Changes in H3 influenza A virus receptor specificity during replication in humans.

Influenza A viruses of the H3 subtype caused the 1968 Hong Kong pandemic, the hemagglutinin (HA) gene being introduced into humans following a reassortment event with an avian virus. Receptor specificity and serum inhibitor sensitivity of the HA of influenza A viruses are linked to the host species. Human H3 viruses preferentially recognize N-acetyl sialic acid linked to galactose by alpha2,6 linkages (Neu5Acalpha2,6Gal) and are sensitive to serum inhibitors, whereas avian and equine viruses preferentially recognize Neu5Acalpha2,3Gal linkages and are resistant to serum inhibitors. We have examined the receptor specificity and serum inhibitor sensitivity of H3 human influenza A viruses from the time they were introduced into the human population to gain insight into the mechanism of viral molecular evolution and host tropism. All of the viruses were sensitive to neutralization and hemagglutination inhibition by horse serum. Early H3 viruses were resistant to pig and rabbit serum inhibitors. Viruses isolated after 1977 were uniformly sensitive to inhibition by pig and rabbit sera. The recognition of Neu5Acalpha2,3Gal or Neu5Acalpha2,6Gal linkages was not correlated with the serum sensitivity. These data showed that the receptor specificity of HA, measured as inhibitor sensitivity, has changed during replication in humans since its introduction from an avian virus.

Coinfection of wild ducks by influenza A viruses: distribution patterns and biological significance.

Coinfection of wild birds by influenza A viruses is thought to be an important mechanism for the diversification of viral phenotypes by generation of reassortants. However, it is not known whether coinfection is a random event or follows discernible patterns with biological significance. In the present study, conducted with viruses collected throughout 15 years from a wild-duck population in Alberta, Canada, we identified three discrete distributions of coinfections. In about one-third of the events, which involved subtypes of viruses that appear to be maintained in this duck reservoir, coinfection occurred at rates either close to or significantly lower than one would predict from rates of single-virus infection. Apparently, the better adapted an influenza A virus is to an avian population, the greater is its ability to prevent coinfections. Conversely, poorly adapted, nonmaintained viruses were significantly overrepresented as coinfectants. Rarely encountered subtypes appear to represent viruses whose chances of successfully infiltrating avian reservoirs are increased by coinfection. Mallards (Anas platyrhynchos) and pintails (A. acuta) were significantly more likely to be infected by a single influenza A virus than were the other species sampled, but no species was significantly more likely to be coinfected. These observations provide the first evidence of nonrandom coinfection of wild birds by influenza A viruses, suggesting that reassortment of these viruses in a natural population does not occur randomly. These results suggest that even though infections may occur in a species, all subtypes are not maintained by all avian species. They also suggest that specific influenza A virus subtypes are differentially adapted to different avian hosts and that the fact that a particular subtype is isolated from a particular avian species does not mean that the virus is maintained by that species.

Analysis of the influenza virus gene pool of avian species from southern China.

Although Southern China has been considered the epicenter of human influenza pandemics, little is known about the genetic composition of influenza viruses in lower mammals or birds in that region. To provide information on the molecular epidemiology of these viruses, we used dot blot hybridization and phylogenetic methods to study the internal genes (PB1, PB2, PA, NP, M, and NS) of 106 avian influenza A viruses isolated from a total of 11,798 domestic ducks, chickens, and geese raised in Southern China including Hong Kong. All 636 genes examined were characteristic of avian influenza viruses; no human or swine influenza genes were detected. Thus, influenza virus reassortants do not appear to be maintained in the domesticated birds of Southeast Asia, eliminating opportunities for further gene reassortment. Phylogenetic analysis showed that the internal genes of these viruses belong to the Eurasian avian lineage, supporting geographical separation of the major avian lineages. The PB1 genes were most similar to A/Singapore/57 (H2N2) and Hong Kong (H3N2) viral genes, supporting an avian origin for the recent human H2N2 and H3N2 pandemic strains. The majority of internal genes from avian influenza viruses in Southern China belong to the Eurasian lineage and are similar to viruses that have recently been transmitted to humans, swine, and horses. This study provides evidence that the transmission of avian influenza viruses and their genes to other species is unidirectional and that the transmission of mammalian influenza virus strains to domestic poultry is probably not a factor in the generation of new pandemic strains.

