The Rescue Of Swine Influenza Virus And "swine-fowl" Influenza Reassortment Virus And The Reseach Of Biologic Property

Swine influenza(SI) is caused by swine influenza virus(SIV),and it can induce an acute zymotic disease of respiratory tract.SIV can not only infects pig,but although fowl and other mammal such as the human being and horse.And pig can be infected by avian influenza virus(AIV) and Human influenza virus,it's the mixing mill for recombination and reassortment of influenza virus genes and the couveuse for the forming of new popular influenza virus,as the important intermediary host for the interspecies transmission of influenza virus between fowl and mammal.The feature of SIV and pig determines their irreplaceable place in the interspecies transmission of influenza virus, and SIV is important for the research of spread and interspecies transmission of influenza virus,so is in public health.In this study,a strain of SIV and two strain of "swine-fowl" influenza reassortment virus were rescued using eight-plasmid rescue system of influenza virus.And the virulence between parent virus and reassortment virus was compared, which supplies information for researching and evaluating the risk of reassortment virus of "swine-fowl" and the probability of interspecies transmission of influenza virus.1.The rescue of H1N1 subtype swine influenza virusThe swine influenza virus(SIV) strain A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04) was rescued successfully by eight-plasmid rescue system.The cDNAs of SIV 8 gene segments were synthesized by RT-PCR and cloned into the RNA polymeraseâ… /â…¡bidirection expression vector pHW2000 independently,resulting in 8 recombinant plasmids.The 8 recombinant plasmids were cotransfected into COS-1 cell,30 hours later TPCK-trypsin was added to 0.5μg/mL.The COS-1 cell and supematant were harvested 48～72 hours after cotransfection and were inoculated into the allantoic cavity of 9-day-old specific-pathgen free(SPF) chicken eggs.The allantoic fluid of dead eggs was harvested and passaged 3 generations in SPF chicken eggs to get infective virus.The successful rescue of A/S/T J/04 SIV was identified by HA,HI assay,sequence analysis and electron microscope observation.The successful rescue of SIV built a platform for the research of the relationship between genome structure and function of SIV,the mechanisms of trans-species transmission of influenza virus and for the generation of new SIV as vaccine. 2.The rescue of "swine-fowl" influenza reassortment virus and the research of biologic propertyReplace the HA gene and both HA and NA genes of SIV A/S/TJ/04 with the corresponding genes of AIV A/chicken/Hubei/327/2004(H5N1)(A/C/HB/04) respectively by eight-plasmid rescue system of influenza virus,resulting in "7+1" type reassortment virus T+H(HA) and "6+2" type reassortment virus T+H(HA/NA).The EID₅₀ of reassortment virus and their parent virus were determined.With BALB/c mice as animal experiment model,mice were challenged with 10⁶EID₅₀ of four kinds of virus.The survival rate and the change of weight of mice were monitored.The heart,lung,spleen, intestinum rectum and brain were obtained on the third,sixth,twelfth day after being challenged,and in which the amount of virus was detected by antigen-catch ELISA test method.The difference of pathogenecity between parent virus and reassortment virus was compared by comparing the data of all challenge group.The result displays that the pathogenicity to mice of reassortment virus are between two parent viruses,more powerful than SIV A/S/TJ/04 and weaker than AIV A/C/HB/04,and the pathogenicity of reassortment virus T+H(HA/NA) is more powerful than T+H(HA) virus.The result indicates,the SIV are stronger after obtaining the HA or HA and NA genes of AIV A/C/HB/04 in pathogenicity and tissue tropism,which have some risk in interspecies transmission and viral prevalence,but there is also some limitation simultaneously.