity patterns that correlated with the 3 main clades of recent isolates identified in the HA gene phylogeny (Table 3 and Figure 1). Viruses from humans and birds in clade 1, represented by VN/1203/04, were found to constitute a relatively homogeneous and distinct antigenic group characterized by poor inhibition by ferret antisera to isolates from other clades (Table 2), in particular by the ferret antiserum raised to HK/213/03 (64-fold reduction compared to the homologous titer). The latter isolate was previously used to develop a vaccine reference strain in response to 2 confirmed H5N1 human infections in February 2003 ( 15 ). These HI results provided the motivation for the development of an updated H5N1 vaccine that would be antigenically similar to 2004–2005 human isolates. The antigenic similarity of VN/1203/04 and the closely related VN/1194/04 to the contemporaneous H5N1 isolates from humans (data not shown) prompted their selection for vaccine reference stock development. Table 2 Antigenic analysis of H5N1 isolates from Asia Virus antigen Clade Reference ferret antisera* HK156 NCVD8 HK213 VN1203 VN04xPR8-rg VN78 VN4207 VN14 VN32321 A/Hong Kong/156/97 3 1,280 320 640 80 320 40 80 80 80 A/ck/Vietnam/NCVD8/03 2 640 160 80 80 160 20 <10 160 40 A/Hong Kong/213/03 1´ 1,280 1,280 2,560 80 640 160 160 640 640 A/Vietnam/1203/04 1 40 20 <10 640 320 40 160 80 40 A/Vietnam/1203/04xPR8-rg 1 80 <10 10 640 320 40 160 160 40 A/Vietnam/1194/04 1 40 20 10 640 320 40 160 160 40 A/Vietnam/JP178/04 1 80 10 <10 1,280 320 80 160 160 80 A/Vietnam/JP4207/05 1 160 40 40 1,280 640 80 320 160 80 A/Vietnam/JP14/05 1 20 <10 10 640 80 20 40 80 40 A/Vietnam/JP30321/05 1 40 40 10 <10 40 10 <10 40 160 *Homologous HI titers are in boldface. Antigenic analysis of human isolates from 2005 provided evidence of antigenic drift among the most recently circulating H5N1 strains (Table 2). For example, VN/JPHN30321/05 showed a reduced HI titer against VN/1203/04 reference serum. This antigenic difference is correlated with 7 amino acid differences between the HA1 domain VN/1203/04 and VN/JPHN30321/05: R53K, N84D, D94N, K140R, L175M, K189R, and V219I (Table 1 and Table A1). Development of Candidate H5N1 Vaccine Reference Stocks Mass vaccination is the most effective approach to reduce illness and death from pandemic influenza. Inactivated influenza vaccines are manufactured from reassortant viruses obtained by transferring the HA and NA genes with the desired antigenic properties into a high-growth strain such as PR8 ( 33 ). However, reassortants with H5-derived HA with a polybasic cleavage site are potentially hazardous for animal health. Because the high pathogenicity of the H5N1 viruses in poultry, mice, and ferrets depends primarily on the polybasic cleavage site in the HA molecule, a derivative with a deletion of this motif was engineered in cloned HA cDNAs. Three high-growth reassortant influenza viruses were developed: NIBRG-14 (NIBSC), VN/04xPR8-rg (SJCRH), and VNH5N1-PR8/CDC-rg (CDC). These candidate vaccine strains, bearing mutant H5 HA, intact NA, and the internal genes from PR8, were generated by a reverse genetics approach ( 12 , 13 , 20 , 34 ) using Vero cells and laboratory protocols compatible with eventual use of the vaccine in human subjects ( 15 , 18 ). These 3 vaccine candidates were characterized genetically (nucleotide sequencing of HA and NA) and antigenically in HI assays to confirm that their antigenicity remained unchanged relative to the wildtype virus (Table 2). The candidate reference stocks had molecular and antigenic properties equivalent to parental H5N1 donor strains and lacked virulence in chicken, mouse, and ferret models. Discussion The growing H5N1 epizootic in eastern Asia could expand the environmental load of virus and cause more infections in mammals ( 35 ), which would increase the probability that a highly transmissible virus will emerge in mammals. We therefore analyzed the medically relevant genes from viruses isolated from the beginning of the outbreak until March 2005 to evaluate parameters relevant to public health. The origin of the HA genes of the 2004–2005 outbreak as well as an earlier isolate from a fatal human infection in Hong Kong in 2003 (clade 1´) can be traced back to viruses isolated in 1997 in Hong Kong (clade 3) and from geese in China (goose/Guangdong/96) (Figure 1A). The phylogeny