Rescue Of H3N2 Subtype Swine Influenza Virus And Recombination Of Different Subtype Influenza Virus

Swine influenza is an acute respiratory disease caused by swine influenza virus(SIV). Once occurs, it may cause great economic losses to swine industry, especially, co-infecting with other respiratory diseases, such as PRRS(Porcine reproductive respiratory syndrome). Pigs are susceptible to avian, equine, human influenza viruses and have been proposed to be intermediate hosts or"mixing vessels"for the generation of pandemic influenza viruses. SIV could infect people without any ressortment. Swine influenza surveillance is of great significance for veterinary and public health.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 RT-PCR and cloned into bidirection expression vector pHW2000 seperately. We cotransfected 8 recombinant plasmids into 293T and MDCK cells and got the rescued virus rgH3N2 that all the eight genes came from parental virus. We can see the typical structures of rgH3N2, with morphological characteristics of influenza virus particles. Particles, a diameter of about 60 ~ 120 nm, were round, oval, some little irregular shape, and we could see the virus envelope and spike clearly by electron microscope observation. The rescued virus rgH3N2, and the wild type virus H3N2 shared similar biological properties, such as in titers of 50% embryo infective, 50% tissue culture infective dose and stability tests. The titer of rescued viruses measured by hemagglutination assay was 24. After several passages in embryonated eggs, the maximum virus titre measured by hemagglutination assay was 28. Titer of rgH3N2 in MDCK cell culture were measured by hemagglutination assay and the maximum virus titre of 26 hemagglutination unit was obtained after infecting MDCK cells for 60 h. The rescued virus being equivalent to that of parental virus could not be isolated in any organ after challengeWe replaced HA, NA of rgH3N2 by HA, NA gene from Human influenza virus A/PR/8/34(H1N1), Avian influenza virus A/Duck/Nanchang/4-165/2000(H4N6), Equine influenza virus A/Equine/ Fuyun/2008/(H3N8). With various combinations of genes from different subtype virus , we successfully generated reassortant viruses rgH1N1, rgH4N6 and rgH3N8. The titer of rescued viruses measured by hemagglutination assay were 211, 24, 27 after one passages in embryonated eggs. rgH1N1, rgH4N6 and rgH3N8 were stable after several passages in embryonated eggs, and the maximum virus titre measured by hemagglutination assay were 211, 29, 210. Meanwhile, rgH1N1, rgH4N6 and rgH3N8 were high-yield in cells, Titer of rgH1N1 and rgH4N6 in MDCK cells culture were measured by hemagglutination assay and the maximum virus titre of 210 and 29 hemagglutination unit were obtained after infecting MDCK cells for 48 h. Titer of rgH3N8 in MDCK cell culture was measured by hemagglutination assay and the maximum virus titre of 29 hemagglutination unit was obtained after infecting MDCK cells for 54 h. rgH1N1, rgH4N6 and rgH3N8 were both high-yield in eggs and MDCK cells and could be used to developing novel vaccines. After challenge rgH3N8 could be only isolated in lung and the maximum virus titre was 211.The successful rescue of reassortment viruses establishes the foundation for the molecular mechanism research on how the swine influenza virus break through the intermediate barriers and the function of HA, NA during transmitting among species, Also it is valuable for developing novel vaccines of influenza in future.