Origin of the pandemic 1957 H2 influenza A virus and the persistence of its possible progenitors in the avian reservoir.

H2N2 influenza A viruses caused the Asian pandemic of 1957 and then disappeared from the human population 10 years later. To assess the potential for similar outbreaks in the future, we determined the antigenicity of H2 hemagglutinins (HAs) from representative human and avian H2 viruses and then analyzed the nucleotide and amino acid sequences to determine their evolutionary characteristics in different hosts. The results of longitudinal virus surveillance studies were also examined to estimate the prevalence of avian H2 isolates among samples collected from wild ducks and domestic poultry. Reactivity patterns obtained with a large panel of monoclonal antibodies indicated antigenic drift in the HA of human H2 influenza viruses, beginning in 1962. Amino acid changes were clustered in two regions of HA1 that correspond to antigenic sites A and D of the H3 HA. By contrast, the antigenic profiles of the majority of avian H2 HAs were remarkably conserved through 1991, resembling the prototype Japan 57 (H2N2) strain. Amino acid changes were distributed throughout HA1, indicating that antibodies do not play a major role in the selection of avian H2 viruses. Phylogenetic analysis revealed two geographic site-specific lineages of avian H2 HAs: North American and Eurasian. Evidence is presented to support interregion transmission of gull H2 viruses. The human H2 HAs that circulated in 1957-1968 form a separate phylogenetic lineage, most closely related to the Eurasian avian H2 HAs. There was an increased prevalence of H2 influenza viruses among wild ducks in 1988 in North America, preceding the appearance of H2N2 viruses in domestic fowl. As the prevalence of avian H2N2 influenza viruses increased on turkey farms and in live bird markets in New York City and elsewhere, greater numbers of these viruses have come into direct contact with susceptible humans. We conclude that antigenically conserved counterparts of the human Asian pandemic strain of 1957 continue to circulate in the avian reservoir and are coming into closer proximity to susceptible human populations.

Analysis of the evolution and variation of the human influenza A virus nucleoprotein gene from 1933 to 1990.

This study examined the evolution and variation of the human influenza virus nucleoprotein gene from the earliest isolates to the present. Phylogenetic reconstruction of the most parsimonious evolutionary path connecting 49 nucleoprotein sequences yielded a single lineage. The average calculated rate of mutation was 3.6 nucleotide substitutions per year (2.3 x 10(-3) substitutions per site per year). Thirty-two percent of these mutations resulted in amino acid substitutions, and the remainder were silent mutations. Analysis of virus isolates from China and elsewhere showed no significant differences in their rate of evolution, genetic diversity, or mean survival time. The nearly constant rate of change was maintained through the two antigenic shifts, and there were no obvious changes in the number or types of mutations associated with the changes in the surface proteins. A detailed comparison of the changes that have occurred on the main evolutionary path with those that have occurred on the side branches of the phylogenetic tree was made. This showed that while 35% of the mutations on the side branches resulted in amino acid changes, only 21% of those on the main path affected the protein sequence. These results suggest that although the rate of change of the human influenza virus nucleoprotein is much higher than that previously described for avian influenza viruses, there are measurable constraints on the evolution of the surviving virus lineage. Comparison of the nucleoproteins of virus isolates adapted to chicken embryos with the nucleoproteins of those grown only in MDCK cells revealed no consistent differences between the virus pairs. Thus, although the nucleoprotein is known to be critical for host specificity, its adaptation to growth in eggs apparently involves no immediate selective pressures, such as are found with hemagglutinin.

Influenza--a model of an emerging virus disease.

