Immune Escape Mechanism Of Key Mutation Sites In HA Gene Of H9N2 Avian Influenza Virus Under Immune Stress

H9N2 subtype avian influenza virus（AIV）is a low pathogenic avian influenza virus and is one of the major subtypes currently prevalent in my country.The virus causes a decline in egg production in birds,immunosuppressive diseases,and often high mortality due to co-infection with other pathogens,causing huge economic losses to the poultry industry.Vaccination is the most effective way to prevent and treat H9N2 subtype avian influenza virus.However,the HA protein antigen of the virus mutates rapidly,and the virus can still escape under the pressure of vaccine immunity,resulting in the failure of vaccine immunity.At present,its immune escape mechanism is still unclear.In the previous study,the laboratory screened several immune escape sites located on the HA gene of the H9N2 subtype avian influenza virus by simulating the immune stress test.In order to explore the immune escape mechanism of these H9N2 subtype avian influenza viruses under immune pressure,this thesis mainly studies the following four aspects,（1）This study successfully rescued the H9N2 subtype avian influenza virus A/Chicken/Shanghai/1/2006（SH1）parental virus and its 8 single-point mutation strains on the HA gene by reverse genetic manipulation of eight plasmids:r SH1-HA_M159I,r SH1-HAD196G,r SH1-HAA198T,r SH1-HAA198V,r SH1-HAV213M,r SH1-HAD201N,r SH1-HAQ234L,r SH1-HA_Q234H and 4 strains of double point mutant strains on the HA gene:r SH1-HA_D196G-Q234L,r SH1-HAA198T-Q234L,r SH1-HAA198V-Q234L,r SH1-HAD201N-Q234L.（2）In order to investigate whether replication capacity is the cause of virus escape,we compared the replication capacity of the parental virus and immune escape strains on MDCK cells,and the results showed that in addition to HA_D201N and HA_D196G mutations at two sites can cause the virus to replicate on MDCK cells In addition to the significant decrease in the replication ability at individual time points,the general replication trend remained unchanged,and the replication ability of other point mutant strains on MDCK cells was not significantly different from that of the parental strain,indicating that the immune escape of these viruses was not related to the change of replication ability.On avian-derived LMH cells,compared with the parental virus,the mutation of HA_D201N,HA_D201N-Q234L and HA_A198T-Q234L resulted in a significant decrease in the in vitro replication ability of SH1.The replication ability of other mutants on LMH cells was similar to that of the parental virus.The strains showed no significant difference.（3）The structural map of HA protein was analyzed by PyMOL software,and it was determined that the target amino acid sites were located in the receptor binding region of the head of HA protein,and then the receptor binding characteristic test was carried out.Tends to bind to humanα-2,6 sialic acid receptors,and the rest of the HA protein point mutants have not changed the receptor binding type,and still prefer avianα-2,3 sialic acid receptors.（4）In order to to investigate whether antigenic variation is the cause of the escape of these viruses,the cross-hemagglutination inhibition test was performed by preparing multi-antibody sera of mutant strains,and the results showed that r SH1-HA_D196G,r SH1-HA_D201N,r SH1-HA_D196G-Q234L and r SH1-HA_D201N-Q234L mutations The R values between the strains and the parental strain r SH1 are all greater than or equal to 2,indicating that the four mutant strains and the parental strain r SH1 have great differences in antigenicity,that is,HA_D196Gand HA_D201N may be important for the H9N2 subtype avian influenza virus.The antigenic site causes the virus antigen to mutate significantly,allowing the virus to escape from immune pressure.In this study,the eight-plasmid reverse genetic technology was used to rescue the parental virus and mutant virus of SH1,and the differences between the parental virus and mutant virus were compared in terms of antigenicity,in vitro replication ability and receptor binding ability of the virus,and to explore the H9N2 AIV’s factors of immune evasion of influenza virus have laid a solid foundation for elucidating the immune evasion mechanism of the virus,and provided a theoretical basis for clarifying the law of antigenic variation of H9N2 subtype avian influenza virus and the further development of vaccines.