Research On Biological Characteristics Of H9N2 Avian Influenza Virus In East China From 2018 To 2019

H9N2 AIV was first isolated in 1966 and first reported in China in 1994.Since then,H9N2 AIVs have been spreading widely among poultry in many countries and regions of the world,causing great economic losses to the poultry industry.H9N2 AIVs were lowly pathogenic to the host,but they had wide host range,high spread speed and many endemic areas,which occasionally breached the species barrier and directly caused human infection.H9N2 viruses could also provide part or all of the internal genes for highly pathogenic viruses to produce more pathogenic recombinant viruses,such as H5N1,H7N9 and so on.At present,the control of H9N2 AIVs still mainly relies on vaccine immunization in China.The mutation and recombination of viruses occurred frequently leading to the diversification of viruses in genes and antigenicity,which made it more difficult to effectively control their transmission.Therefore,to control the spread of H9N2 or all AIVs,it’s important to monitor the prevalent characteristics and genetic evolution of H9N2 AIVs and pay close attention to the mutant viruses with antigenic or pathogenic changes.In this study,436 disease materials or swabs were collected from chicken breeding companies and farmers in Shandong,Jiangsu,Zhejiang,Anhui and Shanghai from 2018 to2019,and 31 purified H9N2 viruses were obtained by three-time limited dilution of eggs from 50 H9 positive samples.Firstly,the whole genome sequencing was performed to analyze homology,genetic evolution and sequence characteristics of these 31 isolates.The results showed that all 31 isolates belonged to G57 genotype,which matched the current epidemic trend of H9N2 AIVs,having low homology with the reference strains such as CK/SD/6/96 and CK/SH/F/98.In addition,the receptor binding sites,potential glycosylation sites and key sites of pathogenicity had a certain degree of changes.According to the sequence characteristics,10 H9N2 AIV strains were selected from 31strains to make highly immuned serum.The antigen map was drawn to analyze their antigenicity by cross-hemagglutination inhibition test.It was found that there were antigenic differences between the early vaccine strains and these isolates.Next,we analyzed the pathogenicity of H9N2 isolates.Among these 10 isolates,4 isolates had obvious pathogenicity to mice,and one of them was lethal to mice.Besides,all 10 strains could replicate in A549 cells and these strains that were pathogenic to mice had stronger replication ability in A549 cells.The above results indicated that the antigenicity of H9N2AIVs had changed greatly in recent years,and they had a potential threat to increase pathogenicity to mammals.Further analysis of CK/SH/49/19 which was lethal to mice,found that there was a single amino acid deletion at position 217 of the HA receptor binding domain and an E627V mutant of PB2 gene.8 genes of CK/SH/49/19 were cloned into the p HH21 plasmid,and the mutant plasmids were constructed by supplementing 217M of HA gene and replacing V by E/K at position 627 of PB2 gene.The wild-type virus and mutant viruses were then rescued by using reverse genetic technology,and mice infection studies,polymerase activity assay,replication kinetic analysis in A549 cells and virus thermal stability assay were performed.We found that mice infected with the viruses which deleted a single amino acid at position 217 of HA gene showed significant losses of body weight.Compared with PB2 627E,627 V/K mutant viruses had stronger pathogenicity,and the lungs of infected mice had higher virus titers and typical pathological changes.On the contrast,mice infected with the mutant viruses which supplemented the amino acid at position 217 of HA gene didn’t perform obvious clinical symptoms and histopathological changes,and PB2 627V/K didn’t significantly enhance the pathogenicity to mice.The MLD₅₀ of the mutant viruses with supplemental amino acid at position 217 of HA gene was higher,causing no death in mice when inoculated with 10⁶ TCID₅₀.The MLD₅₀ of r-49-PB2 627E+HA 217Δwas 3.16×10⁵ TCID₅₀,that of r-CK/SH/49/19 was even lower,only 5.62×10¹ TCID₅₀.Compared with PB2 627E,the polymerase activity of 627V and627K was increased by 5.48 and 5.94 fold changes,respectively.The viruses with the amino acid deletion at position 217 of HA gene got a better replication ability in human A549 cells,without affecting the thermal stability.These results indicated that position 627of PB2 gene was not a key factor in determing the pathogenicity of CK/SH/49/19 to mice,and 627 V/K could help to improve the pathogenicity by increasing polymerase activity.The position 217 of HA gene was a key factor in affecting pathogenicity.Lacking this amino acid would significantly improve the pathogenicity to mice and slightly enhance replication ability in human A549 cells.However,the specific mechanism of the amino acid deletion at position 217 to pathogenicity of H9N2 AIVs remains to be further studied.In summary,this study provides reference value for revealing the prevalence,genetic evolution characteristics and pathogenicity of H9N2 AIVs in East China.