Screening And Validation Of Antigenic Drift Sites Of H9N2 Subtype Avian Influenza Virus From Chickens In China From 2014 To 2020

Avian influenza（AI）is a common zoonotic disease.When infected with avian influenza virus（AIV）,it can cause lesions in various organs of the body,especially the respiratory tract,digestive tract and reproductive system.The infection of low pathogenic avian influenza virus generally does not show obvious symptoms,but it is easy to cause disease in the host due to co-infection with other pathogens,and it is easy to spread across hosts,so it poses a great threat to public health and health security.H9N2 subtype avian influenza virus（H9N2 AIV）has become the main prevalent subtype of AIV in China.Due to antigenic drift and other reasons,existing vaccines cannot play an immune protection role,resulting in immune escape.In order to clarify the key sites of antigenic drift of H9N2AIV in the past five years,this study firstly carried out a genetic evolution analysis of the HA gene of Chinese chicken-derived H9N2 AIV isolates from 2014 to 2020,and preselected the sites of antigenic drift;then,by constructing mutant strains,two-way cross-coagulation inhibition experiments and micro-neutralization experiments were performed to identify the preselected sites.The research results have important reference significance for the evolution of H9N2 AIV and the preparation of new vaccines.The main content of this project was displayed as the follows:1.Screening of antigen drift sites of H9N2 AIV HA gene in Chicken source from2014-2020 in ChinaBy collecting the sequences of HA genes of H9N2 AIV isolates from chicken sources in China from 2014 to 2020,constructing the MCC evolutionary tree and analyzing the amino acid sites and positive selection sites of each branch.The results showed that the sequences all belonged to the clade 15 and evolved into four subgroups（Group 1-4）,and the mean substitution rates was at 1.04×10^-2subs/site/year(95%HPD: 9.30×10^-3～1.15×10^-2).Specific amino acid mutations were present in each subgroup and were mainly concentrated in amino acids in positions 87 to 201.In addition,through the statistics of amino acid mutation rates of isolated strains of HA protein in the past five years,the mutation rate of amino acids in 145-201 sites was high.The AIV antigenic sites are mainly located on the head surface of the HA1 subunit,combined with the high-frequency mutation sites,a total of 6 antigen drift sites（164,168,171,198,200 and201）were pre-selected in this study for later verification,of which selective pressure analysis showed that 168 was a strong positive selection site.2.Analysis of the construction and growth characteristics of mutant strainsIn this study,the 2014 isolate A/Chicken/China/Sichuan CQY/2014（H9N2）（CQY）and the 2020 isolate A/Chicken/China/Sichuan GH/2020（H9N2）（GH）were selected for experiments.In order to verify the effect of HA gene and pre-selected fragments on antigen drift,the HA gene and 164-201 amino acid fragment of GH strain were replaced with the CQY strain as the skeleton,the r CQY-GHHA and r CQY-GHHA（CQY164-201） recombinant strains were rescued.In order to further clarify the key sites causing antigen drift,seven single-point mutant strains（r CQY-R164Q,r CQY-D168N,r CQY-I171T, r CQY-T198A,r CQY-R200T,r CQY-D201G,r CQY-D201G,r CQY-D201A）were rescued.The growth characteristics test results showed that the TCID₅₀and EID₅₀in the MDCK cells and the EID₅₀in the SPF chicken embryos of the 9 mutant strains were lower than the parental strain-CQY,but there was no significant difference.The growth curves of the parental strains and the recombinant strains in MDCK and chicken embryos were basically the same.The results showed that recombinant fragments and site mutations had no significant effect on the proliferation of viruses in MDCK and chicken embryos.3.Effect of mutation points on H9N2 AIV antigenicityVaccines play a very important role in the prevention and treatment of H9N2 AIV in China,in order to study whether the pre-selected sites will cause antigen drift of H9N2 AIV,this study uses cross-hemagglutination inhibition experiments,trace neutralization experiments and animal immunoprotective tests to verify.The results showed that the CQY isolate in 2014 and the GH isolate in 2020 had significant antigenicity differences,and the164-201 amino acid fragments of HA protein had a greater impact on antigenicity.Among them,the substitution of amino acid loci of D168N and D201G can significantly change the antigenicity of H9N2 AIV,and D201G can cause antigenic drift.In summary,from 2014 to 2020,the HA gene in the H9N2 AIV chicken source isolate in China is still mainly dominated by clade 15,and is mainly divided into 4 subgroups,each of which has its own specific amino acid loci.Among them,the D168N and D201G mutations located in the head of the HA protein can significantly change the antigenicity of H9N2 AIV,and D201G can cause antigenic drift.The results of the study provide an important reference for the evolutionary prediction of H9N2 AIV and the development of new vaccines.