A complete analysis of HA and NA genes of influenza A viruses

Background

More and more nucleotide sequences of type A influenza virus are available in public databases. Although these sequences have been the focus of many molecular epidemiological and phylogenetic analyses, most studies only deal with a few representative sequences. In this paper, we present a complete analysis of all Haemagglutinin (HA) and Neuraminidase (NA) gene sequences available to allow large scale analyses of the evolution and epidemiology of type A influenza.

Methodology/Principal Findings

This paper describes an analysis and complete classification of all HA and NA gene sequences available in public databases using multivariate and phylogenetic methods.

Conclusions/Significance

We analyzed 18975 HA sequences and divided them into 280 subgroups according to multivariate and phylogenetic analyses. Similarly, we divided 11362 NA sequences into 202 subgroups. Compared to previous analyses, this work is more detailed and comprehensive, especially for the bigger datasets. Therefore, it can be used to show the full and complex phylogenetic diversity and provides a framework for studying the molecular evolution and epidemiology of type A influenza virus. For more than 85% of type A influenza HA and NA sequences into GenBank, they are categorized in one unambiguous and unique group. Therefore, our results are a kind of genetic and phylogenetic annotation for influenza HA and NA sequences. In addition, sequences of swine influenza viruses come from 56 HA and 45 NA subgroups. Most of these subgroups also include viruses from other hosts indicating cross species transmission of the viruses between pigs and other hosts. Furthermore, the phylogenetic diversity of swine influenza viruses from Eurasia is greater than that of North American strains and both of them are becoming more diverse. Apart from viruses from human, pigs, birds and horses, viruses from other species show very low phylogenetic diversity. This might indicate that viruses have not become established in these species. Based on current evidence, there is no simple pattern of inter-hemisphere transmission of avian influenza viruses and it appears to happen sporadically. However, for H6 subtype avian influenza viruses, such transmissions might have happened very frequently and multiple and bidirectional transmission events might exist.     

The evolution of human influenza viruses.

The evolution of influenza viruses results in (i) recurrent annual epidemics of disease that are caused by progressive antigenic drift of influenza A and B viruses due to the mutability of the RNA genome and (ii) infrequent but severe pandemics caused by the emergence of novel influenza A subtypes to which the population has little immunity. The latter characteristic is a consequence of the wide antigenic diversity and peculiar host range of influenza A viruses and the ability of their segmented RNA genomes to undergo frequent genetic reassortment (recombination) during mixed infections. Contrasting features of the evolution of recently circulating influenza AH1N1, AH3N2 and B viruses include the rapid drift of AH3N2 viruses as a single lineage, the slow replacement of successive antigenic variants of AH1N1 viruses and the co-circulation over some 25 years of antigenically and genetically distinct lineages of influenza B viruses. Constant monitoring of changes in the circulating viruses is important for maintaining the efficacy of influenza vaccines in combating disease.     

Spatiotemporal Structure of Molecular Evolution of H5N1 Highly Pathogenic Avian Influenza Viruses in Vietnam

Background

Vietnam is one of the countries most affected by outbreaks of H5N1 highly pathogenic avian influenza viruses. First identified in Vietnam in poultry in 2001 and in humans in 2004, the virus has since caused 111 cases and 56 deaths in humans. In 2003/2004 H5N1 outbreaks, nearly the entire poultry population of Vietnam was culled. Our earlier study (Wan et al., 2008, PLoS ONE, 3(10): e3462) demonstrated that there have been at least six independent H5N1 introductions into Vietnam and there were nine newly emerged reassortants from 2001 to 2007 in Vietnam. H5N1 viruses in Vietnam cluster distinctly around Hanoi and Ho Chi Minh City. However, the nature of the relationship between genetic divergence and geographic patterns is still unclear.

Methodology/Principal Findings

In this study, we hypothesized that genetic distances between H5N1 viruses in Vietnam are correlated with geographic distances, as the result of distinct population and environment patterns along Vietnam''s long north to south longitudinal extent. Based on this hypothesis, we combined spatial statistical methods with genetic analytic techniques and explicitly used geographic space to explore genetic evolution of H5N1 highly pathogenic avian influenza viruses at the sub-national scale in Vietnam. Our dataset consisted of 125 influenza viruses (with whole genome sets) isolated in Vietnam from 2003 to 2007. Our results document the significant effect of space and time on genetic evolution and the rise of two regional centers of genetic mixing by 2007. These findings give insight into processes underlying viral evolution and suggest that genetic differentiation is associated with the distance between concentrations of human and poultry populations around Hanoi and Ho Chi Minh City.

