Rescue of H3N2 subtype swine influenza virus and substitution of Hemagglutinin, Neuraminidase

Abstract:

Abstract: [Objective] To study the mechanisms of trans-species transmission of influenza virus for developing novel vaccine of influenza in future. [Methods] We rescued H3N2 subtype swine influenza virus strain A/Swine/Henan/S4/01 successfully by a plasmid-base reverse genetics. Eight gene segments were synthesized by reverse transcriptase-PCR and cloned into bidirection expression vector pHW2000. We cotransfected 8 recombinant plasmids into 293T and MDCK cells and got the rescued virus rgH3N2. Then we replaced Hemagglutinin, Neuraminidase of rgH3N2 by Hemagglutinin, Neuraminidase gene from Human influenza virus, Avian influenza virus, Equine influenza virus. [Results] The rescued virus rgH3N2 and the wild type virus shared similar biological properties such as in titers of 50% embryo infective, 50% tissue culture infective dose and stability tests. The rescued virus titer in MDCK cell culture was measured by hemagglutination assay and the maximum virus titre of 1∶64 hemagglutination unit was obtained after infection of MDCK cell for 60 h, The hemagglutination titre was 1:256 after several passages in embryonated eggs. With various combinations of HA, NA genes, we successfully generated high-yield reassortant viruses rgH1N1, rgH4N6 and rgH3N8 in embryonated eggs and MDCK cells. [Conclusion] The successful rescue of reassortment viruses establish the foundation for the molecular mechanism research on how the swine influenza virus breakthrough the intermediate barriers and the function of HA , NA during transmitting among species, Also it is feasible to be used for developing novel vaccine of H3N2 subtype Swine influenza in future.