Influenza A viruses continue to emerge from the aquatic avian reservoir and cause pandemics. Phylogenetic analysis of the nucleotide sequence of all eight influenza A virus RNA segments indicate that all of the influenza viruses in mammalian hosts originate from the avian gene pool. In contrast to the rapid progressive changes in both the nucleotide and amino acid sequences of mammalian virus gene lineages, avian virus genes show far less variation and, in most cases, appear to be in evolutionary stasis. There are periodic exchanges of influenza virus genes or whole viruses between species giving rise to pandemics of diseases in humans, lower animals and birds. The periodic emergence of influenza viruses in mammalian species has been illustrated by the appearance of a new influenza virus in horses in northern China in 1989. Phylogenetic analysis of classical H1N1, avian-like H1N1 and human H3N2 viruses circulating in Italian pigs reveals that genetic reassortment is taking place between avian- and human-like viruses in the European pig population. These studies provide evidence supporting the possibility that pigs serve as a mixing vessel for reassortment between influenza viruses in mammalian and avian hosts and raise the question of whether the next pandemic of influenza will emerge in Europe!

Evolution and ecology of influenza A viruses.

In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.

Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts.

The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.

Composition of the helical internal components of influenza virus as revealed by immunogold labeling/electron microscopy.

The composition of the large helical internal components of influenza virus was investigated by immunogold labeling/electron microscopy with antibodies to the nucleoprotein (NP), matrix protein (M), and polymerase complex (PB1, PB2, and PA) of the virus. The morphologically intact helices, obtained by air-drying of the virions on the electron microscope grid, showed little or no labeling with any of the above antibodies. However, partial to full degradation of the helix by proteinase K (2 ng/ml) prior to immunogold labeling made the helices accessible to all three antibodies. The results are consistent with a model that the helix represents a polymer of M protein enclosing or containing the influenza ribonucleoprotein(s).

Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins.

Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Evolution of influenza A virus nucleoprotein genes: implications for the origins of H1N1 human and classical swine viruses.

A phylogenetic analysis of 52 published and 37 new nucleoprotein (NP) gene sequences addressed the evolution and origin of human and swine influenza A viruses. H1N1 human and classical swine viruses (i.e., those related to Swine/Iowa/15/30) share a single common ancestor, which was estimated to have occurred in 1912 to 1913. From this common ancestor, human and classical swine virus NP genes have evolved at similar rates that are higher than in avian virus NP genes (3.31 to 3.41 versus 1.90 nucleotide changes per year). At the protein level, human virus NPs have evolved twice as fast as classical swine virus NPs (0.66 versus 0.34 amino acid change per year). Despite evidence of frequent interspecies transmission of human and classical swine viruses, our analysis indicates that these viruses have evolved independently since well before the first isolates in the early 1930s. Although our analysis cannot reveal the original host, the ancestor virus was avianlike, showing only five amino acid differences from the root of the avian virus NP lineage. The common pattern of relationship and origin for the NP and other genes of H1N1 human and classical swine viruses suggests that the common ancestor was an avian virus and not a reassortant derived from previous human or swine influenza A viruses. The new avianlike H1N1 swine viruses in Europe may provide a model for the evolution of newly introduced avian viruses into the swine host reservoir. The NPs of these viruses are evolving more rapidly than those of human or classical swine viruses (4.50 nucleotide changes and 0.74 amino acid change per year), and when these rates are applied to pre-1930s human and classical swine virus NPs, the predicted date of a common ancestor is 1918 rather than 1912 to 1913. Thus, our NP phylogeny is consistent with historical records and the proposal that a short time before 1918, a new H1N1 avianlike virus entered human or swine hosts (O. T. Gorman, R. O. Donis, Y. Kawaoka, and R. G. Webster, J. Virol. 64:4893-4902, 1990). This virus provided the ancestors of all known human influenza A virus genes, except for HA, NA, and PB1, which have since been reassorted from avian viruses. We propose that during 1918 a virulent strain of this new avianlike virus caused a severe human influenza pandemic and that the pandemic virus was introduced into North American swine populations, constituting the origin of classical swine virus.

Electron microscopic evidence for the association of M2 protein with the influenza virion.

