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In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine., (Copyright © 2020. Published by Elsevier Ltd.)

Unlabelled: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain., Importance: The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

To improve the hemagglutinin (HA) antigen yield of influenza A(H1N1)pdm09 candidate vaccine viruses, we generated 7:1, 6:2, and 5:3 genetic reassortant viruses between wild-type (H1N1)pdm09 (A/California/7/2009) (Cal7) and a high-yielding master virus, A/Puerto Rico/8/34 (PR8). These viruses contained the HA; HA and neuraminidase (NA); and HA, NA, and M genes, respectively, derived from Cal7, on a PR8 backbone. The influence of the amino acid residue at position 223 in Cal7 HA on virus growth and HA antigen yield differed between these reassortant viruses. NIIDRG-7, a 7:1 virus possessing arginine at position 223, exhibited a 10-fold higher 50% egg infectious dose (EID(50)) (10.0 log(10)EID(50)/ml) than the 5:3 and 6:2 viruses. It also had 1.5- to 3-fold higher protein (13.8 μg/ml of allantoic fluids) and HA antigen (4.1 μg/ml of allantoic fluids) yields than the 5:3 and 6:2 viruses, which possessed identical Cal7 HA proteins. However, the HA antigen yield of the other 7:1 virus, which possessed glutamine at position 223 was 60% of that of NIIDRG-7. In addition, a novel 6:2 virus possessing Cal7 HA and the NA of A/Wisconsin/10/98 (a triple reassortant swine-like H1N1 virus), produced 107% of the HA yield of NIIDRG-7. In this study, we showed that the balance between HA and NA in the influenza A(H1N1)pdm09 virus affects its protein and antigen yield.

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., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Quantum mechanical fragment molecular orbital calculations have been performed for receptor binding of the hemagglutinin protein of the recently pandemic influenza 2009 H1N1, A/swine/Iowa/1930, and A/Puerto Rico/8/1934 viruses to α2-6 linked sialyloligosaccharides, as analogs of human receptors. The strongest receptor binding affinity was observed for the 2009/H1N1pdm. The inter-fragment interaction energy analysis revealed that the amino acid mutation of 2009/H1N1pdm, Ser145Lys, was a major cause of such strong binding affinity. Strong ionic pair interaction between the sialic acid and Lys145 was observed only in the 2009/H1N1pdm, in addition to the hydrogen bond between the sialic acid and Gln226 observed in all the HAs. Therefore, pandemic 2009/H1N1pdm has been found to recognize the α2-6 receptor much stronger than the 1930-swine and 1934-human.

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.

We introduced 38 single-point amino acid changes into the hemagglutinin (HA) protein of the reassortmented A/Duck/Mongolia/54/01 (H5N2) strain by a PCR random mutation method. The percentage of amino acid changes on the HA domain that did not abrogate hemadsorption activity was calculated to be 89%. Changes in the amino acids of the HA2 domain were observed to be about half of those in the HA1 domain of these mutants. We assumed that amino acid changes in the HA1 domain afforded more flexibility in maintaining the functions of the HA protein than did those in the HA2 domain. Changes at two positions allowed the mutants to have same characteristics with respect to HA function despite the difference in the substituted amino acid. The results suggested that the effect on hemadsorption activity of an amino acid change on the HA protein primarily depends on the position rather than the species of substituted amino acid. An amino acid change at residue 122 from Trp to Arg and 179 from His to Arg resulted in the loss of hemadsorption activity of the HA protein. Site 122 is near the antibody binding site A, and site 179 is in the receptor binding domain (RBD) of HSHA. So that we suggest residue position 179 or 122 is very important to maintain the structure of RBD or antigenic site of H5HA. Position 4 in HA1 changed from Cys to Arg and position 148 in HA2 changed from Cys to Tyr also resulted in the loss of hemadsorption activity of the HA protein. Cys plays an important role in maintaining the structure of HA protein by means of S-S bonds. 3 potential glycosylation sites (Asn-X-Ser/Thr) were lost in our experiment that did not lose the hemadsorption activity of HA. Some interesting positions need to be analyzed more finely. Some amino acid changes identified in vitro experiment may serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Starting with nine plaques of influenza A/Kamata/14/91(H3N2) virus, we selected mutants in the presence of monoclonal antibody 203 (mAb203). In total, amino acid substitutions were found at nine positions (77, 80, 131, 135, 141, 142, 143, 144 and 146), which localized in the antigenic site A of the hemagglutinin (HA). The escape mutants differed in the extent to which they had lost binding to mAb203. HA protein with substitutions of some amino acid residues created by site-directed mutagenesis in the escape mutants retained the ability to bind to mAb203. Changes in the amino acid character affecting charge or hydrophobicity accounted for the binding capacity to the antibody of the HA with most of the substitutions in the escape mutants and binding-positive mutants. However, the effect of some amino acid substitutions remained unexplained. A three-dimensional model of the 1991 HA was constructed and used to analyze substituted amino acids in these mutants for the accessible surface hydrophobic and hydrophilic characters. One amino acid substitution in an escape mutant and another amino acid substitution in a binding-positive mutant seemed to be explained by the changes noted on this model.

