Thirty-one strains of human influenza A (H1N1) viruses isolated in Europe, mostly in Finland, from 1978-1992 were compared with respect to their nucleotide sequences coding for the HA1 portion of haemagglutinin. In 1984, at least two sublineages of H1N1 subtype viruses co-circulated in Finland. The viruses isolated after 1986 formed three sequential phylogenetic clusters. Loss of glycosylation sites, on the globular head of the HA1 portion suggests that oligosaccharides at these sites are not necessarily advantageous for the human virus. Isolation of a herald strain in Finland in June 1988 raised the question as to whether the virus was able to survive in Europe throughout the non-epidemic summer period. Demonstration of highly conserved strains, found over two continents in 1988, is further evidence of the existence of infection chains whose viruses have not been subjected to random sampling or selection events.

A single amino acid substitution, Asp-63 to Asn-63, was detected in the hemagglutinin of an antigenic variant of the 1968 Hong Kong (H3) influenza virus that was selected by growth of the wild-type virus in the presence of a monoclonal antibody. The mutation generates an oligosaccharide attachment site, Asn-Cys-Thr at residues 63-65, that is glycosylated. Immunoprecipitation experiments with extracts from variant virus-infected cells prepared in the presence or absence of tunicamycin, which inhibits glycosylation, demonstrate that addition of the new oligosaccharide side chain is required to prevent reaction with the monoclonal antibody. Similar experiments with the virus of the 1969 Hong Kong influenza epidemic, A/England/878/69, which also contains a hemagglutinin glycosylated at residue 63, support this conclusion and provide evidence for the epidemiological significance of carbohydrate-mediated modifications of hemagglutinin antigenicity.

We have determined the structure of the HA of an avian influenza virus, A/duck/Ukraine/63, a member of the same antigenic subtype, H3, as the virus that caused the 1968 Hong Kong influenza pandemic, and a possible progenitor of the pandemic virus. We find that structurally significant differences between the avian and the human HAs are restricted to the receptor-binding site particularly the substitutions Q226L and G228S that cause the site to open and residues within it to rearrange, including the conserved residues Y98, W153, and H183. We have also analyzed complexes formed by the HA with sialopentasaccharides in which the terminal sialic acid is in either alpha2,3- or alpha2,6-linkage to galactose. Comparing the structures of complexes in which an alpha2,3-linked receptor analog is bound to the H3 avian HA or to an H5 avian HA leads to the suggestion that all avian influenza HAs bind to their preferred alpha2,3-linked receptors similarly, with the analog in a trans conformation about the glycosidic linkage. We find that alpha2,6-linked analogs are bound by both human and avian HAs in a cis conformation, and that the incompatibility of an alpha2,6-linked receptor with the alpha2,3-linkage-specific H3 avian HA-binding site is partially resolved by a small change in the position and orientation of the sialic acid. We discuss our results in relation to the mechanism of transfer of influenza viruses between species.

BACKGROUND: Many RNA viruses do not have a single, representative genome but instead form a set of related variants that has been called a quasispecies. The sequence variability of such viruses presents a significant bioinformatics challenge. In order for the sequence information to be understood, the complete mutational spectrum needs to be distilled to a biologically relevant and analyzable representation. RESULTS: Here, we develop a "selection mapping" algorithm?QUASI?that identifies the positively selected variants of viral proteins. The key to the selection mapping algorithm is the identification of particular replacement mutations that are overabundant relative to silent mutations at each codon (e.g., threonine at hemagglutinin position 262). Selection mapping identifies such replacement mutations as positively selected. Conversely, selection mapping recognizes negatively selected variants as mutational "noise" (e.g., serine at hemagglutinin position 262). CONCLUSION: Selection mapping is a fundamental improvement over earlier methods (e.g., dN/dS) that identify positive selection at codons but do not identify which amino acids at these codons confer selective advantage. Using QUASI?s selection maps, we characterize the selected mutational landscapes of influenza A H3 hemagglutinin, HIV-1 reverse transcriptase, and HIV-1 gp120.

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.

Over the last four decades, H3N2 subtype influenza A viruses have gradually acquired additional potential sites for glycosylation within the globular head of the hemagglutinin (HA) protein. Here, we have examined the biological effect of additional glycosylation on the virulence of H3N2 influenza viruses. We created otherwise isogenic reassortant viruses by site-directed mutagenesis that contain additional potential sites for glycosylation and examined the effect on virulence in na�ve BALB/c, C57BL/6, and surfactant protein D (SP-D)-deficient mice. The introduction of additional sites was consistent with the sequence of acquisition in the globular head over the past 40 years, beginning with two sites in 1968 to the seven sites found in contemporary influenza viruses circulating in 2000. Decreased morbidity and mortality, as well as lower viral lung titers, were seen in mice as the level of potential glycosylation of the viruses increased. This correlated with decreased evidence of virus-mediated lung damage and increased in vitro inhibition of hemagglutination by SP-D. SP-D-deficient animals displayed an inverse pattern of disease, such that more highly glycosylated viruses elicited disease equivalent to or exceeding that of the wild type. We conclude from these data that increased glycosylation of influenza viruses results in decreased virulence, which is at least partly mediated by SP-D-induced clearance from the lung. The continued exploration of interactions between highly glycosylated viruses and surfactant proteins may lead to an improved understanding of the biology within the lung and strategies for viral control.

This study describes a method for increasing immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody for effective targeting vaccines to antigen presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on viral envelope hemagglutinin (HA) to carry the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Vaccinating influenza virus expressing alpha-gal epitopes is opsonized in situ by anti-Gal IgG. The Fc portion of opsonizing anti-Gal interacts with Fcgamma receptors on APC and induces effective uptake of the vaccinating virus by APC. APC internalizing the opsonized virus transport it to draining lymph nodes for stimulation of influenza virus specific T cells, thereby eliciting a protective immune response. Efficacy of such flu vaccine is demonstrated in alpha1,3galactosyltransferase (alpha1,3GT) knockout mice which produce anti-Gal and using the influenza virus strain A/Puerto Rico/8/34- H1N1 (PR8). alpha-Gal epitope synthesis on carbohydrate chains of PR8 virus (PR8alphagal) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme synthesizing alpha-gal epitopes in cells of non-primate mammals. Mice immunized with PR8alphagal displayed much higher numbers of PR8 specific CD8+ and CD4+ T cells (intracellular cytokine staining and ELISPOT) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-gal epitopes. Mice immunized with PR8alphagal also displayed a much higher protection than PR8 immunized mice following challenge with lethal dose of live PR8 virus. We suggest that a similar method for increased immunogenicity may be applicable to avian flu vaccines.