Glycosylation and an amino acid insertion in the head of hemagglutinin independently affect the antigenic properties of H5N1 avian influenza viruses

Antigenic drift forces us to frequently update influenza vaccines; however, the genetic basis for antigenic variation remains largely unknown. In this study, we used clade 7.2 H5 viruses as models to explore the molecular determinants of influenza virus antigenic variation. We generated eight monoclonal antibodies(MAbs) targeted to the hemagglutinin(HA) protein of the index virus A/chicken/Shanxi/2/2006 and found that two representative antigenically drifted clade 7.2 viruses did not react with six of the eight MAbs. The E131 N mutation and insertion of leucine at position 134 in the HA protein of the antigenically drifted strains eliminated the reactivity of the virus with the MAbs. We also found that the amino acid N131 in the H5 HA protein is glycosylated. Our results provide experimental evidence that glycosylation and an amino acid insertion or deletion in HA influence antigenic variation.     

Positive selection for gains of N-linked glycosylation sites in hemagglutinin during evolution of H3N2 human influenza A virus

The number of N-linked glycosylation sites in the globular head of hemagglutinin (HA) has increased during evolution of H3N2 human influenza A virus. Here natural selection operating on the gains of N-linked glycosylation sites was examined by using the single-site analysis and the single-substitution analysis. In the single-site analysis, positive selection was not inferred at the amino acid sites where the substitutions generating N-linked glycosylation sites were observed, but was detected at antigenic sites. In contrast, in the single-substitution analysis, positive selection was detected for the amino acid substitutions generating N-linked glycosylation sites. The single-site analysis and the single-substitution analysis appeared to be suitable for detecting recurrent and episodic natural selection, respectively. The gains of N-linked glycosylation sites were likely to be positively selected for the function of shielding antigenic sites from immune responses. At the antigenic sites, positive selection appeared to have operated not only on the radical substitution but also on the conservative substitution in terms of the charge of amino acids, suggesting that the antigenic drift is not a by-product of the evolution of receptor binding avidity in HA of human H3N2 virus.     

Computational Identification of Antigenicity-Associated Sites in the Hemagglutinin Protein of A/H1N1 Seasonal Influenza Virus

The antigenic variability of influenza viruses has always made influenza vaccine development challenging. The punctuated nature of antigenic drift of influenza virus suggests that a relatively small number of genetic changes or combinations of genetic changes may drive changes in antigenic phenotype. The present study aimed to identify antigenicity-associated sites in the hemagglutinin protein of A/H1N1 seasonal influenza virus using computational approaches. Random Forest Regression (RFR) and Support Vector Regression based on Recursive Feature Elimination (SVR-RFE) were applied to H1N1 seasonal influenza viruses and used to analyze the associations between amino acid changes in the HA1 polypeptide and antigenic variation based on hemagglutination-inhibition (HI) assay data. Twenty-three and twenty antigenicity-associated sites were identified by RFR and SVR-RFE, respectively, by considering the joint effects of amino acid residues on antigenic drift. Our proposed approaches were further validated with the H3N2 dataset. The prediction models developed in this study can quantitatively predict antigenic differences with high prediction accuracy based only on HA1 sequences. Application of the study results can increase understanding of H1N1 seasonal influenza virus antigenic evolution and accelerate the selection of vaccine strains.     

Patterns of predicted T-cell epitopes associated with antigenic drift in influenza H3N2 hemagglutinin

Antigenic drift allowing escape from neutralizing antibodies is an important feature of transmission and survival of influenza viruses in host populations. Antigenic drift has been studied in particular detail for influenza A H3N2 and well defined antigenic clusters of this virus documented. We examine how host immunogenetics contributes to determination of the antibody spectrum, and hence the immune pressure bringing about antigenic drift. Using uTOPE™ bioinformatics analysis of predicted MHC binding, based on amino acid physical property principal components, we examined the binding affinity of all 9-mer and 15-mer peptides within the hemagglutinin 1 (HA1) of 447 H3N2 virus isolates to 35 MHC-I and 14 MHC-II alleles. We provide a comprehensive map of predicted MHC-I and MHC-II binding affinity for a broad array of HLA alleles for the H3N2 influenza HA1 protein. Each HLA allele exhibited a characteristic predicted binding pattern. Cluster analysis for each HLA allele shows that patterns based on predicted MHC binding mirror those described based on antibody binding. A single amino acid mutation or position displacement can result in a marked difference in MHC binding and hence potential T-helper function. We assessed the impact of individual amino acid changes in HA1 sequences between 10 virus isolates from 1968–2002, representative of antigenic clusters, to understand the changes in MHC binding over time. Gain and loss of predicted high affinity MHC-II binding sites with cluster transitions were documented. Predicted high affinity MHC-II binding sites were adjacent to antibody binding sites. We conclude that host MHC diversity may have a major determinant role in the antigenic drift of influenza A H3N2.     

