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Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production. IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines., (Copyright © 2017 American Society for Microbiology.)

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.)

Ab initio electronic-state calculations for influenza virus hemagglutinin (HA) trimer complexed with Fab antibody were performed on the basis of the fragment molecular orbital (FMO) method at the second and third-order Møller-Plesset (MP2 and MP3) perturbation levels. For the protein complex containing 2351 residues and 36,160 atoms, the inter-fragment interaction energies (IFIEs) were evaluated to illustrate the effective interactions between all the pairs of amino acid residues. By analyzing the calculated data on the IFIEs, we first discussed the interactions and their fluctuations between multiple domains contained in the trimer complex. Next, by combining the IFIE data between the Fab antibody and each residue in the HA antigen with experimental data on the hemadsorption activity of HA mutants, we proposed a protocol to predict probable mutations in HA. The proposed protocol based on the FMO-MP2.5 calculation can explain the historical facts concerning the actual mutations after the emergence of A/Hong Kong/1/68 influenza virus with subtype H3N2, and thus provides a useful methodology to enumerate those residue sites likely to mutate in the future., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human influenza A viruses evolve more rapidly than influenza B viruses. To clarify the cause of this difference, we have evaluated the mutation rate of the nonstructural gene as revealed by the genetic diversity observed during the growth of individual plaques in MDCK cells. Six plaques were studied, representing two strains each of type A and B viruses. A total of 813,663 nucleotides were sequenced, giving rates of 2.0 x 10(-6) and 0.6 x 10(-6) mutations per site per infectious cycle, which, when extended to 1 year, agree well with the published annual evolutionary rates.

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.

Sera from 27 children and eight older persons, which had been collected in 1998 and 1999 and showed haemagglutination-inhibition (HI) activity against influenza A/Sydney/5/97 (H3N2) strain, were characterized with a binding assay using chimeric haemagglutinin (HA) proteins between A/Aichi/2/68 (A/AI/68) and A/Sydney/5/97 (A/SD/97) strains. Sera from the young children had a tendency to recognize only the antigenic site B1 of the HA1 region. On the other hand, sera of the older individuals were fully reactive to all antigenic sites of HA1 except antigenic site D. Recent epidemic strains, A/Panama/2007/99 (A/PM/99)-like viruses have differences in amino acids in antigenic sites A, C, and B2 but not B1. However, human antisera obtained even from young children had HI activity to Panama-like viruses. The limited epidemic of A/PM/99-like viruses may have been due to the existence of antibody against B1, which had been produced in response to infection by the A/SD/97-like viruses.

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.

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.)

In order to conduct a survey of influenza viruses entering Japan via travellers arriving by airplanes, gargle solutions were collected from passengers who reported to the quarantine station of Nagoya International Airport complaining of respiratory symptoms. From 504 samples collected between August 1996 and March 1999, 30 influenza virus strains were isolated. Twenty-eight of the isolates were influenza A (H3N2) viruses and two were influenza B viruses. No H1N1 virus was isolated. Among 28 isolates of H3N2 virus, 3 strains were obtained outside the influenza season. Nucleotide sequences of the haemagglutinin (HA) genes of these isolates along with those from domestic patients were analysed in order to determine the influence of imported influenza viruses by travellers on epidemics in Japan. From the phylogenetic and chronological aspects, the possibility was suggested in one case in 1997/8 and two in the 1998/9 season that imported virus by travellers may have influenced the domestic influenza epidemics.

Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the pathogenicity and even for the neurovirulence of influenza A viruses. WSN, a neurovirulent virus, adapted to mouse brain, grew in vitro in several types of cells including neuroblastoma cells in the absence of trypsin. When mice were intracerebrally inoculated with WSN, the viral antigen was found in the substantia nigra zona compacta and hippocampus. The mice inoculated with viruses isolated from children with acute encephalopathy associated with an influenza virus infection, on the other hand, showed no neurological symptoms. Furthermore, these viruses did not grow in the human neuroblastoma and glioblastoma cells. Since 1991, most of the human influenza A viruses have not agglutinated chicken erythrocytes. Whether this altered receptor binding specificity is related to the occurrence of influenza encephalitis and encephalopathy is now under investigation.

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.

