Development Of High-Yielding H9N2 Subtype Avian Influenza Virus Vaccine Strain And Construction Of Avian Influenza Virus Sensitive Cell Line

The H9N2 subtype avian influenza virus(AIV)has a wide range of prevalence and rapid spread.Under the dual effects of the uncorrected function of the RNA-dependent RNA polymerase carried by the virus itself and immune pressure,frequent virus mutations lead to frequent outbreaks,which seriously threaten the development of the poultry industry and public health safety.Therefore,the development of high-yield and efficient avian influenza vaccine is an important means to control the H9N2 influenza virus epidemic.In this study,based on the analysis of the growth characteristics of the H9N2 recombinant virus,this study aims to select a candidate vaccine strain with strong replication ability and good immunogenicity.At the same time,a sensitive cell line of avian influenza virus will be constructed.The development of this research provides technical reserves for the development of high-yield and high-efficiency H9N2 subtype avian influenza vaccine to effectively prevent and control the occurrence of this subtype of influenza epidemic.1.Screening of high-yielding H9N2 subtype vaccine strains and evaluation of immune effectA/chicken/Fujian/11/2020(Fj11 strain for short)was a H9N2 subtype avian influenza virus new variant strain isolated in 2020 in our laboratory.In this study,the 6 internal genes of the A/chicken/Henan/HP/1998,A/chicken/Anhui/LH99/2017,H9-HY backbone#1,H9-HY backbone #2,and A/Puerto Rico/8/1934 strain were used as the virus backbone.Infectious clones were constructed with the HA and NA genes of the Fj11 strain.And 5 recombinant viruses rFj11-HP,rFj11-LH99,rFj11-HY #1,rFj11-HY #2,rFj11-PR8 were rescued using reverse genetic technology.After mixing these 5 recombinant strains in equal proportions,competitive reassortment of viruses was used to screen out more competitive superior strains,and the screened superior strains were purified and genomewide sequencing.The results showed that the HA and NA genes were consistent with wildtype Fj11.The gene of NP,PA,PB1,PB2,and NS were from H9-HY backbone #2,and the M gene was from H9-HY backbone #1.The results showed that the donor gene combination had a clear competitive advantage in the competitive reassortment of recombinant viruses,and the selected dominant strain was named rFj11-New HY Backbone(referred to as rFj11-New HY).The five strains rescued by reverse genetics and the screened rFj11-New HY were tested for the half-infectivity of chicken embryos and the replication kinetics analysis.The results showed that the proliferation effect of rFj11-HY#2,rFj11-HY #1,rFj11-New HY on chicken embryos was better than that of rFj11-LH99,rFj11-PR8,and rFj11-HP.The rescued strains and the screened strains were further made into inactivated vaccines to immunize SPF chickens.And the antibody level in the serum was evaluated by the hemagglutination inhibition test.It was found that the inactivated vaccine prepared by rFj11-New HY produced a higher,stable and lasting antibody level compared to other strains.Therefore,it was finally determined that the rFj11-New HY strain with strong proliferation characteristics and good immunogenicity on chicken embryos was an ideal H9N2 subtype AIV vaccine candidate strain.2.Construction of Avian Influenza Virus sensitive cell lineThe products encoded by α-2,3 sialyltransferase I(ST3GALI)and transmembrane serine protease 2(TMPRSS2)genes can increase the sensitivity of avian influenza virus to host cells.In this study,we used the PLOX-IRES-neo vector to construct a stable MDCK cell line that overexpresses the chicken ST3 GALI gene.Through q PCR identification,the ST3 GALI m RNA expression level of MDCK-D8 cells was higher than that of other monoclonal cell lines screened.The retrovirus vector PMX-TMPRSS2-Blast was further used to package the retrovirus carrying the TMPRSS2 gene to infect MDCK-D8 cells.Identification by western blot,the TMPRSS2 protein was expressed in MDCK cells.The genetic stability of MDCK cells overexpressing foreign genes was further tested.And the results showed that the cell line could still overexpress ST3 GALI and TMPRSS2 genes stably after the 13 th generation.The H9N2 recombinant virus in the first part of the work infected MDCK cells,MDCK-D8 cells,MDCK-C6 cells and MDCK-ANP32 A cells with the same dose.The results showed that the titer of the avian influenza virus proliferated on MDCK-C6 cells was higher than that of ordinary MDCK cells.High-level avian influenza virus receptor ST3 GALI and host cell protease TMPRSS2 more effectively promoted the replication ability of the tested H9N2 strain on MDCK-C6 cells.In summary,the rFj11-New HY recombinant virus screened in this study was a vaccine candidate strain with good proliferation characteristics on chicken embryos and strong antigenicity.At the same time,this experiment successfully constructed avian influenza virus sensitive cell line MDCK-C6 that stably overexpressed the ST3 GALI and TMPRSS2 double genes for efficient proliferation of avian influenza virus.The development of this research has laid the foundation for the development of high-yield and high-efficiency avian influenza vaccine strains and a cell proliferation platform that provides vaccine strains,thereby providing technical reserves for the effective prevention and control of avian influenza epidemics.