| The H3N2 subtype influenza virus has been present in the population since the pandemic in 1968,and it causes seasonal influenza commonly around the world.This subtype influenza virus is also extensively present in mammals and poultry.Domestic birds would display no symptoms or death after being infected with H3N2 virus,which creates a stealthy condition for the circulation and evolution and brings a burden for the detection,control and prevention of the H3N2 virus in poultry.Based on the previous epidemiological surveillance work in our laboratory,a total of 21 H3N2 avian influenza viruses isolated in poultry farms in our country between February 2019 and April 2021 were selected to sequence the genome and construct phylogenetic tree.The results showed that HA genes of 21 viruses shared 85.7%～98.9% identity at the nucleotide level and formed 8 different groups in the phylogenetic tree.The NA genes shared 88.1%～99.1% identity at the nucleotide level and formed 5groups.The other six genes,namely PB2,PB1,PA,NP,M and NS shared 86.6%～99.3%,89.3～98.9%,88.8%～99%,90.2%～98.7%,92.2%～99.8% and 70.6%～99.6%,respectively,and formed 5,6,7,3,3 and3 groups,respectively.According to the 95% sequence identity cut-offs and branch of the phylogenetic tree,the 21 H3N2 viruses could form 19 genotypes.To investigate the characteristics of receptor-binding and antigenicity of 21 H3N2 viruses isolated from poultry farms in the last three years as well as the property of their replication and pathogenicity in mammals,the BALB/c mice were inoculated intranasally to examine the viral replication and pathogenicity.The results showed that all 21 viruses could replicate in mice and showed low pathogenicity in mice.In addition,seven strains of viruses that replicated well in the nasal turbinate and lung of mice were selected and their receptor-binding property was investigated by using erythrocyte agglutination assay.The results of the serum cross-hemagglutination inhibition assay showed that the antigenicity of the 21 viruses was highly variable.In order to investigate whether the viruses acquired key amino acid mutations after replication in mice,the mice were inoculated intranasally with 21 viruses and the amino acid mutations in HA and PB2 genes of viruses were analyzed after replication.The results showed that no amino acid mutations were detected in the HA gene of any of the 21 viruses.After replication in mice,the CK/AH/S1354/20 virus from nasal turbinate and the DK/GX/S40365/20 virus from lung showed an E to K mutation at position627 of PB2 gene,which enhanced the pathogenicity of both mutant viruses in mice.In summary,this study investigated the genetic evolution and biological properties of H3N2 avian influenza viruses isolated from poultry farms in different provinces in China between 2019 and 2021.The results showed that the H3N2 avian influenza viruses present in poultry farms in our country are evolutionarily diverse.Animal study proved that these viruses show low pathogenicity in mice with different replication ability.Some viruses could acquire PB2 E627 K mutation after replication in mice,and the mutant viruses have enhanced pathogenicity in mice.Therefore,close monitoring of H3N2 subtype avian influenza viruses in poultry is needed to prevent potential threats to public health safety from new mutant strains. |
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