| The H9N2 subtype avian influenza virus(Avian Influenza Virus,AIV)was first discovered and isolated in North American turkey in 1966,and then spread to all parts of the world,it seriously harms the survival and development of the poultry industry and is one of the main subtypes currently popular worldwide.In China,H9N2 subtype AIV was first isolated in Guangdong in 1994.Subsequently,the prevalence of this subtype virus in mainland China gradually expanded,causing huge economic losses to the poultry industry.At present,vaccine immunization is still the main strategy for prevention and control of H9N2 subtype AIV,and the use of vaccine strains(such as A/chicken/Shanghai/F/98,A/chicken/Beijing/1/94)has played an important role.However,with the widespread and frequent use of vaccines,the virus has accelerated its evolution and antigenic mutations under the pressure of immunity,causing the epidemic of H9N2 subtype AIV to intensify in recent years.The purpose of this study is to carry out the screening of vaccine candidate strains based on the analysis of HA gene antigen variation characteristics of H9N2 subtype AIV isolates in East China.1.Epidemiological survey of H9N2 subtype avian influenza virus in parts of East ChinaIn this study,a total of 1,007 samples and conditioned samples from a breeding company from December 2018 to November 2019 were subjected to virus isolation,identification and analysis,and a total of 292 H9 subtype AIV positive samples were determined,and the detection rate About 29%.The results of rheology suggest that the detection rate of H9 subtype AIV positive samples has obvious seasonal differences,in which the detection rate is generally higher in winter and spring,and generally lower in summer.Subsequently,61 of the H9N2 subtype AIV strains from 2018 to 2019 were selected for HA gene sequencing,and the DNA gene,CLC,MEGA7.0 and other software were used to perform splicing and comparison on the HA gene sequencing results,draw the HA gene genetic evolution tree,and The key sites were compared and analyzed to reveal the antigenic variation of these H9N2 isolates.The results showed that the 61 strains selected in this study were isolated from the H9N2 epidemic strains in 2018-2019,and all belong to the h9.4.2.5 branch.The nucleotide homology among these 61 epidemic strains reached 91.0%-100.0%,and the amino acid homology was 90.4%-100.0%,which showed a close relationship.Analysis of the changes of glycosylation sites of HA protein in these 61 epidemic strains revealed that all epidemic strains had the characteristic change of 218G-/313G+,which proved the tendency of H9N2 subtype AIV antigen mutation in East China.And stability.Analysis of the changes in the HA protein receptor binding sites of these 61 popular strains revealed that the mutations at amino acids 198 and 234 were the most obvious.All popular strains mutated 198V to 198A or 198T;and all had Q234L,which The mutation greatly increases the ability of AIV to infect mammals.2.Screening of H9N2 vaccine strain and evaluation of immune effectThe second part of the experiment is the antigenicity analysis and cross-protection analysis of the HA mutant library strain of vaccine strain A/chicken/Anhui/BR1-99/2016(#99)and 14 H9N2 subtype AIV epidemic strains.In this study,two important escape mutant viruses were screened from the#99 mutant virus library:99TB-15 and 99TB-D,which have 207NTT glycosylation mutation sites and T197A amino acid mutation sites,respectively.At the same time,comprehensive antigenicity analysis,crosshemagglutination inhibition test and immune challenge protection test results found that A/chicken/Changzhou/193/2019(CZ193)had little difference in antigens from other branches of the strain and had good cross-protection ability.In the immune challenge protection test.the inactivated vaccine prepared with CZ193 produced high,stable,and long-lasting antibody levels after immunizing SPF chickens,and completely resisted the attack of the#99 virus,with a protection rate of 100%;However,the antibodies produced by the 99TB-15 and 99TB-D escape mutant viruses are not completely protective against the#99 virus,and the significance of their mutations needs further study.Therefore,the final analysis found that the CZ193 strain is an ideal candidate for the H9N2 subtype AIV vaccine.Compared with the H9N2 subtype AIV detoxification experiment,the application of the inactivated vaccine shortened the detoxification time of infected SPF chickens by about 4 days,and the response speed of the immune system was also significantly accelerated,proving the effectiveness and effectiveness of vaccine immunization.necessity.In summary,the results of this study indicate that the epidemic strain of H9N2 subtype AIV in parts of East China in the past two years was mainly a virus branched from h9.4.2.5.Comprehensive antigenicity analysis,virus value-added efficiency,stimulation of host antibody levels,and cross-immune protection capabilities,it is recommended to use the A/chicken/Changzhou/193/2019(H9N2)strain as a vaccine candidate. |
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