| The H9N2 subtype avian influenza virus(AIV)was first discovered in China in 1992,and the virus persisted for more than 20 years,and gradually became the most prevalent avian influenza subtype in China.Although H9N2 subtype avian influenza is a low pathogenic avian influenza,after infection,it will cause a decline in egg production and respiratory symptoms in avian,and secondary infections often occur,aggravating the morbidity and mortality,and causing huge economic losses to the poultry industry.In order to understand the antigenic variation of H9N2 subtype AIV in China from 2019 to 2020,this study conducted a molecular epidemiological investigation on 1152 H9N2 subtype AIV isolates in some areas of China from2019 to 2020 in our laboratory to study the evolutionary variation of the virus strain.using serological test technology to analyze the antigenicity of the selected strains,select vaccine candidate strains,and evaluate the immune efficacy of the vaccine candidate strains.1.Molecular epidemiological investigation of H9N2 subtype AIVThe H9N2 subtype AIV isolated in some parts of China from 2019 to 2020 was sequenced and its genetic evolution characteristics were analyzed.The results showed that all viruses belonged to the h9.4.2 subline,and the isolates were relatively concentrated on the phylogenetic tree.All belong to the h9.4.2.5 clade,but multiple separate clade have been formed.Except for3 strains,the HA proteins of the other strains have the Q226 L mutation,which has the potential to infect humans.The NA protein is obviously divided into two types according to whether the deletion of the neck amino acid occurs.Due to the deletion of the neck,the potential glycosylation site of the 61 amino acid of the NA protein is deleted.The neck deletion strain is similar to the Y280-Like.The potential glycosylation site types of the strains were consistent,and the strains without neck deletion were consistent with the potential glycosylation site types of the A/Duck/Hong Kong/Y439/97(H9N2)strain.Among the 110 viruses analyzed,8 viruses had the potential glycosylation site at position 61.After the internal gene sequencing analysis of 11 representative strains,the genetic distance between 3 viruses and the other 8 viruses is farther,they belong to a separate branch,and they are more similar to other subtypes(H10N3,H7N9,H10N8),and H9N2 The internal gene can serve as a donor for other subtype internal genes,such as H7N9,which can cause severe respiratory disease.2.Analysis of antigenicity of H9N2 subtype AIVThrough amino acid homology comparison and genetic evolution analysis,9 strains of viruses were selected to analyze their antigenicity.After 9 strains immunized chickens,the antibody level was high(antibody titer ? 8log2)and had good immunogenicity;the results of neutralization test showed that the two strains H2049 and J1443 only produced relatively high levels of antibodies against themselves and each other.High protective power,but low protection against other strains,and the R values between H2049,J1443 strains and other strains are all small,indicating that antigenic differences between antigens are significant.Combining the results of the cross-hemagglutination inhibition assay and the neutralization assay,it was demonstrated that there were two antigen groups.Based on the results of the crosshemagglutination inhibition test and neutralization test,we selected a Y2432 strain with good antigenicity and genetic stability as the vaccine candidate strain to immunize the chickens.After21 days,the chickens were challenged with viruses from the two antigen groups.,the flocks did not detoxify,proving that the vaccine can completely protect the flocks.The prepared candidate inactivated vaccine immunized SPF chickens were continuously monitored for 20 weeks,and the regularity of antibody fluctuation was observed.High-level antibodies could be produced within 14 days,and the highest average antibody could reach 14.4 log2,and the antibody could be maintained for more than 140 days.A virus was successfully rescued by reverse genetic technology,and the similarities and differences between the parental virus and the recombinant virus were compared.The results showed that the HI difference between the recombinant virus and the parental virus did not exceed 1 log2,and the gap was small.MDT results showed that the lethality of recombinant virus to chicken embryos was lower than that of wild virus.The construction of the recombinant virus provides a platform for the follow-up molecular targeted research,and also provides ideas for the new H9N2 subtype avian influenza vaccine. |
PDF Full Text Request