Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses

Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens.     

Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

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.     

Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses

Summary The hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.     

Minor changes in the hemagglutinin of influenza A(H1N1)2009 virus alter its antigenic properties

Background

The influenza A(H1N1)2009 virus has been the dominant type of influenza A virus in Finland during the 2009–2010 and 2010–2011 epidemic seasons. We analyzed the antigenic characteristics of several influenza A(H1N1)2009 viruses isolated during the two influenza seasons by analyzing the amino acid sequences of the hemagglutinin (HA), modeling the amino acid changes in the HA structure and measuring antibody responses induced by natural infection or influenza vaccination.

Methods/Results

Based on the HA sequences of influenza A(H1N1)2009 viruses we selected 13 different strains for antigenic characterization. The analysis included the vaccine virus, A/California/07/2009 and multiple California-like isolates from 2009–2010 and 2010–2011 epidemic seasons. These viruses had two to five amino acid changes in their HA1 molecule. The mutation(s) were located in antigenic sites Sa, Ca1, Ca2 and Cb region. Analysis of the antibody levels by hemagglutination inhibition test (HI) indicated that vaccinated individuals and people who had experienced a natural influenza A(H1N1)2009 virus infection showed good immune responses against the vaccine virus and most of the wild-type viruses. However, one to two amino acid changes in the antigenic site Sa dramatically affected the ability of antibodies to recognize these viruses. In contrast, the tested viruses were indistinguishable in regard to antibody recognition by the sera from elderly individuals who had been exposed to the Spanish influenza or its descendant viruses during the early 20^th century.

Conclusions

According to our results, one to two amino acid changes (N125D and/or N156K) in the major antigenic sites of the hemagglutinin of influenza A(H1N1)2009 virus may lead to significant reduction in the ability of patient and vaccine sera to recognize A(H1N1)2009 viruses.     

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.     

Reactivity of human convalescent sera with influenza virus hemagglutinin protein mutants at antigenic site A

How the antibodies of individual convalescent human sera bind to each amino acid residue at the antigenic sites of hemagglutinin (HA) of influenza viruses, and how the antigenic drift strains of influenza viruses are selected by human sera, is not well understood. In our previous study, it was found by a binding assay with a chimeric HA between A/Kamata/14/91 (Ka/91) and A/Aichi/2/68 that convalescent human sera, following Ka/91 like (H3N2) virus infection, bind to antigenic site A of Ka/91 HA. Here using chimeric HAs possessing single amino acid substitutions at site A, it was determined how those human sera recognize each amino acid residue at antigenic site A. It was found that the capacity of human sera to recognize amino acid substitutions at site A differs from one person to another and that some amino acid substitutions result in all convalescent human sera losing their binding capacity. Among these amino acid substitutions, certain ones might be selected by chance, thus creating successive antigenic drift. Phylogenetic analysis of the drift strains of Ka/91 showed amino acid substitutions at positions 133, 135 and 145 were on the main stream of the phylogenetic tree. Indeed, all of the investigated convalescent sera failed to recognize one of them.     

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.     

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.     

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.     

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.     

Highly pathogenic avian influenza virus subtype H5N1 escaping neutralization: more than HA variation

Influenza A viruses are one of the major threats in modern health care. Novel viruses arise due to antigenic drift and antigenic shift, leading to escape from the immune system and resulting in a serious problem for disease control. In order to investigate the escape process and to enable predictions of escape, we serially passaged influenza A H5N1 virus in vitro 100 times under immune pressure. The generated escape viruses were characterized phenotypically and in detail by full-genome deep sequencing. Mutations already found in natural isolates were detected, evidencing the in vivo relevance of the in vitro-induced amino acid substitutions. Additionally, several novel alterations were triggered. Altogether, the results imply that our in vitro system is suitable to study influenza A virus evolution and that it might even be possible to predict antigenic changes of influenza A viruses circulating in vaccinated populations.     

Antigenic drift in H5N1 avian influenza virus in poultry is driven by mutations in major antigenic sites of the hemagglutinin molecule analogous to those for human influenza virus

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.     

Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein.

The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product.     

Hemagglutinin mutations related to antigenic variation in H1 swine influenza viruses.