During protein evolution the amino acid substitutions accumulate with time. However, the effect of accumulation of the amino acid substitutions to structural changes has not been estimated well. We will propose that the discordance of amino acid substitution on the HA protein of influenza A virus is useful for the assessment of structural changes during evolution. Discordance value can be obtained from the experimental data of tolerance or intolerance by introducing site directed mutagenesis at the homologous positions of two HA proteins holding the same amino acid residues. The value of discordance correlated to the number of amino acid differences among proteins. In the H3HA discordance rate was calculated to be 0.45% per one amino acid change. Furthermore, discordance of amino acid substitutions suggests that tolerable amino acid substitutions in different order have a probability of promoting irreversible divergence of the HA protein to different subtypes.

The C-terminal sequence of the cytoplasmic tail (CT) of influenza B haemagglutinin (BHA) consists of strictly conserved, hydrophobic amino acids, and the endmost C-terminal amino acid of the CT is Leu. To elucidate the role of this amino acid in the fusion activity of BHA (B/Kanagawa/73), site-specific mutant HAs were created by replacing Leu at this position with Arg, Lys, Ser, Try, Val or Ile or by the deletion of Leu altogether. All mutants were expressed at the cell surface, bound to red blood cells, were cleaved properly into two subunits and could be acylated like the wild-type (wt) HA. The membrane-fusion ability of these mutants was examined with a lipid (R18) and aqueous (calcein) dye-transfer assay and quantified with a syncytium-formation assay. All mutant HAs showed no measurable effect on lipid mixing or fusion-pore formation. However, mutant HAs with a hydrophobic value of the C-terminal amino acid lower than that of Leu had a reduced ability to form syncytia, whereas mutants with a more hydrophobic amino acid (Val or Ile) promoted fusion to the extent of the wt HA. On the other hand, the mutant HA with the deletion of Leu supported full fusion. These results demonstrate that Leu at the endmost portion of the C terminus of the BHA-CT is not essential for BHA-mediated fusion, but that the hydrophobicity of the single amino acid at this position plays an important role in syncytium formation.

In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited.

We studied the effects of an increase in the hydrophobicity of the transmembrane domain (TM) and cytoplasmic tail (CT) of influenza B virus hemagglutinin (BHA) on fusion activities. For this purpose, we created mutant HAs with novel acylation site(s) in the TM and/or CT. All mutants were able to induce hemifusion and to form fusion pores as well as could wild type (wt) BHA. However, the ability of these mutants to form syncytia was impaired, indicating that the increase in the hydrophobicity of these domains (especially the CT) affected fusion pore dilation.

The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.

We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.

In an effort to find efficient DNA vaccine candidates, cDNA of influenza A virus hemagglutinin (HA) gene and several derived mutants were injected into mice using a gene gun. Mice immunized with HA1 DNA, with or without a membrane domain, showed a humoral immune response and the survival rate against homologous virus challenge was comparable to that of mice injected with HA DNA. In order to analyze epitopes recognized by antibodies induced by gene gun immunization, we used a binding assay employing the chimeric HA protein method. Serum antibodies of mice immunized with HA DNA recognized the HA1 domain but not the HA2 domain. In addition, antisera obtained from mice immunized with HA1 DNA reacted with each of the known antigenic sites on the HA1 domain, similar to the results obtained with HA DNA immunization.

It was reported previously that haemadsorption by the haemagglutinin (HA) protein of influenza B virus required that the protein must undergo desialidation. When MDCK and COS cells were infected with influenza B/Kanagawa/73 virus in the presence of a neuraminidase (NA) inhibitor, Zanamivir, haemadsorption on MDCK cells was inhibited but that on COS cells was not. The activity of the NA protein of the two types of infected cells was similar and both were inhibited by Zanamivir in a dose-dependent manner. A comparison of the desialidation of the HA protein was made on MDCK and COS cells in the presence of bacterial NA and both cells were found to have similar sensitivity. On the accumulation of the HA and NA proteins in the trans-Golgi network of MDCK cells by means of low-temperature treatment, desialidation of the HA protein in the presence of Zanamivir was detected by two-dimensional gel electrophoresis. Because this agent was reported to be unable to penetrate cells, these data suggest that, in MDCK cells, desialidation of the HA protein occurs on the cell surface but, in COS cells, the HA and NA proteins might accumulate in the trans-Golgi network, thus allowing NA desialidation before their migration to the cell surface.

