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The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses. [ABSTRACT FROM AUTHOR]

To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity. [ABSTRACT FROM AUTHOR]

The change in the phenotypic properties resulting from amino acid substitutions in the hemagglutinin (HA) molecule is an important link in the evolutionary process of influenza viruses. It is believed to be one of the mechanisms of the emergence of highly pathogenic strains of influenza A viruses, including subtype H5N1. Using the site-directed mutagenesis, we introduced mutations in the HA gene of the H5N1 subtype of influenza A virus. The obtained virus variants were analyzed and compared using the following parameters: optimal pH of conformational transition (according to the results of the hemolysis test), specificity of receptor binding (using a set of synthetic analogues of cell surface sialooligosaccharides), thermoresistance (heat-dependent reduction of hemagglutinin activity), virulence in mice, and the kinetics of replication in chicken embryos, and reproductive activity at different temperatures (RCT-based). N186I and N186T mutations in the HA protein increased the virulence of the original virus in mice. These mutations accelerated virus replication in the early stages of infection in chicken embryos and increased the level of replication at late stages. In addition, compared to the original virus, the mutant variants replicated more efficiently at lower temperatures. The obtained data clearly prove the effect of amino acid substitutions at the 186 position of HA on phenotypic properties of the H5N1 subtype of influenza A. [ABSTRACT FROM AUTHOR]