Conclusions/Significance

The results show that genetic evolution of H5N1 viruses in Vietnamese domestic poultry is highly correlated with the location and spread of those viruses in geographic space. This correlation varies by scale, time, and gene, though a classic isolation by distance pattern is observed. This study is the first to characterize the geographic structure of influenza viral evolution at the sub-national scale in Vietnam and can shed light on how H5N1 HPAIVs evolve in certain geographic settings.     

Glycosylation site alteration in the evolution of influenza A (H1N1) viruses

Influenza virus typically alters protein glycosylation in order to escape immune pressure from hosts and hence to facilitate survival in different host environments. In this study, the patterns and conservation of glycosylation sites on HA and NA of influenza A/H1N1 viruses isolated from various hosts at different time periods were systematically analyzed, by employing a new strategy combining genome-based glycosylation site prediction and 3D modeling of glycoprotein structures, for elucidation of the modes and laws of glycosylation site alteration in the evolution of influenza A/H1N1 viruses. The results showed that influenza H1N1 viruses underwent different alterations of protein glycosylation in different hosts. Two alternative modes of glycosylation site alteration were involved in the evolution of human influenza virus: One was an increase in glycosylation site numbers, which mainly occurred with high frequency in the early stages of evolution. The other was a change in the positional conversion of the glycosylation sites, which was the dominating mode with relatively low frequency in the later evolutionary stages. The mechanisms and possibly biological functions of glycosylation site alteration for the evolution of influenza A/H1N1 viruses were also discussed. Importantly, the significant role of positional alteration of glycosylation sites in the host adaptation of influenza virus was elucidated. Although the results still need to be supported by experimental data, the information here may provide some constructive suggestions for research into the glycosylation of influenza viruses as well as even the design of surveillance and the production of viral vaccines.     

Unseasonal Transmission of H3N2 Influenza A Virus During the Swine-Origin H1N1 Pandemic

The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.The recent emergence of swine-origin H1N1 influenza A virus (pandemic H1N1/09) in humans has heightened awareness of how the burden of morbidity and mortality due to influenza is associated with the appearance of new genetic variants (5) and of the genetic and epidemiological determinants of viral transmission (8). The emergence of pandemic H1N1/09 is also unprecedented in recorded history as it means that three antigenically distinct lineages of influenza A virus—pandemic H1N1/09 and the seasonal H1N1 and H3N2 viruses— currently cocirculate within human populations.Although the presence of multiple subtypes of influenza A virus may place an additional burden on public health resources, it also provides a unique opportunity to compare the patterns and dynamics of evolution in these viruses on a similar time scale. Indeed, one of the most interesting secondary effects of the current H1N1/09 pandemic has been an increased vigilance for cases of influenza-like illness and hence an intensified sampling of seasonal H1N1 and H3N2 viruses during the typical influenza “off-season” (i.e., spring-summer) in the northern hemisphere. Because the influenza season in the northern hemisphere generally runs from November through March, with a usual peak in January or February, influenza viruses sampled outside of this period are of special interest.The current model for the global spatiotemporal dynamics of influenza A virus is that the northern and southern hemispheres represent ecological “sinks” for this virus, with little ongoing viral transmission during the summer months (9). In contrast, more continual viral transmission occurs within the tropical “source” population (13) that is most likely centered on an intense transmission network in east and southeast Asia (10). However, the precise epidemiological and evolutionary reasons for this major geographic division, and for the seasonality of influenza A virus in general, remain uncertain (1, 4). Evidence for this “sink-source” ecological model is that viruses sampled from successive seasons in localities such as New York State do not usually form linked clusters on phylogenetic trees, indicating that they are not connected by direct transmission through the summer months (7). Similar conclusions can be drawn for the United States as a whole and point to multiple introductions of phylogenetically distinct lineages during the winter (6), followed by complex patterns of spatial diffusion (14). However, despite the growing epidemiological and phylogenetic data supporting this model, it is also evident that there is relatively little sequence data from seasonal influenza viruses that are sampled from April to October in the northern hemisphere. Hence, it is uncertain whether extended chains of transmission can occur during this time period, even though this may have an important bearing on our understanding of influenza seasonality.To address these issues, we examined the evolutionary behavior of seasonal H1N1 and H3N2 viruses as they cocirculated during a single time period—(late) April to June 2009—within a single locality (New York State). Not only are levels of influenza virus transmission in the northern hemisphere usually very low during this time period, but in this particular season the human host population was also experiencing the emerging epidemic of pandemic H1N1/09.     