Human H3N2 influenza A viruses were known to preferentially bind to sialic acid (SA) in alpha2,6Gal linkage on red blood cells (RBC). However, H3N2 viruses isolated in MDCK cells after 1992 did not agglutinate chicken RBC (CRBC). Experiments with point-mutated hemagglutinin (HA) of A/Aichi/51/92, one of these viruses, revealed that an amino acid change from Glu to Asp at position 190 (E190D) was responsible for the loss of ability to bind to CRBC. A/Aichi/51/92 did not agglutinate CRBC treated with alpha2, 3-sialidase, suggesting that SAalpha2,3Gal on CRBC might not inhibit the binding of the virus to SAalpha2,6Gal on CRBC. However, the virus agglutinated derivatized CRBC resialylated with SAalpha2, 6Galbeta1,4GlcNAc. These findings suggested that the E190D change might have rendered the HA able to distinguish sialyloligosaccharides on the derivatized CRBC containing the SAalpha2,6Galbeta1,4GlcNAc sequence from those on the native CRBC., (Copyright 2000 Academic Press.)

Eight convalescent human sera obtained from patients aged 3 to 14 years old, who were infected with influenza A(H3N2) virus during the 1990/1991 influenza season, were characterized by a binding assay with chimeric hemagglutinin (HA) proteins between influenza virus A/Aichi/2/68 and A/Kamata/14/91(H3N2) strains. These sera did not recognize the HA protein of the A/Aichi/2/68 strain but recognized that of the A/Kamata/14/91 strain. The binding assay revealed that these sera recognized only the HA1 domain of A/Kamata/14/91 HA protein. A further assay of the binding of these sera to the chimeric proteins of the HA1 domain revealed that three sera (A-1, A-2, and A-3) from very young patients bound only to region 150-170 (site B1) and one serum (Y-1) bound to regions 96-150 (site A) and 96-170 (sites A and B1). These four sera showed reduced hemagglutination inhibition (HI) activity with the 203v2 strain, a monoclonal variant of the A/Kamata/14/91 strain with two amino acid changes in the HA protein at antigenic sites A and B1. The other four sera (Y-2, G-1, G-2, and A-4) bound to regions 1-96 (site C/E), 96-150 (site A), 96-170 (sites A and B1), and 170-200 (site B2), two of which further bound to region 240-306 (site C); these sera were all fully reactive with the 203v2 strain. All eight sera showed reduced HI reactivity to a drift strain A/Aichi/4/93. Amino acid changes of the A/Aichi/4/93 strain from the A/Kamata/14/91 strain were located at antigenic sites A, B1, B2, and C. We propose a possible model for the emergence of a drift strain A/Aichi/4/93 from an A/Kamata/14/91-like strain by sequential changes during reinfections of individuals starting from A-1-like, next to Y-1-like, and then to Y-2-like populations., (Copyright 2000 Academic Press.)

We analysed the role of neuraminidase (NA) on haemadsorption by the haemagglutinin (HA) protein of influenza B virus. The influenza B virus mutant ts-7 has a temperature-sensitive mutation in the NA protein. At high temperature, cells infected with this virus did not exhibit haemadsorption activity, but the addition of bacterial neuraminidase (bNA) restored haemadsorption activity. COS cells transfected with HA cDNAs of B/Kanagawa/73 or B/Lee/40 virus showed no evidence of haemadsorption. However, with the addition of bNA or co- transfection with NA cDNA of the B/Lee/40 virus, haemadsorption was observed. Experiments with point-mutated HA cDNAs of B/Lee/40 virus showed that two N-acetyl glycosylation sites at amino acid residues 160 and 217 were responsible for the inability of the HA protein to adsorb to erythrocytes. These results indicated that haemadsorption by the HA protein of influenza B virus required the involvement of NA. Because the NA inhibitor Zanamivir was reported not to penetrate cells, we investigated the action of this inhibitor and found that Zanamivir inhibited haemadsorption on MDCK cells infected with B/Kanagawa/73 or B/Lee/40 virus. After removing Zanamivir by washing, the addition of bNA restored the haemadsorption activity on the infected cells. Scanning electron microscopy indicated that at 0.4 microM Zanamivir, HA protein did not adsorb to erythrocytes but retained the ability to aggregate virions. However, at 4 microM Zanamivir, distinct virion formation could not be observed.

