Stepaniuk S. Molecular genetic diagnostics of influenza virus А(H5N1) and А(H1N1) pandemic strains.

The dissertation is devoted to the study of biological and molecular genetic features of influenza virus pandemic strains: Ukrainian isolate of avian influenza A (H5N1) isolated in the Crimea and California strains of the influenza A(H1N1)pdm09 virus. The purpose of the study is the development of high-sensitivity test systems based on the PCR method in real time for the diagnosis and genotyping of these viruses. It was established that the Crimean isolate of the influenza A virus (H5N1) – LA-NK-21205, which caused large-scale epizootics in the Crimea in 2005, belongs to the group of highly pathogenic HPA / H5N1 influenza viruses. It contains multiple injections of arginine and lysine in a site for cutting hemagglutinin H5 (RRRKKR at position 1050-1065), which has a 100% homology of this gene site to modern high-pathogenic strains. Molecular genetic analysis of sequenced genes of the Crimean isolate of the influenza A /H5N1 – LA-NK-21205 virus and the comparison with the nucleotide sequences of modern HPA / H5N1 strains has identified the unique areas of 3 genes (M-gene, H5 and N1) with a homology of 98–100%. The comparative molecular genetic analysis of California strains of pandemic influenza A(H1N1)pdm09 virus revealed an homology of 95% of the 3 genes (M gene, NA1 and NA1) with the sequences of strain A/California/07/09 strains (H1N1), which has been reported by WHO as a pandemic strain. The high-conservative areas detected by us in the genomes of Californian strains of the virus were unique and have a low homology with the seasonal influenza virus strains (less than 50%). The selected areas of the HPA/H5N1 and A(H1N1)pdm09 influenza viruses were used to develop differential primers and TaqMan probes and a two-stage multiplex Real-Time RT-PCR assay. On the basis of the conducted researches, test systems for diagnosing pandemic strains of the НРА/H5N1 («DIA-Real Avian Influenza») and influenza А(H1N1)pdm09 («DIA Influenza H1N1») have been developed. The test systems have successfully passed governmental tests in the Ministry of Agrarian and Industrial Complex (registered certificate No. 9533/2010 dated July 1, 2006) and in the Ministry of Health of Ukraine (registered certificate No. 9533/2010 dated July 1, 2011).