Influenza as a model system for studying the cross-species transfer and evolution of the SARS coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) moved into humans from a reservoir species and subsequently caused an epidemic in its new host. We know little about the processes that allowed the cross-species transfer of this previously unknown virus. I discuss what we have learned about the movement of viruses into humans from studies of influenza A, both how it crossed from birds to humans and how it subsequently evolved within the human population. Starting with a brief review of severe acute respiratory syndrome to highlight the kinds of problems we face in learning about this viral disease, I then turn to influenza A, focusing on three topics. First, I present a reanalysis of data used to test the hypothesis that swine served as a "mixing vessel" or intermediate host in the transmission of avian influenza to humans during the 1918 "Spanish flu" pandemic. Second, I review studies of archived viruses from the three recent influenza pandemics. Third, I discuss current limitations in using molecular data to study the evolution of infectious disease. Although influenza A and SARS-CoV differ in many ways, our knowledge of influenza A may provide important clues about what limits or favours cross-species transfers and subsequent epidemics of newly emerging pathogens.     

Reassortment and Insertion-Deletion Are Strategies for the Evolution of Influenza B Viruses in Nature

The evolution of influenza B viruses is poorly understood. Reassortment of influenza B viruses in nature as a means of genetic variation has not been considered to be a major contributor to their evolution. However, the current practice of assigning evolutionary relationships by antigenic analysis of the hemagglutinin of influenza B viruses would fail to detect reassortants. In this study, influenza B viruses isolated within the past 10 years from sites in the United States and China were studied by nucleotide sequencing of the hemagglutinin and neuraminidase genes and construction of phylogenetic trees to assess evolutionary relationships. A group of viruses represented by B/Houston/1/92 possess a hemagglutinin derived from a B/Yamagata/16/88-like strain and a neuraminidase derived from a B/Victoria/2/87-like strain. A second reassortment event between the hemagglutinin of a B/Yamagata/16/88-like virus closely related to the B/Beijing/184/93 strain and the neuraminidase of a B/Victoria/2/87-like strain is represented by a single virus, B/Memphis/3/93. The neuraminidase of the reassortant viruses is most closely related to that of B/Victoria/2/87-like viruses currently circulating in Nanchang, China. A pattern of insertions and deletions in the hemagglutinin and the neuraminidase of different strains of influenza B viruses is observed. Reassortment plays a role in the evolution of influenza B viruses and may necessitate a change in the methods used to assess and identify new influenza viruses.     

Reassortant between Human-Like H3N2 and Avian H5 Subtype Influenza A Viruses in Pigs: A Potential Public Health Risk

Background

Human-like H3N2 influenza viruses have repeatedly been transmitted to domestic pigs in different regions of the world, but it is still uncertain whether any of these variants could become established in pig populations. The fact that different subtypes of influenza viruses have been detected in pigs makes them an ideal candidate for the genesis of a possible reassortant virus with both human and avian origins. However, the determination of whether pigs can act as a “mixing vessel” for a possible future pandemic virus is still pending an answer. This prompted us to gather the epidemiological information and investigate the genetic evolution of swine influenza viruses in Jilin, China.

Methods

Nasopharyngeal swabs were collected from pigs with respiratory illness in Jilin province, China from July 2007 to October 2008. All samples were screened for influenza A viruses. Three H3N2 swine influenza virus isolates were analyzed genetically and phylogenetically.

Results

Influenza surveillance of pigs in Jilin province, China revealed that H3N2 influenza viruses were regularly detected from domestic pigs during 2007 to 2008. Phylogenetic analysis revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations.

Conclusions

The present study reports for the first time the coexistence of wholly human-like H3N2 viruses and double-reassortant viruses that have emerged in pigs in Jilin, China. It provides updated information on the role of pigs in interspecies transmission and genetic reassortment of influenza viruses.     