Influenza A virus is unique among human viruses in its capacity to alter the antigenic phenotype with relative ease and evade neutralizing antibodies. This property is ascribed to the accumulation of a series of amino acid changes in the antigenic regions of hemagglutinin (HA) molecule. Neutralizing antibodies against HA prevent the early stage of infection, whereas neuraminidase (NA) antibodies mediate the antiviral effect by restricting spread of viruses in the host cells after infection. Thus the understanding of antigenic structure on those protective antigen is significant. Sequence analysis of natural variants and escape mutants selected by monoclonal antibodies allowed us to assign each antigenic sites and epitopes to a particular region on the three dimensional structure of HA and NA molecules.

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.

Influenza A viruses are suitable for the analysis of virus evolution because the genes of the viruses are well analyzed. The origin of the present human influenza A viruses are deduced to be direct descendent of the viruses which caused Spanish flu in 1918. The analysis of NS gene shows the branching point between avian and human viruses are early 1900. By comparison of the amino acid sequences, HA serotypes could be divided into two groups, i.e., an H1 and an H3 groups. The branching between subtypes H1 and H2 occurred fairly recently. The HA genes of influenza A viruses evolve causing two kinds of antigenic variation, saift and drift, which are caused by different mechanisms.

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.

We determined the sequences of 7 serotypes (H4, H6, H8, H9, H11, H12, and H13) of hemagglutinin (HA) genes, which have not been reported so far. The coding regions consisted of 1692 nucleotides in H4, 1698 in H6, 1695 in H8, 1680 in H9, 1695 in H11, 1692 in H12, and 1698 in H13, and specified 564, 566, 565, 560, 565, 564, and 566 amino acids, respectively. By comparison of amino acid sequences, 13 HA serotypes could be divided into two families, i.e., an H1 group (H1, H2, H5, H6, H8, H9, H11, H12, and H13) and an H3 group (H3, H4, H7, and H10). The relationship was essentially similar to that reported by Air from the comparison of 80 amino-terminal amino acid sequence of 12 HA serotypes (G.M. Air, 1981, Proc. Natl. Acad. Sci. USA 78, 7639-7643). Though a considerable amino acid sequence difference exists between certain HA serotypes, several amino acid residues in fusion peptides (HA2(1-11)) and receptor-binding sites (HA1(98), -134, -138, -153, -183, and -195) were shown to be conserved among the 13 HA serotypes. Human H1 and avian H3, H4, H8, and H10 viruses preferentially bound NeuAc alpha 2,3Gal sequences, whereas human H2 and H3 and avian H6 and H9 viruses bound NeuAc alpha 2,6Gal sequences, although the amino acid residues at position 226 of human H2 and avian H6 and H9 serotype HAs are glutamine. These results show that the amino acid residue at position 226 is not necessarily a determinant of receptor specificity for all serotypes.

We have studied changes of epitopes on the haemagglutinin molecule (HA) of H1N1 influenza viruses isolated between 1977 and 1986. For this purpose monoclonal antibodies (MAbs) were raised against the HA of the influenza A/England/333/80 and A/Yamagata/120/86 strain viruses. In order to define the amino acid residues responsible for the change of epitopes, we prepared several HA cDNAs modified by site-directed mutagenesis and cloned them into a simian virus 40 expression vector (SVHA). The substitution of glycine with serine at position 125c (suffix indicates presence in H1 but not in H3 subtype HAs) on the HA of the influenza A/USSR/90/77 strain virus resulted in the loss of epitope 110 (epitopes were named after MAbs) and created new epitopes 139 and 15, which were observed on the HA of A/England/333/80 and a few isolates from 1983. These new epitopes disappeared from the HA in some of the isolates in 1983 and most of the isolates in 1984 and 1986. The disappearance of epitopes 139 and 15 seems to be associated with the loss of epitope W18, which was identified on the HA of A/USSR/90/77. We suggested previously that amino acid residue 189 was involved in epitope W18. We therefore expressed an HA protein with two amino acid substitutions at positions 189 and 125c and found that the conversion of glutamine to lysine at position 189 in SVHA-67 prevented the expression of epitopes 139 and 15.

We compared the nucleotide sequences of the NS genes of 13 animal influenza viruses belonging to human, swine, avian, and equine viruses for the study of the genetic relatedness of the NS genes in animal influenza viruses. The NS genes of three virus strains A/chicken/Brescia/02, A/equine/Prague/56, and A/equine/Miami/63 were newly sequenced. The base sequence homologies between the NS genes of avian, human, swine, and the A/equine/Miami/63 viruses were 87.8% or higher. On the other hand, the base sequence of the NS gene of the A/equine/Prague/56 virus differed widely from those of other viruses analyzed in the present study. We constructed a model of the genetic tree of the NS genes of avian and equine influenza viruses by a modified Farris method. For comparison of the NS genes between human and avian viruses, we estimated the speed of the nucleotide substitutions of the avian influenza NS genes. It was roughly constant, even though the substitutions did not occur sequentially. The nucleotide substitution rate of the NS genes of avian influenza viruses was one-third to one-fourth that of human influenza viruses. We deduced the time of separation between the NS genes of human and avian influenza viruses during evolution.