also shows that viruses with HK/97-like HA may have circulated in avian hosts continuously after 1997, without causing any reported human infections until the 2 confirmed cases in Hong Kong in February 2003 ( 28 ). The 2004–2005 H5N1 isolates are sensitive to 2 neuraminidase inhibitors that are recommended for prophylactic or therapeutic intervention against human infections with recent H5N1 strains. Rapidly testing potentially pandemic influenza viruses for their susceptibility to licensed drugs is essential to establish appropriate control measures. An effective H5N1 vaccine is a public health priority and the cornerstone for pandemic prevention and control. Reverse genetics approaches allow the rapid production of high-growth PR8 reassortant viruses by engineering a virus with a homologous HA gene lacking the polybasic amino acids associated with high virulence. These candidate H5N1 pandemic vaccine viruses have been made available to vaccine manufacturers to produce pilot lots for clinical trials and are available for possible large-scale manufacturing should the need arise. Genetic and antigenic analyses have shown that, compared to previous H5N1 isolates, 2004–2005 isolates share several amino acid changes that modulate antigenicity and perhaps other biological functions. Furthermore, our molecular analysis of the HA from isolates collected in 2005 suggests that several amino acids located near the receptor-binding site are undergoing change, some of which may affect antigenicity or transmissibility. For example, an isolate (VN/JP12-2/05) showed a change from serine to asparagine at position 223 of the HA1 (S223N) that may affect receptor-binding specificity ( 36 ). The VN/30321/05 isolate demonstrated considerable antigenic drift from VN/04-like isolates, which have been selected as the candidate vaccine antigens. Further surveillance to determine the prevalence of such variants in poultry will be critical to determine if these variants compromise the efficacy of the candidate vaccine or increase the efficiency of transmission. The phylogenies of the 8 genomic segments from the clade 1 and 2 isolates from 2004–2005 showed that all genes are of avian origin. All H5N1 isolates from both clades belong to 1 of the genotypes recently circulating in Eastern and Southern Asia, e.g., genotypes V and Z ( 1 , 37 ). The influenza virus genome has remarkable plasticity because of a high mutation rate and its segmentation into 8 separate RNA molecules. This segmentation allows frequent genetic exchange by segment reassortment in hosts co-infected with 2 different influenza viruses. No evidence has been seen that the 2004–2005 H5N1 isolates have acquired nonavian influenza genes by reassortment. However, continued surveillance is important because genetic reassortment may facilitate the evolution of viruses with increased virulence or expanded host range. The currently circulating H5N1 viruses were reported to infect domestic or wild captive felids, such as tigers, feeding on infected bird carcasses, and the infected cats can transmit H5N1 to pen mates ( 38 ). Furthermore, circumstantial evidence indicates that tiger-to-tiger transmission of H5N1 has occurred at a zoo in Thailand ( 39 ). Recent evidence of person-to-person transmission and the clustering of H5N1 cases raise the level of concern for a pandemic of H5N1 influenza ( 3 ). Therefore, sustained and aggressive efforts to control H5N1 circulation in poultry are mandatory to avoid possible catastrophic public health consequences.
Author and article information
Journal
Journal ID (nlm-ta): Emerg Infect Dis
Journal ID (publisher-id): EID
Title:
Emerging Infectious Diseases
Publisher:
Centers for Disease Control and Prevention
ISSN
(Print):
1080-6040
ISSN
(Electronic):
1080-6059
Publication date
(Print):
April
2006
Volume: 12
Issue: 4
Pages: 638-646
Affiliations
Author notes
Address for correspondence: David A. Stenger, Center for Bio/Molecular Science and
Engineering, Code 6900, Naval Research Laboratory, Washington, DC 20375, USA; fax:
202-767-9598; email:
dstenger@
123456cbmse.nrl.navy.mil
Article
Publisher ID: 05-1441DOI: 10.3201/eid1204.051441
PMC ID: 3294710
PubMed ID: 16704813
SO-VID: e1a213dc-d2a1-43c1-a0a6-fd29359c75b1
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Research
Subject:
Research
ScienceOpen disciplines: Infectious disease & Microbiology