| Avian influenza virus(AIV)is one of the main pathogens that harm the poultry industry.Among them,the H9N2 subtype is the most widespread in my country’s poultry,causing a serious economic burden on the poultry industry.Because H9N2 often undergoes antigenic variation,the vaccine cannot provide complete and effective protection for poultry.The amino acid at the head of the glycoprotein HA on the surface of the influenza virus is prone to mutations,which causes the affinity of the virus and the antibody to decrease and antigenic drift occurs.Changes in the structure of HA protein antigens include mutations at Nglycosylation sites.Changes in glycosylation sites will affect the antigenicity and the virulence of AIV,the effectiveness of the vaccine,and its pathogenicity and transmission ability to mammals.In this study,two H9N2 subtype AIV strains isolated from a chicken farm in Jiangsu Province were taken as the research objects,named A/chicken/Jiangsu/75/2018(JS/75)and A/chicken/Jiangsu/76/2018(JS/76).After continuously purified by the chicken embryo limited dilution method,the two viruses were subjected to whole-genome sequencing analysis.The results showed that the internal genes and NA genes of the two strains were highly similar,and only three amino acid differences appeared in the HA gene,which were127,183 and 212 amino acids.Genetic evolution analysis of the HA gene showed the evolutionary distance of the HA gene of the two isolates had changed.Sequence analysis revealed that the JS/76 strain added a potential glycosylation site in the form of NGT at position 127.The different amino acids of the two viruses are not only located in the epitope of the HA head but also located in the receptor-binding region.Evolutionary analysis showed that two viruses have antigenic mutations.Reverse genetic technology was used to rescue the virus(r-JS/75 and r-JS/76)and immune protection tests were used to evaluate the immune effect of existing vaccines against the virus.The results showed that chickens detoxified on the 3th and 5th day after the challenge,and the virus isolation rate of the r-JS/76 group was higher than that of the r-JS/75 group on the 5th day.It showed that the current commercial vaccines had poor immune protection effects on r-JS/75 and r-JS/76.On the other hand,compared with r-JS/75,r-JS/76 had a more obvious immune escape phenomenon.The amino acid differences in the HA sequence might be the reason for the different degrees of antigen escape between the two viruses.In order to reveal the key sites that determine the antigenic variation,PR8 was used as the background to rescue the JS/75 and JS/76 recombinant viruses,and the antigen map was drawn through the HI assay to determine the antigenic characteristics between the recombinant viruses.Three different antigen groups were formed between the recombinant virus and the classic reference strain.Among them,the HI titer between the virus of antigen group C and the antiserum of the vaccine strain decreased by 16-64 times,and significant antigenic drift occurred.It was worth noting that the substitution of amino acids at position 127 of r-75/PR8 and r-76/PR8 changed the antigenic distance between the recombinant virus and the vaccine strain.The results showed that the recombinant viruses were divided into different antigen groups due to antigen variation,r-76/PR8 antigen showed significant antigen escape than r-75/PR8.Then through animal experiments to explore the immune protective effect of the recombinant virus inactivated vaccine on chickens,it was found that the r-76/PR8 vaccine had the best protective effect on the virus.After being infected with r-JS/76,the virus isolation rate in the throat of chicks was higher.Compared with the r-76/PR8 immunization group,the amino acid change at position 127 increased the virus isolation rate of chickens in the r-76-HAN127D/PR8 immunization group,indicating that the 127 amino acid substitution changed the antigenicity of the r-76/PR8 virus.Part of the H9N2 subtype AIV has been shown to possess both human and avian receptor affinity.Receptor affinity test results showed that,except for r-75-HA-D127N/PR8,other recombinant viruses already had the affinity characteristics of α-2,6 sialic acid receptors.r-75/PR8 had a higher affinity for α-2,6 sialic acid receptors than r-76/PR8.To explore the replication ability of recombinant virus in human cells,viral replication kinetics results showed r-75/PR8 virus replication in A549 cells was higher than the level of r-76/PR8.Compared with the recombinant wild virus,the replication level of the recombinant mutant virus was lower,and r-75-HA-D127N/PR8 and r-75/PR8 viruses had significant differences in the replication level in A549 cells.The r-75-HA-G183R/PR8 and r-76-HA-L212R/PR8 mutant viruses had poor growth effects in A549 cells.To further analyze the pathogenicity of the recombinant virus to mice,the result showed that r-75/PR8 was lethal to mice while r-76/PR8 only caused weight loss in mice.The two viruses had significant differences in lethality in mice.In addition,the introduction of the D127 N mutation of the HA gene into the r-75/PR8 virus weakened the pathogenicity.On the contrary,after the N127 D mutation was introduced into the r-76/PR8 virus,the survival rate of mice in the r-76-HA-N127D/PR8 group decreased(the survival rate dropped from 40% to20%).Furthermore,evaluate the replication ability of the recombinant virus to replicate in mice lung tissue.The results showed that the r-75/PR8 recombinant virus was more pathogenic to mice than the r-76/PR8 recombinant virus.Inducing the D127 N mutation of the r-75/PR8 virus reduced the virulence and replication ability of the mutant virus in the lungs of mice.Statistical analysis showed that the virus titers of the two viruses were significantly different in the lungs of mice.Finally,it was verified by the Western Blot test that 127 N of the HA sequence of JS/76 virus was a functional glycosylation site.This study successfully isolated two H9N2 subtype AIV strains through the genetic evolution analysis of the HA gene and a series of in vitro and in vivo experiments.It proved that the HA sequence 127 N glycosylation and the substitution of epitope amino acids(183and 212)affect the biological characteristics of the H9N2 virus,especially the introduction of 127 N glycosylation reduces the virus replication ability and pathogenicity.It plays an important role in the in-depth study of the antigenicity and pathogenicity of influenza A virus,and also provides a reference for the prediction of H9N2 subtype AIV antigenic variation and subsequent vaccine development. |
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