Phylogenetic analysis was carried out for genes encoding hemagglutinin (HA) (24 new and 25 previously reported sequences) and nonstructural proteins (NS) (22 new and 14 previously reported sequences) of influenza B virus isolates obtained from 1940 to 1999. Two antigenically and genetically distinct HA lineages are presently known to exist. Divergence into these two lineages was estimated to have occurred around 1969. Phylogenetic analysis of NS genes revealed that their phylogenetic relationships were not linked to the two HA lineages but suggested that reassortment of viral genes between the viruses of two HA lineages had occurred. In addition two distinct NS lineages which were not linked to the two HA lineages were observed. Viruses isolated after 1997 formed their own lineage in combination with B/Houston/84 while other virus isolates obtained from 1973 to 1995 comprised the other NS lineage.

From the reassortment experiments between A/Aichi/4/92 and A/WSN/33 (WSN) (H1N1) viruses, two different phenotype viruses which contained the haemagglutinin (HA) gene from A/Aichi/4/92 virus and the neuraminidase (NA) gene from WSN virus were obtained. PW13 and PW15 viruses agglutinated chicken red blood cells (CRBC), while PW10 and PW70 viruses did not. However, the expressed HA proteins of these viruses did not adsorb CRBC. The difference in gene constellation between PW13, PW15 and PW10, PW70 viruses was the membrane protein (M) gene. The former two had the M gene from A/Aichi/4/92 virus and the latter two had that from WSN virus. In PW15-infected cells, haemadsorption of CRBC was observed 30 min later than that of goose red blood cells and the M1 protein migrated from the nucleus to the cytoplasm 30 min earlier than adsorption of CRBC was observed. On the other hand, in PW10-infected cells, haemadsorption of CRBC was not observed through the virus replication and the M1 protein stayed in the nucleus after HA and NA activities reached maximum levels. Co-expression of the M and the HA proteins of A/Aichi/4/92 virus did not help the HA protein gain the ability to adsorb CRBC. However, neuraminidase treatment of COS cells expressing the HA protein of A/Aichi/4/92 virus or MDCK cells infected by PW10 virus restored the ability to adsorb CRBC. We discussed the possibility that the M1 protein helped the NA protein in its role to modify the HA protein on the cell surface.

The hemagglutinin(HA) of influenza virus is a major glycoprotein and plays a crucial role in the early stage of virus infection: HA is responsible for binding of the virus to cell surface receptors, and it mediates liberation of the viral genome into the cytoplasm through membrane fusion. The essential component of the receptor for influenza viruses has been considered to be the sialic acid. Influenza A and B viruses recognize N-acetylneuraminic acid, whereas influenza C virus specifically recognizes N-acetyl-9-O-acetylneuraminic acid as the receptor. Influenza A viruses are subdivided into 15 subtypes by their antigenic differences, but several amino acid residues composing functional domains (receptor binding site and fusion peptide) are shown to be conserved among HAs.

Recent strains of influenza A but not B viruses have lost the ability to agglutinate chicken red blood cells (CRBC). The H1N1 viruses isolated in Japan during the 1991/92 season could be divided into two groups. Group 1 viruses (A/Aichi/4/92 and A/Aichi/7/92) agglutinated goose red blood cells (GRBC) and CRBC, while group 2 viruses (A/Aichi/24/92 and A/Aichi/26/92) did not agglutinate CRBC. There were no amino acid differences between them in the haemagglutinin (HA) polypeptide. Reassortment experiments between a group 1 virus (A/Aichi/4/92) or a group 2 virus (A/Aichi/24/92) and the A/WSN/33 influenza A (H1N1) virus strain suggested that the HA gene products of the viruses of both groups had lost the capacity to agglutinate CRBC. The HA proteins expressed on Cos cells by transfecting the cDNAs of the virus HA gene of A/Aichi/4/92 and A/Aichi/24/92 agglutinated GRBC but not CRBC. These experiments indicated that the HA proteins of H1N1 viruses of both groups isolated in 1992 had lost the ability to agglutinate CRBC even though the group 1 virions showed haemagglutinating capacity with CRBC. By using the cDNAs of the HA gene of seven natural isolates obtained from 1977 to 1992, it was found that the expressed HA proteins of influenza A (H1N1) viruses isolated since 1988 had lost the ability to agglutinate CRBC. Experiments with chimeric and point-mutated HA cDNAs of A/Aichi/24/92 showed that an amino acid change at residue 225, which occurred after 1986, and a cluster of amino acid changes at residues 193, 196 and 197, which occurred before 1986, were responsible for loss of the ability to agglutinate CRBC. Egg-adapted virus derived from A/Aichi/24/92 had one amino acid change at residue 225 compared to the parental virus.