We compared the amino acid sequences of the NS1 proteins of human, equine, and avian influenza viruses. The ratios of the amino acid substitutions per nucleotide substitutions in the NS1 proteins were about 27-45%, suggesting the existence of constraints on the amino acid changes of the NS1 protein in evolution. As a measure of constraints exerted on the regions of a protein, a changeability index is proposed. There was a highly conserved region between amino acid residues 30 and 50. The C-terminal region of amino acid residue 165 was a continuously changeable region. We have either introduced several nucleotide substitutions to the NS cDNA of the A/Udorn/72 virus in vitro or constructed the recombinant NS cDNAs between the A/Udorn/72 and A/chick/Japan/24 viruses, and then expressed them in animal cells. We have found that the amino acid substitutions introduced to the low-conserved region of the NS1 protein affected the stability and nuclear localization of the NS1 protein. One of the chimeric proteins between the A/Udorn/72 and A/chick/Japan/24 viruses did not move to the nucleus of the cell and remained in the cytoplasm.

Full-length cDNA of HA gene derived from B-1-23 virus, a variant of A/USSR/90/77 (A/USSR/77) (H1N1) which had been selected with monoclonal antibody (mAb) 264 and contained an amino acid change at position 190 of the HA1 region, was cloned into the SV40 expression vector. Hemabsorbing or fusion activities of the HA protein expressed on CV-1 cells infected with the recombinant virus were indistinguishable from those of the authentic HA protein on B-1-23 virus infected cells. The antigenicity of the expressed HA protein was examined with five monoclonal antibodies to the HA of A/USSR/77 virus. The HA protein reacted with all mAbs except for mAb 264. To define the area of epitope 264, we prepared seven HA cDNAs modified by site-specific mutagenesis and cloned them into the SV40 expression vector (SVHA). The single base change from G to A at position 642 of B-1-23 HA cDNA replaced Asn at position 190 of the HA protein with Asp. The resulting HA protein, the amino acid sequence of which was the same as that in A/USSR/77, regained the binding activity with mAb 264. F. L. Raymond et al. (1984, In "Segmented Negative Strand Viruses" (R. W. Compans and D. H. L. Bishop, Eds.), pp. 253-258, Academic Press, Orlando; 1986, Virology 148, 275-287) and A. P. Kendal et al. (1984, In "Modern Approaches to Vaccines," pp. 151-157, Harvard Univ. Press, Cambridge) showed that changed amino acid residues 219(Lys) and 227(Glu) in A/Brazil/11/78 and 189(Lys) and 225(Asp) in A/Lackland/3/78 were presumably included in the area of epitope 264 by the nucleotide sequence analysis. Of those amino acids, our results show it is the residues 190 and 219 and possibly residue 189 that are involved in the epitope 264, but neither residue 225 nor 227.

Human influenza A virus isolates bearing antigenically different H1 (A/PR/8/34), H2 (A/Japan/305/57), and H3 (A/Aichi/2/68, A/X-31) hemagglutinin serotypes caused extensive hemagglutination, low pH fusion, and hemolysis of asialoerythrocytes reconstituted with gangliosides. Sialylparaglobosides (IV3NeuAc-nLc4Cer, IV6NeuAc-nLc4Cer), I-active and i-active (VI3NeuAc-nLc6Cer) gangliosides, and GM3-NeuAc commonly exhibited significant specific receptor activity toward the viruses. A/PR/8/34 recognized IV3NeuAc-nLc4Cer containing the NeuAc alpha 2-3Gal sequence preferentially over IV6NeuAc-nLc4Cer containing NeuAc alpha 2-6Gal, whereas the other two recognized the NeuAc alpha 2-6Gal sequence preferentially over NeuAc alpha 2-3Gal. Responsiveness of erythrocytes labeled with gangliosides containing NeuGc to the viruses used was considerably lower than that of erythrocytes labeled with gangliosides containing NeuAc. The activities of GM1a, GM2, and GD1b bearing NeuAc on inner galactose of the ganglio series core were also very low. These results indicate that sialyloligosaccharides of IV3NeuAc-nLc4Cer, IV6NeuAc-nLc4Cer, I-active ganglioside, and VI3NeuAc-nLc6Cer in addition to GM3-NeuAc and GM1b-NeuAc (Suzuki, Y., Matsunaga, M., and Matsumoto, M. (1985), J. Biol. Chem. 260, 1362-1365; Suzuki, Y., Matsunaga, M., Nagao, Y., Taki, T., Hirabayashi, Y., and Matsumoto, M. (1985) Vaccine 3, 201-203) are functional receptor determinants toward hemagglutinin of human influenza A viruses, and the viruses differentiate microdomains of the gangliosides, such as the sialic acid species (NeuAc, NeuGc) and the sequence of sialic acid linkages (NeuAc alpha 2-3Gal, NeuAc alpha 2-6Gal).