新甲型H1N1(2009)病毒的早期分子特征

摘要：【目的】本世纪首次流感大流行的病原属于甲型H1N1流感病毒,在遗传特性和抗原等方面都有别于人群中流行多年的季节性H1N1流感病毒。为了深入了解病毒的遗传特性,跟踪病毒的演化趋势,及时发现具有流行病学意义的变异株,本研究对早期分离的甲型H1N1(2009)病毒的分子特性进行了详细分析。【方法】通过GenBank的流感资源中心下载相关毒株的基因组信息, 序列分析采用DNAStar软件包的EditSeq和MegAlign比较与病毒致病性和宿主特异性相关的氨基酸变化情况。以A/California/07/2009（H1N1）作为新甲型H1N1(2009)的代表株进行详细的分子特征分析。【结果】A/California/07/2009不具备高致病性流感病毒的分子特征;病毒编码的11个蛋白大部分保留有猪流感病毒的分子特征,同时也具有一些禽和人流感病毒的特征;PB1-F2在11aa,57aa和87aa后发生断裂,具有古典猪H1N1和人H1N1双重特点,这是甲型H1N1（2009）病毒一个特有的分子特征。【结论】首次详细分析了新甲型H1N1（2009）病毒的分子特征。随着病毒在人群中的进一步适应和持续存在,这些分子特征将发生变化,应该特别关注这些变化对病毒的传播力和致病性的影响。     

Evidence of intra-segmental homologous recombination in influenza A virus

The evolution of influenza viruses is remarkably dynamic. Influenza viruses evolve rapidly in sequence and undergo frequent reassortment of different gene segments. Homologous recombination, although commonly seen as an important component of dynamic genome evolution in many other organisms, is believed to be rare in influenza. In this study, 256 gene segments from 32 influenza A genomes were examined for homologous recombination, three recombinant H1N1 strains were detected and they most likely resulted from one recombination event between two closely rated parental sequences. These findings suggest that homologous recombination in influenza viruses tends to take place between strains sharing high sequence similarity. The three recombinant strains were isolated at different time periods and they form a clade, indicating that recombinant strains could circulate. In addition, the simulation results showed that many recombinant sequences might not be detectable by currently existing recombinant detection programs when the parental sequences are of high sequence similarity. Finally, possible ways were discussed to improve the accuracy of the detection for recombinant sequences in influenza.     

Influenza genome diversity and evolution

The influenza viruses contain highly variable genomes and are able to infect a wide range of host species. Large-scale sequencing projects have collected abundant influenza sequence data for assessing influenza genome diversity and evolution. This work reviews current influenza sequence databases characteristics and statistics, as well as recent studies utilizing these databases to unravel influenza virus diversity and evolution. Also discussed are the newest deep sequencing methods and their applications to influenza virus research.     

Antigenic and genetic evolution of swine influenza A (H3N2) viruses in Europe

In the early 1970s, a human influenza A/Port Chalmers/1/73 (H3N2)-like virus colonized the European swine population. Analyses of swine influenza A (H3N2) viruses isolated in The Netherlands and Belgium revealed that in the early 1990s, antigenic drift had occurred, away from A/Port Chalmers/1/73, the strain commonly used in influenza vaccines for pigs. Here we show that Italian swine influenza A (H3N2) viruses displayed antigenic and genetic changes similar to those observed in Northern European viruses in the same period. We used antigenic cartography methods for quantitative analyses of the antigenic evolution of European swine H3N2 viruses and observed a clustered virus evolution as seen for human viruses. Although the antigenic drift of swine and human H3N2 viruses has followed distinct evolutionary paths, potential cluster-differentiating amino acid substitutions in the influenza virus surface protein hemagglutinin (HA) were in part the same. The antigenic evolution of swine viruses occurred at a rate approximately six times slower than the rate in human viruses, even though the rates of genetic evolution of the HA at the nucleotide and amino acid level were similar for human and swine H3N2 viruses. Continuous monitoring of antigenic changes is recommended to give a first indication as to whether vaccine strains may need updating. Our data suggest that humoral immunity in the population plays a smaller role in the evolutionary selection processes of swine H3N2 viruses than in human H3N2 viruses.     

Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis

H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G). Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.     

Prevalence of epistasis in the evolution of influenza A surface proteins

The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with , which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs.     

Close relationship between the 2009 H1N1 virus and South Dakota AIV strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.     

Detailed Report on 2014/15 Influenza Virus Characteristics,and Estimates on Influenza Virus Vaccine Effectiveness from Austria’s Sentinel Physician Surveillance Network

Background

Influenza vaccine effectiveness (VE) is influenced by the antigenic similarity between vaccine- and circulating strains.

