The aim of study was to carry out comparative investigation of biological properties of site-specific mutants of Influenza A virus and variant of live cold-adapted (CA) influenza reassortant vaccine.
Materials and methods. The genetic stability of site-specific mutants (SSM) of the A/WSN/33 (H1N1) strain with ts (temperature sensitive)-mutations in polymerase genes was studied using a stress-test in MadinDarby Canine Kidney (MDCK) culture. A comparative study of immunogenicity of U2 and M26 mutants with the high genetic stability and the CA-reassortant with similar surface proteins was carried out. The increase in the antibody titer was investigated using enzyme-linked immunosorbent assay and the reaction of delayed hemagglutination. Ability of the studied viruses to induce type 1 interferon in A549 cells was determined using real-time polymerase chain reaction (real-time PCR).
Results. It was shown that U2 and M26 mutants, which have 3 ts-mutations or more in polymerase genes have high genetic stability. It was found that U2 and M26 mutants induced a higher antibody titers than the CA reassortant in mice following the intranasal immunization. The ability of site-specific mutants and CA reassortant to induce type 1 interferon was also investigated. Mutants U2 and M26 increased the level of interferon to a greater extent than the CA-reassortant.
Conclusion. The data obtained indicate that SSM U2 and M26 with 3 ts-mutations or more in the genome have a significant level of genetic stability. Mutants U2 and M26 have a higher immunogenicity and a higher ability to induce interferon in comparison with the CA reassortant. These facts allow us to conclude that SSM of the influenza virus with a set of mutations in polymerase genes can be considered as promising candidates for live influenza vaccines.
Aim. Study the possibility of obtaining attenuated variants of influenza virus by including specially selected site-specific mutations into a conservative sequence of PA-gene (terminal segment of COOH-domain of the PA-gene) of a virulent strain. Materials and methods. А/ WSN/33 - a virulent strain of influenza virus was used in the study. I nclusion of site-specific mutations into PA-gene of the A/WSN/33 virulent strain was carried out using a two-step mutation PCR. Cloning was carried out using GoldenGate reaction. 8-plasmid transfection system based on pHW2000 vector was used. Transformation was carried out in rubidium competent bacterial cells of DH5a strain. Transfection was done using Lipofectamine LTX (Invitrogen) reagent in a 293T and MDCK cells’ co-culture. Results. Transfectants with F658A substitution in the COOH-domain of the PA-gene were shown to acquire ts-phenotype and sharply reduce the ability to reproduce in mice lungs. Introduction of F658A substitution into COOH-domain of the PA-gene in combination with introduction of ts-mutations from ca influenza virus strains into the genome of the virulent strain resulted in obtaining transfectants that have phenotypic characteristics typical for live influenza vaccine candidates. Conclusion. The ability to obtain attenuated variants of influenza viruses by introducing specially selected site-specific mutations into conservative sequence of the PA-gene is shown.
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