Screening Of H9N2 Subtype Avian Influenza Vaccine Strain And Preliminary Development Of Bivalent Inactivated Vaccine

H9N2 subtype avian influenza virus(AIV)is a low pathogenic virus.it was first isolated from poultry in China in 1994.subsequently,this subtype virus spread all over the country.co-infection with other pathogens in poultry can lead to a high mortality rate that seriously affects the development of poultry industry in China.Later,some studies have also confirmed that human beings can also be infected with H9N2 subtype AIV,and that H9N2 subtype AIV is closely related to the outbreak of highly pathogenic influenza virus.the prevention and control of the disease is of great significance in public health.Vaccination is one of the main ways to prevent and control the disease.Since 2012,disease caused by H9N2 subtype AIV sometime still occur after getting current vaccine.From 2012 to 2017,the Avian Influenza Research Group of South China Agricultural University has isolated and identified several strains of H9N2 subtype AIV from Guangdong,Hunan,Fujian,Chongqing,Shandong,Anhui,Henan,Yunnan and other provinces.15 strains selected as the research object were used in genomic HA sequence analysis in oder to understand the genetic and evolutionary differences of H9N2 subtype AIV and provide scientific basis for the prevention and control of H9N2 subtype avian influenza.According to the classification and analysis standards of antigen variation of H9N2 subtype AIV from the World Organization for Animal Health and some laboratories in China,in recent years,the epidemic strains in South China,East China and Southwest China were mainly located in the h9.4.2.5 branch(BJ/94-like or Y280/G9-like)of h9.4.2 branch,and h9.4.2.5 branching viruses have gradually become the main epidemic evolutionary branch.According to genetic evolution,we selected representative isolated strains located in h9.4.2.3 branch,h9.4.2.5 branch and h9.4.2.6 branch of h9.4.2 branch to make oil emulsion inactivated vaccine.Serological cross-reaction test,neutralizing antibody cross-reaction test and cross-challenge protection test were carried out.The results showed that there were differences in antigens among epidemic strains of different branches and there was significant difference in antigenicity among the epidemic strains of h9.4.2.5,h9.4.2.6 and h9.4.2.3 branches in BJ/94-like(R=0.14～1.0).Even the epidemic strains located in the same branch of h9.4.2.5 were significantly divided into two different antigen groups.the antigenic difference between the two antigen groups of the h9.4.2.5 branch can reach 16 to 64 times.Chicken embryo neutralization test showed that A/Chicken/Hunan/HN/2015(H9N2)and A/Chicken/Shandong/SD/2014(H9N2)strains located in the h9.4.2.5 evolutionary branch had better cross-reactions with HI antibodies of most strains of the same branch or different branches,with only 2 times difference.In conclusion,A/Chicken/Hunan/HN/2015(H9N2)and A/Chicken/Shandong/SD/2014(H9N2)strains with great antigenicity of h9.4.2.5 branch were selected as candidate vaccine strains.The two strains and the classical A/Chicken/Guangdong/SS/94(H9N2)vaccine strain on the market were used to evaluate the immunogenicity of the two strains.The results showed that the vaccine strains on the market and the candidate vaccine strains had great immunogenicity,and the protection rate of the vaccine against its own strain could reach more than 93%.However,the protection rate of classical vaccines on the market against candidate vaccine strains is only 66%.It was preliminarily revealed that A/Chicken/Hunan/ HN/2015(H9N2)and A/Chicken/Shandong/SD/2014(H9N2)as candidate vaccines have potential protective effects on the prevalent H9N2 subtype AIV.In order to meet the needs of production,the bivalent inactivated vaccines against A/Chicken/Hunan/HN/2015(H9N2)and A/Chicken/Shandong/SD/2014(H9N2)strains were designed.The results showed that the protective effect of bivalent vaccines were consistent with that of single vaccine and the lowest dose of immunization(0.1 m L)can reach more than 92% protection rate.The effective protective antibody can last up to 24 weeks.And the immune quality and immune protection effect are great,which can meet the requirements of clinical application.Therefore the bivalent vaccine can effectively prevent the epidemic of H9N2 subtype avian influenza.