Changing selective pressure during antigenic changes in human influenza H3

The rapid evolution of influenza viruses presents difficulties in maintaining the optimal efficiency of vaccines. Amino acid substitutions result in antigenic drift, a process whereby antisera raised in response to one virus have reduced effectiveness against future viruses. Interestingly, while amino acid substitutions occur at a relatively constant rate, the antigenic properties of H3 move in a discontinuous, step-wise manner. It is not clear why this punctuated evolution occurs, whether this represents simply the fact that some substitutions affect these properties more than others, or if this is indicative of a changing relationship between the virus and the host. In addition, the role of changing glycosylation of the haemagglutinin in these shifts in antigenic properties is unknown. We analysed the antigenic drift of HA1 from human influenza H3 using a model of sequence change that allows for variation in selective pressure at different locations in the sequence, as well as at different parts of the phylogenetic tree. We detect significant changes in selective pressure that occur preferentially during major changes in antigenic properties. Despite the large increase in glycosylation during the past 40 years, changes in glycosylation did not correlate either with changes in antigenic properties or with significantly more rapid changes in selective pressure. The locations that undergo changes in selective pressure are largely in places undergoing adaptive evolution, in antigenic locations, and in locations or near locations undergoing substitutions that characterise the change in antigenicity of the virus. Our results suggest that the relationship of the virus to the host changes with time, with the shifts in antigenic properties representing changes in this relationship. This suggests that the virus and host immune system are evolving different methods to counter each other. While we are able to characterise the rapid increase in glycosylation of the haemagglutinin during time in human influenza H3, an increase not present in influenza in birds, this increase seems unrelated to the observed changes in antigenic properties.     

Role of Positive Selection Pressure on the Evolution of H5N1 Hemagglutinin

The surface glycoprotein hemagglutinin (HA) helps the influenza A virus to evade the host immune system by antigenic variation and is a major driving force for viral evolution. In this study, the selection pressure on HA of H5N1 influenza A virus was analyzed using bioinformatics algorithms. Most of the identified positive selection (PS) sites were found to be within or adjacent to epitope sites. Some of the identified PS sites are consistent with previous experimental studies, providing further support to the biological significance of our findings. The highest frequency of PS sites was observed in recent strains isolated during 2005-2007. Phylogenetic analysis was also conducted on HA sequences from various hosts. Viral drift is almost similar in both avian and human species with a progressive trend over the years. Our study reports new mutations in functional regions of HA that might provide markers for vaccine design or can be used to predict isolates of pandemic potential.     

Molecular evolution of influenza A (H3N2) viruses circulated in Fujian Province, China during the 1996–2004 period

We studied the genetic and epidemic characteristics of influenza A (H3N2) viruses circulated in human in Fujian Province, south of China from 1996 to 2004. Phylogenetic analysis was carried out for genes encoding hemagglutinin1 (HA1) of influenza A virus (14 new and 11 previously reported reference se-quences). Our studies revealed that in the 8 flu seasons, the mutations of HA1 genes occurred from time to time, which were responsible for about four times of antigenic drift of influenza H3N2 viruses in Fujian, China. The data demonstrated that amino acid changes were limited to some key codons at or near antibody binding sites A through E on the HA1 molecule. The changes at the antibody binding site B or A or sialic acid receptor binding site 226 were critical for antigenic drift. But the antigenic sites might change and the key codons for antigenic drift might change as influenza viruses evolve. It seems important to monitor new H3 isolates for mutations in the positively selected codons of HA1 gene in south of Asia.     