Material and Methods

This paper presents data obtained by the Austrian sentinel surveillance system on the evolution of influenza viruses during the season 2014/15 and its impact on influenza vaccine effectiveness in primary care in Austria as estimated by a test-negative case control design. VE estimates were performed for each influenza virus type/subtype, stratified by underlying diseases and adjusted for age, sex and calendar week of infection.

Results

Detailed genetic and antigenic analyses showed that circulating A(H3N2) viruses were genetically distinct from the 2014/15 A(H3N2) vaccine component indicating a profound vaccine mismatch. The Influenza A(H1N1)pdm09 viruses were antigenically conserved and matched the respective vaccine component. Influenza B viruses were lineage-matched B/Yamagata viruses with a clade-level variation. Consistent with substantial vaccine mismatch for the A(H3N2) viruses a crude overall VE of only 47% was estimated, whereas the VE estimates for A(H1N1)pdm09 were 84% and for influenza B viruses 70%. Increased VE estimates were obtained after stratification by underlying diseases and adjustment for the covariates sex and age, whereby the adjustment for the calendar week of infection was the covariate exerting the highest influence on adjusted VE estimates.

Conclusion

In summary, VE data obtained in this study underscore the importance to perform VE estimates in the context of detailed characterization of the contributing viruses and also demonstrate that the calendar week of influenza virus infection is the most important confounder of VE estimates.     

Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.     

乙型流行性感冒病毒两大谱系的起源及其演变特征

测定1972－2000年间在中国分离并保存的乙型流行性感冒（流感）病毒一些毒株的HA1区核苷酸序列,结合GenBank中其它毒株的相关序列以及流行病学资料进行分析。结果提示,当前在世界上流行的乙型流感病毒两大谱系起源于20世纪70年代中期,而不是以前推测的60年代末,并且其中一个谱系起源于中国。结果还提示,乙型流感病毒的演变同时受到很强的正选择与负选择的作用,而以前人们认为选择对乙型流感病毒的演变作用甚微。还修正了乙型流感病毒变异速率的计算方法。     

A context-free encoding scheme of protein sequences for predicting antigenicity of diverse influenza A viruses

Background

The evolution of influenza A viruses leads to the antigenic changes. Serological diagnosis of the antigenicity is usually labor-intensive, time-consuming and not suitable for early-stage detection. Computational prediction of the antigenic relationship between emerging and old strains of influenza viruses using viral sequences can facilitate large-scale antigenic characterization, especially for those viruses requiring high biosafety facilities, such as H5 and H7 influenza A viruses. However, most computational models require carefully designed subtype-specific features, thereby being restricted to only one subtype.

Methods

In this paper, we propose a Context-FreeEncoding Scheme (CFreeEnS) for pairs of protein sequences, which encodes a protein sequence dataset into a numeric matrix and then feeds the matrix into a downstream machine learning model. CFreeEnS is not only free from subtype-specific selected features but also able to improve the accuracy of predicting the antigenicity of influenza. Since CFreeEnS is subtype-free, it is applicable to predicting the antigenicity of diverse influenza subtypes, hopefully saving the biologists from conducting serological assays for highly pathogenic strains.

Results

The accuracy of prediction on each subtype tested (A/H1N1, A/H3N2, A/H5N1, A/H9N2) is over 85%, and can be as high as 91.5%. This outperforms existing methods that use carefully designed subtype-specific features. Furthermore, we tested the CFreeEnS on the combined dataset of the four subtypes. The accuracy reaches 84.6%, much higher than the best performance 75.1% reported by other subtype-free models, i.e. regional band-based model and residue-based model, for predicting the antigenicity of influenza. Also, we investigate the performance of CFreeEnS when the model is trained and tested on different subtypes (i.e. transfer learning). The prediction accuracy using CFreeEnS is 84.3% when the model is trained on the A/H1N1 dataset and tested on the A/H5N1, better than the 75.2% using a regional band-based model.

Conclusions

The CFreeEnS not only improves the prediction of antigenicity on datasets with only one subtype but also outperforms existing methods when tested on a combined dataset with four subtypes of influenza viruses.