Immunogold electron microscopy revealed that site-specific antibodies elicited by a synthetic peptide representing the N-terminal sequence (residues 2-10) of influenza virus M2 protein were capable of binding to the surface of virions. Antibody binding was observed with two human influenza virus strains but not with an avian virus strain which has amino acid substitutions in the appropriate sequence of M2. These results provide direct evidence for the presence of M2 in the influenza virion.

Balloon dilation of prostatic urethra.

Retrograde balloon catheter dilation of the prostatic urethra was performed for the management of bladder outlet obstruction secondary to prostatic hyperplasia. Of the 55 patients in our series, 43 were treated entirely as outpatients and 12 were inpatients for unrelated conditions. The patient selection was limited to older, high-risk patients who were poor surgical candidates for transurethral resection of the prostate or suprapubic prostatectomy because of underlying medical problems. Twenty-two of these patients had Foley catheters for relief of their outflow obstructions. The procedures were performed under local anesthesia or intravenous sedation. Successful results were noted in 46 of 55 patients with relief of symptoms for up to twenty-six months. In 9 cases the procedures were unsuccessful and transurethral resection of the prostate was required.

Evolution of the nucleoprotein gene of influenza A virus.

Nucleotide sequences of 24 nucleoprotein (NP) genes isolated from a wide range of hosts, geographic regions, and influenza A virus serotypes and 18 published NP gene sequences were analyzed to determine evolutionary relationships. The phylogeny of NP genes was determined by a maximum-parsimony analysis of nucleotide sequences. Phylogenetic analysis showed that NP genes have evolved into five host-specific lineages, including (i) Equine/Prague/56 (EQPR56), (ii) recent equine strains, (iii) classic swine (H1N1 swine, e.g., A/Swine/Iowa/15/30) and human strains, (iv) gull H13 viruses, and (v) avian strains (including North American, Australian, and Old World subgroups). These NP lineages match the five RNA hybridization groups identified by W. J. Bean (Virology 133:438-442, 1984). Maximum nucleotide differences among the NPs was 18.5%, but maximum amino acid differences reached only 10.8%, reflecting the conservative nature of the NP protein. Evolutionary rates varied among lineages; the human lineage showed the highest rate (2.54 nucleotide changes per year), followed by the Old World avian lineage (2.17 changes per year) and the recent equine lineage (1.22 changes per year). The per-nucleotide rates of human and avian NP gene evolution (1.62 x 10(-3) to 1.39 x 10(-3) changes per year) are lower than that reported for human NS genes (2.0 x 10(-3) changes per year; D. A. Buonagurio, S. Nakada, J. D. Parvin, M. Krystal, P. Palese, and W. M. Fitch, Science 232:980-982, 1986). Of the five NP lineages, the human lineage showed the greatest evolution at the amino acid level; over a period of 50 years, human NPs have accumulated 39 amino acid changes. In contrast, the avian lineage showed remarkable conservatism; over the same period, avian NP proteins changed by 0 to 10 amino acids. The specificity of the H13 NP in gulls and its distinct evolutionary separation from the classic avian lineage suggests that H13 NPs may have a large degree of adaptation to gulls. The presence of avian and human NPs in some swine isolates demonstrates the susceptibility of swine to different virus strains and supports the hypothesis that swine may serve as intermediates for the introduction of avian influenza virus genes into the human virus gene pool. EQPR56 is relatively distantly related to all other NP lineages, which suggests that this NP is rooted closest to the ancestor of all contemporary NPs. On the basis of estimation of evolutionary rates from nucleotide branch distances, current NP lineages are at least 100 years old, and the EQPR56 NP is much older.(ABSTRACT TRUNCATED AT 400 WORDS)

What is the potential of avirulent influenza viruses to complement a cleavable hemagglutinin and generate virulent strains?

