Genetic Evolution Analysis Of H7N9 Avian Influenza Virus And Preliminary Study Of Candidate Vaccine Strains

In 2017,lowly pathogenic H7N9 avian influenza virus changed to high pathogenic one.In the same year,H5 + H7 bivalent inactivated vaccine(H7-Re1 strain)effectively controlled the spread of H7N9 virus between poultry and between human in china.In2018,H7-Re1 vaccine strain was replaced by H7-Re2 vaccine strain and updated to H7-Re3 vaccine strain in 2020.However,since 2019,H7N9 virus has been continuously mutated,resulting in cases in some areas.As an important anti-epidemic tool,vaccines play a crucial role in the prevention and control of H7N9 avian influenza.However,H7N9 virus is still in continuous evolution and variation,and the pressure level of prevention and control may not decrease but increase.H7N9 avian influenza viruses to keep tracking and monitoring the virus variation and carry out vaccines-related research for the scientific reserves.In this study,13 H7N9 avian influenza viruses were isolated from January 2020 to April 2021.HA sequence analysis showed that the amino acid homology of 13 viruses was97.2%–98.0%,and there was a mutation combination of Q226 L and G186 V in HA.According to the single gene fragment homology >95%,13 H7N9 viruses were divided into two genotypes,of which 12 belonged to G2 genotype.The nucleotide homology of NS and NP genes of CK/TS0328/21 strain with other 12 viruses was 93.40% – 95.3% and93.0%–93.6%,respectively.The genetic evolution showed that the NS and NP genes of CK/TS0328/21 strain were from the internal gene of H9N2,which was different from the other 12 viruses and occupied a group alone.It is suggested that CK/TS0328/21 strain may be a new genotype(G11)of H7N9 and H9N2 virus internal gene recombination.Antigenicity analysis showed that 13 strains of H7N9 virus had poor reactivity with H7-Re3 antiserum,and the reactivity with corresponding serum was different in different years.The 13 strains of H7N9 virus were divided into three antigen groups.The H7-Re3 vaccine strain was in the first group,although in the same antigen group,the H7-Re3 antiserum reacted with other H7N9 viruses,and the HI antibody titer was 2 log2 different from the homologous strain.According to the antigen map analysis,one H7N9 strain was selected from each antigen group,and the immune efficacy of H7-Re3 vaccine strain was evaluated.It was found that H7-Re3 vaccine strain could only provide 70%-80% lethal protection to the challenged virus.Based on the HI test results,A/chicken/Shanxi/HY1027/2020 was selected as the parental virus,and the internal gene of A /Puerto Rico /8/34(PR8)was used as the skeleton.A recombinant H7N9 virus was successfully rescued by reverse genetic operation technology,named as r HY1027 strain.The F6 generation of this strain was selected to prepare the inactivated vaccine for the immune efficacy test.The results showed that the HI antibodies of SPF chickens immunized with r HY1027 inactivated vaccine were all around 8log2 at 21 d after immunization,and still above 5log2 at 150 d after immunization.The results of challenge protection test showed that the chickens inoculated with H7N9 strain of different antigen groups had no disease and no virus shedding,and could form100 % protection.This study provides basic data and technical reserves for effective prevention and control of H7N9 avian influenza.