Evolution of H3N2 influenza virus in a guinea pig model

Studies of influenza virus evolution under controlled experimental conditions can provide a better understanding of the consequences of evolutionary processes with and without immunological pressure. Characterization of evolved strains assists in the development of predictive algorithms for both the selection of subtypes represented in the seasonal influenza vaccine and the design of novel immune refocused vaccines. To obtain data on the evolution of influenza in a controlled setting, naïve and immunized Guinea pigs were infected with influenza A/Wyoming/2003 (H3N2). Virus progeny from nasal wash samples were assessed for variation in the dominant and other epitopes by sequencing the hemagglutinin (HA) gene to quantify evolutionary changes. Viral RNA from the nasal washes from infection of naïve and immune animals contained 6% and 24.5% HA variant sequences, respectively. Analysis of mutations relative to antigenic epitopes indicated that adaptive immunity played a key role in virus evolution. HA mutations in immunized animals were associated with loss of glycosylation and changes in charge and hydrophobicity in and near residues within known epitopes. Four regions of HA-1 (75–85, 125–135, 165–170, 225–230) contained residues of highest variability. These sites are adjacent to or within known epitopes and appear to play an important role in antigenic variation. Recognition of the role of these sites during evolution will lead to a better understanding of the nature of evolution which help in the prediction of future strains for selection of seasonal vaccines and the design of novel vaccines intended to stimulated broadened cross-reactive protection to conserved sites outside of dominant epitopes.     

Antigenic drift in influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and sequential amino acid changes at key antigenic sites

Surveys of the antigenic properties of a wide range of variants of the H3N2 (Hong Kong) influenza virus subtype have revealed complex patterns of variants cocirculating during each of the main epidemic eras of the subtype. We determined hemagglutinin (HA) gene sequences for 14 isolates chosen to give the wildest possible spread of variant types. The addition of these data to existing HA gene sequence information for other variants provides a comprehensive picture of HA gene evolution during antigenic drift among H3N2 subtype viruses. The data reveal the existence of multiple evolutionary pathways during at least one period of development of the subtype and strikingly demonstrate that amino acid changes are limited to a small number of locations on the HA molecule during antigenic drift. The occurrence of sequential amino acid changes at key positions within these variable regions suggests that the HA structure has remained constant during subtype evolution so that only limited possibilities remain for further antigenic drift among H3N2 viruses.     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

Subtype- and antigenic site-specific differences in biophysical influences on evolution of influenza virus hemagglutinin

ABSTRACT: BACKGROUND: Influenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge. Methods: To understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures. Results: Areas of HA showing the highest frequency of changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties. Conclusions: The biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.     

Genetic Requirement for Hemagglutinin Glycosylation and Its Implications for Influenza A H1N1 Virus Evolution

Influenza A virus has evolved and thrived in human populations. Since the 1918 influenza A pandemic, human H1N1 viruses had acquired additional N-linked glycosylation (NLG) sites within the globular head region of hemagglutinin (HA) until the NLG-free HA head pattern of the 1918 H1N1 virus was renewed with the swine-derived 2009 pandemic H1N1 virus. Moreover, the HA of the 2009 H1N1 virus appeared to be antigenically related to that of the 1918 H1N1 virus. Hence, it is possible that descendants of the 2009 H1N1 virus might recapitulate the acquisition of HA head glycosylation sites through their evolutionary drift as a means to evade preexisting immunity. We evaluate here the evolution signature of glycosylations found in the globular head region of H1 HA in order to determine their impact in the virulence and transmission of H1N1 viruses. We identified a polymorphism at HA residue 147 associated with the acquisition of glycosylation at residues 144 and 172. By in vitro and in vivo analyses using mutant viruses, we also found that the polymorphism at HA residue 147 compensated for the loss of replication, virulence, and transmissibility associated with the presence of the N-linked glycans. Our findings suggest that the polymorphism in H1 HA at position 147 modulates viral fitness by buffering the constraints caused by N-linked glycans and provide insights into the evolution dynamics of influenza viruses with implications in vaccine immunogenicity.     

2017年河北省蛋鸡养殖场H9N2亚型禽流感病毒的遗传演化和抗原性分析

【背景】H9N2亚型禽流感病毒在鸡群中广泛流行,引起巨大损失。【目的】了解河北省蛋鸡养殖场H9N2亚型禽流感病毒(avian influenza virus,AIV)的基因序列和抗原性的变异情况,为该病原的科学防控提供理论依据。【方法】于2017年从河北省部分蛋鸡养殖场分离鉴定出7株H9N2亚型AIV,对其HA基因进行序列测定,并进行遗传演化、关键氨基酸位点及抗原性分析。【结果】7株分离毒株HA基因同源性在95.5%?97.2%之间;与2016年前的流行毒株相比,分离病毒HA裂解位点均为典型低致病性AIV特征,在受体结合区域出现变异,潜在糖基化位点无明显差异;抗原分析结果显示分离毒株与早期分离株相比抗原性发生了变异,形成了新的抗原群;抗原性相关位点分析显示,分离毒株在9个位点发生了较为明显的突变,可能是导致抗原性变异的分子基础。【结论】河北省蛋鸡养殖场H9N2亚型AIV中的流行毒株在关键功能区发生基因突变,并且抗原性发生变异,提示应持续监测H9N2亚型AIV的遗传变异情况,并及时更换疫苗株。     

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.     

Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody

We explored the molecular basis of antigenic variation by comparing two H9N2 subtype avian influenza viruses, A/Chicken/Shandong/6/96 (CK/SD/6) and A/Chicken/Guangxi/10/99 (CK/GX/10), that react differently to a monoclonal antibody C/B3. To assess the genetic basis for this antigenic difference, we used reverse genetics to generate a series of chimera and mutants of these two viruses. We found that a single-amino-acid substitution of asparagine for serine at position 145 (S145N) in the HA protein prevents the reaction of CK/SD/6 virus with C/B3. Substitution of serine for asparagine at the same position (N145S) enables the CK/GX/10 to react with C/B3 in hemaglutinin inhibition, immunofluorescence and neutralization assays. We further demonstrated that the amino acid N145 in the H9 HA protein is glycosylated. Our results provide experimental evidence that the glycosylation of HA oligosaccharide attachment sites implicated in antibody binding could have a role in antigenic variation.     

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the p _epitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012–2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6–93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years.     

N-Linked Glycosylation of the Hemagglutinin Protein Influences Virulence and Antigenicity of the 1918 Pandemic and Seasonal H1N1 Influenza A Viruses

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06), led to increased virulence in mice. The addition of glycosylation sites to 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 viruses also caused changes in viral antigenicity based on cross-reactive hemagglutinin inhibition antibody titers with antisera from mice infected with wild-type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 viruses directly contribute to differences in virulence and are partially responsible for their distinct antigenicity.     

Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry

Amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large branched polysaccharide molecules on proteins, can modulate the function of HA and shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to quantitatively measure glycosylation changes in variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages, and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 (H3N2): WT and mutant, each expressed in embryonated chicken eggs and Madin–Darby canine kidney cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence variations and changing viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster genomic surveillance already being done, for a more complete understanding of IAV evolution.     

南京市2011年乙型流感血凝素基因分子特征分析

[目的]分析2011年南京市乙型流感病毒的血凝素(HA)分子学特征.[方法]选择7株2011年南京市不同时间段有代表性的乙型流感毒株进行HA基因序列测定,通过生物信息学方法对HA分子学特征进行分析.[结果]7株乙型流感毒株分为两个系,4株为Victoria,3株为Yamagata;与2011年度疫苗株相比,Victoria和Yamagata系毒株分别在抗原位点146、197和116、198发生了氨基酸替换;其中197和198位点分别是Victoria和Yamagata毒株的受体结合位点,由于上述位点的替换使得Victoria系/Yamagata系毒株分别在197/196位增加了一个潜在的糖基化位点.[结论]2011年南京市乙型流感Victoria 系和Yamagata系病毒同时存在,Victoria/Yamagata毒株197/198位点的氨基酸替换,值得做进一步的探讨.     

Evolutionary analyses on the HA gene ofpandemic H1N1/09: early findings

The HA protein is responsible for influenza virus attachment and the subsequent fusion of viral and cellular membranes. Antigenic drift is driven by an accumulation of point mutations in the HA. And, the receptor-binding specificity of HA is responsible for the host range restriction of the virus. In April 2009, large outbreaks of novel H1N1 influenza in human population were reported from North America. The pandemic H1N1 virus originated from swine influenza virus. Evolutionary process of the pandemic virus after its introduction to human population remains to be clarified. We conducted phylogenetic analyses constructing a phylogenetic tree for and calculating site-by-site selective pressures in the HA gene. Phylogenetic tree showed that pandemic viruses were not clustered clearly by their geographical location or isolation time in the phylogenetic tree. The virus has been circulating the globe extensively with multiple introductions into most geographical areas. We found 3 sites positively selected in the HA gene for pandemic H1N1 virus. Among them, position 206 is located in an antigenic site. We did not find significant negative selection on any of the receptor binding sites. The virus has been evolving under unique selective pressure.