Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions.

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes.

Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants.

Reassortants between a low-pathogenic avian influenza virus strain A/Duck/Primorie/2621/2001 (H5N2) and a high-yield human influenza virus strain A/Puerto Rico/8/34 (H1N1) were generated, genotyped and analyzed with respect to their yield in embryonated chicken eggs, pathogenicity for mice, and immunogenicity. A reassortant having HA and NA genes from A/Duck/Primorie/2621/2001 virus and 6 genes from A/Puerto Rico/8/34 virus (6:2 reassortant) replicated efficiently in embryonated chicken eggs, the yields being intermediate between the yields of the avian parent virus and those of the A/Puerto Rico/8/34 parent strain. The reassortant having the HA gene from A/Duck/Primorie/2621/2001 virus and 7 genes from A/Puerto Rico/8/34 virus (7:1 reassortant) produced low yields.

Postreassortment changes in a model system: HA-NA adjustment in an H3N2 avian-human reassortant influenza virus.

In our previous studies we described the postreassortment changes in the hemagglutinin (HA) of H2N1, H3N1, H4N1 and H13N1 influenza A virus reassortants with HAs derived from avian viruses and low-functional neuraminidase (NA) of a human parent virus A/USSR/90/77 (H1N1). The changes involved amino acid substitutions that increased the negative local charge in the vicinity of the receptor-binding pocket and decreased the affinity of HA to sialic acid receptors. In the present report we describe the studies performed with H3N2 reassortant viruses having HA of A/Duck/Ukraine/1/63 (H3N8) virus and NA of A/Aichi/2/68 (H3N2) virus.

Antigenically relevant amino acid positions as revealed by reactions of monoclonal antibodies with the nucleoproteins of closely related influenza A virus strains.

The nucleoproteins (NP) of a group of influenza A virus strains were analyzed with the use of a panel of anti-NP monoclonal antibodies (Mabs) in the radioimmunosorbtion reaction with subsequent polyacrylamide gel electrophoresis of the immune complexes, and in the immunoblotting. The group included 2 pairs of closely related viruses having minimal amino acid differences in NP sequence. The analysis of the results of the immune reactions in comparison with the known amino acid sequences of NP allowed us to suggest that the Mab 150/4 recognizes the epitope containing amino acid residues in positions 196 and/or 290, whereas the amino acid residue in position 353 participates in the formation of the antigenic epitope reacting with the Mab 7/3.

Influenza H5 virus escape mutants: immune protection and antibody production in mice.

Avian H5N1 influenza A viruses are considered to be of high pandemic potential as they are able to cross the avian-human species barrier and cause disease in humans. In the present study we assessed the impact of amino acid substitutions in the hemagglutinin (HA) of antigenic escape mutants of influenza A/Mallard/Pennsylvania/10218/84 (H5N2) (Mld/PA/84-MA) virus on the level of neutralizing antibodies and the ability to protect mice against challenge with the wild type H5 influenza virus. beta-Propiolactone-inactivated vaccines prepared from eight different H5 escape mutants could be separated into two groups based on levels of protection.

Prevention and treatment of bronchopneumonia in mice caused by mouse-adapted variant of avian H5N2 influenza A virus using monoclonal antibody against conserved epitope in the HA stem region.

The effects of monoclonal antibody (MAb) C179 recognizing a conformational epitope in the middle of the hemagglutinine (HA) stem region were examined in a mouse model in the experiments of prevention and treatment of lethal bronchopneumonia caused by influenza A virus of H5 subtype. To model the lethal infection, avian nonpathogenic strain A/mallard duck/Pennsylvania/10218/84 (H5N2) was adapted to mice. This resulted in highly pathogenic pneumovirulent mouse-adapted (MA) variant, which was characterized.