     

Mixed Infection and the Genesis of Influenza Virus Diversity

The emergence of viral infections with potentially devastating consequences for human health is highly dependent on their underlying evolutionary dynamics. One likely scenario for an avian influenza virus, such as A/H5N1, to evolve to one capable of human-to-human transmission is through the acquisition of genetic material from the A/H1N1 or A/H3N2 subtypes already circulating in human populations. This would require that viruses of both subtypes coinfect the same cells, generating a mixed infection, and then reassort. Determining the nature and frequency of mixed infection with influenza virus is therefore central to understanding the emergence of pandemic, antigenic, and drug-resistant strains. To better understand the potential for such events, we explored patterns of intrahost genetic diversity in recently circulating strains of human influenza virus. By analyzing multiple viral genome sequences sampled from individual influenza patients we reveal a high level of mixed infection, including diverse lineages of the same influenza virus subtype, drug-resistant and -sensitive strains, those that are likely to differ in antigenicity, and even viruses of different influenza virus types (A and B). These results reveal that individuals can harbor influenza viruses that differ in major phenotypic properties, including those that are antigenically distinct and those that differ in their sensitivity to antiviral agents.Influenza viruses (family Orthomyxoviridae) possess a negative-strand segmented RNA genome and enveloped virions. Genetic diversity in influenza virus is the result of a high rate of mutation associated with replication using low-fidelity RNA polymerase and of the reshuffling (or reassortment) of segments among coinfecting strains. Although the 13.5-kb genome of influenza A virus is composed of eight segments coding for 11 known proteins, these viruses are typically categorized by their two surface antigens, hemagglutinin (HA), of which there are 16 subtypes (H1 to H16), and neuraminidase (NA), of which there are 9 (N1 to N9) (9). All known subtypes are present in aquatic birds of the orders Anseriformes and Charadriformes, and a smaller number circulate in some mammalian species. The HA plays a major role in the attachment of the virus to the host cell surface by binding to the sialic acid moiety of host receptors and facilitating the fusion of the viral envelope with host cell membranes. It is also the major viral antigen against which neutralizing antibodies are directed. The NA is important for mobility of the virions by cleaving the sialic acid residues from the viral hemagglutinin, which facilitates both entry of the virus into the cell and release of the viruses during budding (11).Most discussions of influenza virus evolution have focused on the process of antigenic drift in which mutations accumulate—most likely by natural selection—in the antigenic sites of the HA and NA, thereby allowing evasion of the host populations’ acquired immunity to previously circulating strains. Such antigenic variation occurs primarily in the HA1 domain and is clustered into five main epitope regions (19, 20, 22). Although antigenic drift clearly plays a key role in the seasonal evolution of influenza A virus, recent studies making use of large data sets generated by the Influenza Genome Sequencing Project (IGSP) suggest that reassortment may also be important in the generation of antigenically novel isolates by placing diverse HAs in compatible genetic backgrounds (6, 8, 10, 14).Segment reassortment is also central to the process of cross-species transmission and emergence of pandemic influenza virus. In particular, the segmented nature of the influenza virus genome allows reassortment of gene segments to occur between diverse influenza A virus strains when they coinfect a single host, including those derived from different species. This can result in subtle changes within a subtype, or dramatic changes that occur when different subtypes mix, leading to the generation of novel viruses expressing surface glycoproteins to which a specific host immune system has little if any serological cross-reactivity. Such antigenic shift is believed to have led to the emergence of global human influenza A virus pandemics in 1957 (A/H2N2) and in 1968 (A/H3N2), with new segments ultimately derived from the avian reservoir pool reassorting into human influenza viruses (17).Given the potential for emerging viruses such as influenza virus to adversely affect the health of human and other animal populations, it is essential to determine the factors that allow viruses to acquire the mutations they need to adapt to new host populations. As a large number of point mutations are thought to be required for an avian influenza virus such as A/H5N1 to evolve sustained transmission in human populations (5), one likely scenario for successful emergence is through the acquisition of genetic material from a viral subtype already adapted to humans, such as A/H1N1 or A/H3N2. This would require that viruses of both subtypes coinfect the same cells, thereby generating a mixed infection, and then exchange genomic segments through reassortment, as was the case in 1957 and 1968. As a consequence, it is crucial to determine the frequency with which mixed infection naturally occurs in influenza A virus as well as its phenotypic consequences. To address these questions we undertook, for the first time, in-depth sequencing of multiple viral genome sequences sampled from individual influenza patients. These studies were performed with approval of the New York State (study numbers 04-103 and 02-054) and University of Pittsburgh (08-110400) institutional review boards.