H9N2Subtype Avian Influenza Virus Cells In The Development Of The Inactivated Vaccine And Their Immune Effect Valuation

Avian influenza(AI)is an infectious disease syndrome caused by type A influenza virus, whichincluding highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI).H9N2subtype of avian influenza is low-pathogenicity avian influenza. In China’s mainland, H9N2avian influenza virus was firstly isolated from chickens in Guangdong province in1994, and thenH9N2AIV circulated widely in the domestic poultry and had caused great economic losses topoultry industry in our country.Vaccination is the most effective means for preventing the occurrence and spread of avianinfluenza. Restricted by the economic and technological factors, avian influenza cellular vaccinehas not been in market. Therefore,this research mainly used veterinary research institute, Chineseacademy of agricultural sciences’ Shanghai avian influenza epidemiological lab MDCK HA-2cellculture Contains H9subtype avian influenza virus (AIV) of the HA and NA genesSH441HANAPR8(NS mutant) recombinant virus. The culture environment is37℃, the volumefraction of5%CO2incubator. We first determine the best dose of virus infected cells, and then usedirect inoculation method and virus adsorption to inoculate virus, after inoculating virus wecompare the two methods of virus HA titer. Then we use2%NCS DMEM cell culture medium andserum-free medium to raise the cells of inoculating virus. Under the same conditions, we comparedthe two different maintaining fluid produced by virus HA titer. Finally we use the MDCK andMDCK HA-2two cells to inoculate virus under the same conditions, we compared their virus HAtiter after inoculating virus. After the virus best breeding conditions are determined, we preparedthe high titer of virus strains, and to expand culturing. To be inactivated and emulsification for thepreparation of inactivated vaccines and use the inactivated vaccine to vaccinate3weeks SPFchickens, and inspection of inactivated vaccine immune effect and its inoculation of challengeprotection rate of vaccine.According to the results, we use30000TCID50virus dose to vaccinate MDCK HA-2cells withdirect inoculation method and use2%NCS DMEM culture instead of serum-free culture medium.Without adding TPCK pancreatic enzymes, Infection after48hours, we harvest the virus andmeasured virus HA titer is210. It was the best to mix SH441HANAPR8(NS mutant) withformaldehyde to a final concentration of0.1%and inactivated at24h.The inactivated SH441HANAPR8(NS mutant) viruses were mixted with adjuvant Montanide ISA70VG, andemulsified to inactivated vaccine. By chest muscles pathway,3-week-age specific pathogen free(SPF) chickens were immunized with H9avian influenza virus inactivated vaccineSH441HANAPR8(NS mutant) in different doses of0.05,0.1,0.2and0.3mL per chicken. After21days post-immunization, these chickens were infected with avian influenza virusA/Chicken/Shanghai/441/2009(H9N2). The results demonstrated that minimum immune dose ofthe vaccine was0.2mL per chicken. In addition，when HI titer was equal or greater than25,challenge protection rate of vaccine was100%for chicken.All in all, the institute of avian influenza epidemiology laboratory preparation of H9N2AIVcells inactivated vaccine can provide a better immune protection in chickens, has great businessdevelopment potential.