The Establishment Of Reverse Genetics System For H1Subtype Swine Influenza Virus And Basic Research Of Inactivated Reassortant H1N1Vaccine

Swine influenza is an acute respiratory disease caused by swine influenza virus(SIV)，which leadsto a rapid onset of high fever, dullness, loss of appetite, labored abdominal breathing and coughing andthus harms the swine industry all over the world. As a long time，the research of SIV has not been thinkhighly of，for SIV infected alone could not cause pandemic or the death of swinery.However,SIV hasplayed a significant role in pubic health,since pigs are considered as “mixing vessels” for thegeneration of novel pandemic influenza viruses through reassortment,so the research of swine influenzabegin to be considered highly of.In2007，a virus strain was isolated from tissue samples collected in pig with respiratory tractproblems in a pig farm of south China．After propagation in SPF embryonated chicken’s eggs andpudfication，the virus was determined to be a H1N2subtype SIV by HI and NI，designatedA/swine/Shanghai/1/07（H1N2）(abbreviated as S12). The eight segments of S12were amplified byRT-PCR，and then all the genes were respectively cloned into pMD18-T vector and sequenced．It wasfound that S12was a novel ressortant H1N2influenza virus containing genes from the classical swine(HA, M, NP and NS), human (NA and PB1) and avian (PB2and PA) lineages. Several pandemic2009H1N1influenza（pdm2009） strains were then chosen as reference strain to perform genomic sequencealignment with S12. Results demonstrated that six gene segments (HA, PB1, PB2, PA, NP and NS) ofS12have high homology with the those of pdm2009，while another two segments（NA and M）are quitedifferent from the corresponding gene segments of pdm2009.Based on the result of sequence similarity and nucleotide evolution analysis, the eight segments ofS12were respectively cloned into the bidirection transcriptional vector PBD, eight plasmids whichcould replicate and express in mammalian cells were constructed. After the eight plasmids wereco-transfected into293T cells, the cell suspension was collected and injected into SPF embryonatedeggs, the virions (rS12) that could agglutinate erythrocytes were rescued, and all the genes were derivedfrom the eight plasmids by necleotide sequencing. Then the two gene segments (NA and M) werereplaced by the corresponding genes of pdm2009, a reassortant virus (r09SZ) was rescued using thesame method, according the result of nucleotide evolution analysis, the source of the eight segement ofr09SZ are exactly the same as that of pdm2009. Comparing the pathogenicity to mice of the threeviruses (S12, rS12, r09SZ), we found that there is no remarkable difference between rS12and r09SZ，but the pathogenesis of them was both weaker than the parental strain S12. So, the two replacedsegments (NA and M) are not the key factors associated with the pathogenicity of pdm09in mice.Although the gene segments of the rescued reassortant virus r09SZ are originally from Swineinfluenza S12and pdm09, the virus could passage stably in embryonated egg. Immunization for micewith inactivated r09SZ vaccine could provoke complete protection against challenge with H1N1influenza viruses, and also provoked high titer of r09SZ-specific IgG antibodies, strain-specifichemagglutinin inhibition (HI) antibodies and neutralization antibodies. According to the results, reversegenetics system could be a feasible means to construct vaccine strain for influenza viruses.