The genotype of laboratory reassortants produced by co-cultivation of virulent A/USSR/2/85 (H3N2) virus and multi-passaged attenuation donor A/Leningrad/9/46 virus (H1N1) was determined by two different methods: a method of competitive dot hybridization (CDH) and polyacrylamide gel electrophoresis (PAGE) with virion RNAs urea. The results of the two methods were shown to be completely identical which confirmed the potential of using CDH method for genetic typing of influenza A virus reassortant strains.

A new method for genetic typing of influenza viruses using molecular hybridization of DNA-RNA was developed which consisted in addition to the hybridization solution, apart from the radioactively labeled probe, of RNA of a virus with known gene homologous to the plasmid DNA used as the probe but belonging to a different serosubtype of influenza A virus, other than cloned kDNA, (within the range of H1N1, H2N2, and H3N2). This competitive RNA (RNAc) is added in considerable excess with regard to both molecular probe and to RNA immobilized on the filter. Therefore hybrids of molecular probe DNA with RNAc are rapidly formed from which RNAc may be replaced only by those bRNA immobilized on the filter whose homology to the probe is higher than that of RNAc.

As a result of two successive recombinations of influenza A viruses: A/Leningrad/0139/76 (H3N2) with A/PR/8/34 (H1N1) strain and the resulting reassortant A/Leningrad/0139/76, R (H3N2) with a field isolate A/Kiev/59/79 (H1N1), a virus. A/Kiev/59/79, R (H1N1) was obtained. It was established by electrophoresis in polyacrylamide gel and sequencing of kDNA fragments of its genome that the genes PB2, PBI and NP of this virus belonged to serosubtype H3N2, while genes PA, M, and NS to serosubtype H1N1 (like A/PR/8/34). The surface glycoproteins HA and NA belonged to serosubtype H1N1/like A/USSR/90/77.

As a result of two successive recombinations of influenza A viruses: A/Leningrad/0139/76 (H3N2) with A/PR/8/34 (H1N1) strain and the resulting reassortant A/Leningrad/0139/76, R (H3N2) with a field isolate A/Kiev/59/79 (H1N1), a virus. A/Kiev/59/79, R (H1N1) was obtained. It was established by electrophoresis in polyacrylamide gel and sequencing of kDNA fragments of its genome that the genes PB2, PB1 and NP of this virus belonged to serosubtype H3N2, while genes PA, M, and NS to serosubtype H1N1 (like A/PR/8/34). The surface glycoproteins HA and NA belonged to serosubtype H1N1/like A/USSR/90/77.

Simultaneous circulation of different subtypes of influenza A viruses provides conditions for reassortant strains formation. A comparative investigation of genome of 47 influenza A virus strains (H1N1, H2N2, and H3N2) was carried out by competitive dot hybridization technique and sequence analysis of some of cDNA-copies of the virus genes. All the genes of 43 strains encoding nonglycolysed proteins corresponded to the serum subtype of surface glycoproteins. The reassortant pattern of genome for some genes of core proteins was revealed in 4 viruses. All the dot hybridization data were completely confirmed by sequence analysis of the genes.

A molecular analysis was made of genomes of influenza A (H1N1) virus strains, the causative agents of an epidemic in Leningrad, 1986. The primary structure of hemagglutinin gene of two of these strains, A/Leningrad/624/86 and A/Leningrad/621/86, was established, as well as partial primary structure of PB1 gene of certain current strains of the A (H1N1) subtype. A hypothesis of a "shift" of PB1 gene in 1950-1957 is suggested.

The hemagglutinin gene primary structure of influenza virus A/Riga/9977/86 (H3N2) belonging to the "Coen/84" antigenic subgroup was determined by primer sequencing. A comparative analysis confirmed that the reversions of amino acids in the late stages of the H3 influenza virus subtype antigenic drift became more frequent and the antigenic variants remained in epidemic circulation longer. The possible role of some mutations is discussed.

Electrophoresis in polyacrylamide gel of the reference influenza A/Victoria/35/72 (H3N2) virus and its persisting variants (PV) showed that the PV isolated on the 158th day from the moment of persistence modelling (PV158) had mutation in the gene of hemagglutinin (HA). This mutation is manifested by incomplete HA synthesis at 40 degrees C and increase of mobility of the light HA subunit (HA2). Analysis of nucleotide sequence of the greater part of HA gene of PV158 virus revealed 5 nucleotide substitutions four of which were significant. Three substitutions were found in Hal: 219 (Ser----Phe), 220 (Arg----Gly), 226 (Leu----Gln) and one in HA2: 156 (Thr----Asn). The importance of these mutations in the determination of the PV phenotype is discussed.

A molecular analysis was made of genomes of influenza A (H1N1) virus strains, the causative agents of an epidemic in Leningrad, 1986. The primary structure of hemagglutinin gene of two of these strains, A/Leningrad/624/86 and A/Leningrad/621/86, was established, as well as partial primary structure of PB1 gene of certain current strains of the A (H1N1) subtype. A hypothesis of a "shift" of PB1 gene in 1950-1957 is suggested.

The reproduction activity in human embryo kidney cell culture (HEK) of epidemic influenza A viruses (H3N2 serosubtype) isolated in different years and with a certain level of virulence for man was studied. The cells were inoculated with the viruses at a multiplicity of infection of 0.001 EID50/cell, and the reproductive activity was judged by the levels of infectious and hemagglutinating activity during 96 hours of observation. There was a clear-cut correlation between the virus virulence level and the reproductive activity in HEK cell culture. Epidemic influenza A (H1N1) viruses of the A/Chile/1/83 variant were found to be heterogeneous by the marker of their reproductive activity in HEK cells. It is presumed that the observed differences in the infectious activity of the viruses of this group are also associated with different levels of their virulence for man.

The two variants of influenza A/Victoria/35/72 (H3N2) virus resistant simultaneously to remantadine, deitiforin, adapromine and amantadine were obtained while passaging the virus in presence of remantadine or deitiforin. Both variants differed from the parental strain in optimal pH for hemolysis, transcriptase activity and in amino acid sequence of M2 protein. Maximal hemolytic activity of the parental strain is registered at pH 5.2, for the variants cultured in the presence of remantadine or deitiforin at pH 5.5 and 5.8, respectively. In contrast to NH4OH, remantadine and deitiforin do not exert inhibition of virus-induced hemolysis. Transcriptase activity of resistant variants is about 50% higher as compared with parental strain (enzyme source--whole virus particles or RNP). The M2 protein of the remantadine variant has 2 amino acid substitutions: 31 (Ser----Asn) and 59 (Met----Leu); the deitiforin variant has 3 substitutions: 14 (Met----Leu), 30 (Ala----Val) and 59 (Met----Leu). The phenotypic resistance of the virus seems to be determined by the mutations in the hydrophobic protein region (30,31); the other substitutions (14,59) may modify conformational structure and functional activity of the viral proteins.