A large pool of avirulent influenza viruses are maintained in the wild ducks and shorebirds of the world, but we know little about their potential to become virulent. It is well established that the hemagglutinin (HA) is pivitol in determining virulence and that a constellation of other genes is also necessary (R. Rott, M. Orlich, and C. Scholtissek, 1976, J. Virol. 19, 54-60). The question we are asking here is the ability of avirulent influenza viruses to provide the gene constellation that will complement the HA from a highly virulent virus and for the reassortant to be virulent. Reassortant influenza viruses were prepared between ultraviolet treated A/Chicken/Pennsylvania/1370/83 (H5N2) [Ck/Penn] and influenza viruses from natural reservoirs. These viruses included examples of the predominant subtypes in wild ducks, shorebirds, and domestic poultry. Attention was given to the influenza viruses from live poultry markets, for it is possible that these establishments may be important in mixing of influenza genes from different species and the possible transmission to domestic and mammalian species. The reassortants were genotyped by partial sequencing of each gene and were tested for virulence in chickens. Each of the reassortants contained the hemagglutinin and matrix (M) genes from Ck/Penn and a majority of genes from the viruses from natural reservoirs indicating a preferential association between the HA and M genes. The reassortants containing multiple genes from wild ducks and a cleavable HA were avirulent indicating that the gene pool in ducks may not have a high potential to provide genes that are potentially virulent. In contrast, a disproportionate number of viruses from shorebirds and all avirulent H5N2 influenza viruses from city markets provided a gene constellation that in association with cleavable H5 HA were highly virulent in chickens. An evolutionary tree based on oligonucleotide mapping established that the H5N2 influenza viruses from birds in city markets are closely related.

Biologic potential of amantadine-resistant influenza A virus in an avian model.

Amantadine has been accepted for both the treatment and prophylaxis of influenza A virus infections. Although amantadine-resistant mutants have been shown to be readily generated both in the laboratory and in children treated with rimantadine, little is known about their biologic properties, such as genetic stability, transmissibility, or pathogenicity, compared with the parental virus. This study examined these properties using an avian influenza virus, A/chicken/Pennsylvania/1370/83 (H5N2). Variants that were amantadine-resistant, virulent, and capable of competing with wild-type virus for transmission to susceptible hosts in the absence of the drug were selected. These amantadine-resistant variants were also genetically stable, showing no reversion to wild-type after six passages in birds over a period of greater than 20 d. Thus, these virus variants had no detectable biologic impairment. The mutations conferring drug resistance were in the M2 polypeptide and were identical to mutations previously described in human amantadine-resistant virus. These results suggest that resistant mutants may have the potential to threaten the effective use of amantadine and rimantadine for the control of epidemic influenza.

Distinct lineages of influenza virus H4 hemagglutinin genes in different regions of the world.

To understand the determinants of influenza virus evolution, phylogenetic relationships were determined for nine hemagglutinin (HA) genes of the H4 subtype. These genes belong to a set of viruses isolated from several avian and mammalian species from various geographic locations around the world between 1956 and 1985. We found that the HA gene of the H4 subtype is 1738 nucleotides in length and is predicted to encode a polypeptide of 564 amino acids. The connecting peptide, which is removed from the precursor polypeptide by peptidases to yield the mature HA1 and HA2 polypeptides, contains only one basic amino acid. This type of connecting peptide is a feature of all avian avirulent HAs. On the basis of pairwise nucleotide sequence homology comparisons the genes can be segregated into two groups: influenza virus genes isolated in North America and those isolated from other parts of the world. A high degree of homology exists between pairs of genes from viruses of similar geographic origin. The nucleotide sequences within a group differ by 1.5 to 10.6%; in contrast, between groups the differences range from 15.8 to 19.4%. An evolutionary tree for the nine sequences suggests that North American isolates have diverged extensively from those circulating in other parts of the world. Geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. The lack of correlation between date of isolation and evolutionary distance suggests that different H4 HA genes cocirculate in a fashion similar to avian H3 HA genes (H. Kida et al., 1987, Virology 159, 109-119) and influenza C genes (D. Buonagurio et al., 1985, Virology 146, 221-232) implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. In contrast to avian influenza virus genes, human influenza virus genes evolve rapidly under the selective pressure of antibody.