The nucleotide sequences of the M and NS1 genes of influenza virus A/Swine/Iowa/15/30 (A/SW/IW/30)(H1N1) were determined with cloned DNAs and compared with reported sequences of human and avian influenza viruses. A/SW/IW/30 virus was found to be closely similar to A/PR/8/34(H1N1) virus in the nucleotide sequences of the M and NS1 genes, the base differences between the two strains being 64 out of 1027 nucleotides in the M gene and 52 out of 740 in the NS1 gene. Based on the assumptions that these two viruses were derived from a common ancestor and that the rate of base changes per year was the same in man and in swine, it was estimated that the progenitor virus was in circulation during the period from 1915 to 1920. This estimation was compatible with the epidemiological findings suggesting that the progenitor of the swine influenza virus was the agent of the 1918 influenza pandemic. Furthermore, the M and NS1 gene sequences of A/FPV/Rostock/34(H7N6) virus were much closer to those of A/SW/IW/30 and A/PR/8/34 viruses than to A/duck/Alberta/60/76(H12N5) virus, but not as close as the A/SW/IW/30 virus was to A/PR/8/34 virus.

The nucleotide sequences of the NS genes of avian influenza A viruses, A/Chicken/Japan/24, A/Duck/England/56, A/Tern/South Africa/61, A/Duck/Ukraine/1/63, and A/Mynah/Haneda-Thai/76, were determined and compared among themselves and with two reported NS sequences of the avian viruses, A/FPV/Rostock/34 and A/Duck/Alberta/60/76. Thirty-six to two hundred forty base differences in the NS genes were found in pairwise comparisons among the viruses. The numbers of base differences in the NS genes increased with time, except A/Duck/Alberta/60/76 virus. However, the NS genes of the avian viruses did not change sequentially with time and were arranged in separate evolutionary lineages. When the NS genes of avian viruses employed in the present study were compared with those of human viruses, sequence similarity was confirmed (M. Baez, R. Taussig, J. J. Zarza, J. F. Young, P. Palese, A. Reisfield, and A. M. Skalka, 1980, Nucleic Acids Res. 8, 5845-5858). The numbers of base differences in the NS genes between avian viruses and the A/PR/8/34 virus were 61 to 83, and the NS gene of the oldest avian isolate, A/Chicken/Japan/24, was most closely related to that of the A/PR/8/34 virus. It was hypothesized that NS genes of human influenza viruses and those of some avian influenza viruses had been derived from a common ancestor gene.

The receptor binding site of the hemagglutinin (HA) molecule of type A influenza virus A/USSR/90/77 (H1N1) has been studied. Site-specific mutagenesis has been used to introduce base changes into the sequence that codes for the amino acid residue at position 226 on the HA molecule, and mutant sequences replaced the wild-type sequence of the HA gene of the SV40-HA recombinant virus (SVHA). Mutant HA proteins were expressed in African green monkey kidney cells and analyzed for receptor binding and fusion activities. Two mutant HA proteins containing single amino acid substitutions of Asn and Met for Gln at position 226 retained their receptor binding activity, but others with amino acid substitutions Glu, His, Leu, Val, and Thr for Gln at position 226 lost this activity. All the mutant proteins retained their fusion activity. On the other hand, another four mutants containing single amino acid substitutions at positions other than 226 retained the receptor binding and fusion activities, despite the drastic change in charge or polarity to the respective amino acids. These results suggest that amino acid residue 226 of the H1 subtype of HA is critical for receptor binding activity of the HA protein. Our results also show that the lack of receptor binding activity of the HA protein does not affect fusion activity.

Antigenic drift of the haemagglutinin (HA) molecule of type A influenza viruses has been thought to occur by the accumulation of a series of point mutations in the antigenically important regions of the molecule. In order to study the antigenic sites on the HA molecule, an attempt was made to use the site-specific mutagenesis method.