The hemagglutinin (HA) of a recent swine influenza virus, A/Sw/IN/1726/88 (H1N1), was shown previously to have four antigenic sites, as determined from analysis of monoclonal antibody (MAb)-selected escape mutants. To define the HA mutations related to these antigenic sites, we cloned and sequenced the HA genes amplified by polymerase chain reaction of parent virus and MAb-selected escape mutants. The genetic data indicated the presence of four amino acid changes. After alignment with the three-dimensional structure of H3 HA, three changes were located on the distal tip of the HA, and the fourth was located within the loop on the HA. We then compared our antigenic sites, as defined by the changed amino acids, with the well-defined sites on the H1 HA of A/PR/8/34. The four amino acid residues corresponded with three antigenic sites on the HA of A/PR/8/34. This finding, in conjunction with our previous antigenic data, indicated that two of the four antigenic sites were overlapping. In addition, our previous studies indicated that one MAb-selected mutant and a recent, naturally occurring swine isolate reacted similarly with the MAb panel. However, their amino acid changes were different and also distant on the primary sequence but close topographically. This finding indicates that changes outside the antigenic site may also affect the site. A comparison of the HA amino acid sequences of early and recent swine isolates showed striking conservation of genetic sequences as well as of the antigenic sites. Thus, swine influenza viruses evolve more slowly than human viruses, possibly because they are not subjected to the same degree of immune selection.     

Distinct glycoprotein inhibitors of influenza A virus in different animal sera.

Normal horse and guinea pig sera contain the glycoprotein inhibitor alpha 2-macroglobulin, which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. In the current study, the presence of inhibitors of influenza A virus in pig and rabbit sera was investigated. Variants of influenza virus type A/Los Angeles/2/87(H3N2) that were resistant to horse, pig, or rabbit serum were isolated. Analysis of the variant viruses with anti-hemagglutinin (HA) monoclonal antibodies revealed that antigenic changes occurred with the development of serum inhibitor resistance. Characterization of the inhibitors in pig and rabbit sera by using periodate and receptor-destroying enzyme demonstrated that carbohydrate is an important constituent of the active portion of both inhibitor molecules and that sialic acid is involved in the interaction of the inhibitors with influenza virus HA. Nucleotide sequence analysis of the HA molecule revealed that the serum-resistant variants each acquired a different set of amino acid alterations. The multiply resistant variants maintained the original amino acid changes and acquired additional changes. Sequence modifications in the HA involved the conserved amino acids within the receptor binding site (RBS) at position 137 and the second-shell RBS residues at positions 155 and 186. Amino acid changes also occurred within antigenic site A (position 145) and directly behind the receptor binding pocket (position 220). Amino acid alterations resulted in the acquisition of a potential glycosylation site at position 128 and the loss of potential glycosylation sites at positions 246 and 248. The localization of the amino acid changes in HA1 to the region of the RBS supports the concept of serum inhibitors as receptor analogs. The unique set of mutations acquired by the serum inhibitor-resistant variants strongly suggests that horse, pig, and rabbit sera each contain distinct glycoprotein inhibitors of influenza A virus.     

Two-dimensional antigenic dendrogram and phylogenetic tree of avian influenza virus H5N1

The hemagglutination-inhibition (HI) titers of a panel of 25 mouse monoclonal antibodies (MAbs) against 44 isolates of highly pathogenic avian influenza virus H5N1 were determined. A two-dimensional antigenic dendrogram was constructed by hierarchical clustering of HI titers. Viruses with similar reactivity patterns were clustered horizontally, whereas MAbs were clustered vertically. In this 2-D dendrogram, with 40% similarity as a cut-off, four virus clusters and four MAbs clusters were identified. A phylogenetic tree based on the deduced amino acid sequence of the hemagglutinin gene of tested viruses was constructed and its topology was compared to the antigenic dendrogram. Interestingly, viruses with high genetic homology in the phylogenetic tree also had high similarity in their reactivity patterns, as indicated by their relatedness in the tree and close clustering in the dendrogram, respectively. However, the reverse and the converse were also true. Of the five pairs of viruses in the dendrogram with bootstrap values higher than 75, four pairs were in concordance with their genetic relatedness. However, one pair contained viruses belong to two distinct genetic clades. These results were discussed in the context of antigenic variation, genetic polymorphism, and the potential application of MAbs in antigenic analysis.     

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.     

Correlation of Influenza Virus Excess Mortality with Antigenic Variation: Application to Rapid Estimation of Influenza Mortality Burden

The variants of human influenza virus have caused, and continue to cause, substantial morbidity and mortality. Timely and accurate assessment of their impact on human death is invaluable for influenza planning but presents a substantial challenge, as current approaches rely mostly on intensive and unbiased influenza surveillance. In this study, by proposing a novel host-virus interaction model, we have established a positive correlation between the excess mortalities caused by viral strains of distinct antigenicity and their antigenic distances to their previous strains for each (sub)type of seasonal influenza viruses. Based on this relationship, we further develop a method to rapidly assess the mortality burden of influenza A(H1N1) virus by accurately predicting the antigenic distance between A(H1N1) strains. Rapid estimation of influenza mortality burden for new seasonal strains should help formulate a cost-effective response for influenza control and prevention.     

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.