Isolation,Identification And Sequence Analysis Of H9N2 Subtype Influenza Virus From Raccoon Dogs And Foxes

The H9N2 subtype influenza virus is currently a prevalent low pathogenic influenza virus in China.It has a wide range of hosts and can infect poultry,pigs,horses and other mammals,resulting in decreased host production performance and immunosuppression diseases.The H9N2 subtype influenza virus may also cause high mortality due to co-infection with other pathogens,causing huge economic losses to the aquaculture industry.At the same time,the H9N2 subtype influenza virus has a strong ability to spread and it can break through the species barrier to infect mammals and humans without going through an intermediate host,which greatly threatens public health and safety.In 2013,the laboratory isolated H9N2 subflu virus A/mink/Shangdong/F6/2013 and A/mink/Shangdong/F10/2013 from lung tissues with respiratory symptoms of blisters in the central and eastern regions of Shandong Province.The laboratory isolated H9N2 subtype influenza virus from lung tissues with respiratory symptoms of blisters in the central and eastern regions of Shandong Province.Eight strains of influenza virus were isolated from this laboratory in August 2016.They were identified as H9N2 subtype influenza virus and named as A/Raccoon dog/Shandong/G1/2016,A/Raccoon dog/Shandong/G5/2016,A/Raccoon dog /G7/Shandong/2016,A/Raccoon dog/Shandong/G13/2016,A/Raccoon dog/Shandong/G31/ 2016,A/Raccoon dog/Shandong/G41/2016,A/Raccoon dog/Shandong/G55/2016,A/Fox/ Shandong/G60/2016.According to the gene sequence of the H9N2 subtype influenza virus published by Genbank,specific primers for each gene fragment were designed,and the HA,NA,PA,PB1,PB2,NS,and NP of the eight strains were amplified by RT-PCR method.Each segment of M gene was homologously compared with the sequence of influenza virus in GenBank.The phylogenetic tree of 8 gene segments was drawn.Homology analysis showed that the homology of the eight gene fragments of the eight strains was 91.1% to 100%.Molecular phylogenetic analysis showed that HA gene belongs to Y280-like branch,M gene belongs to G1-like branch,NP belongs to Y439-like branch,NA,NS,PA and PB1 genes belong to F/98-like branch,PB2 belongs to Korea-like branch.From this,it can be seen that the branches of each gene of the eight isolates are not uniform,and it can be inferred that the eight influenza virus strains of H9N2 subtype isolated in this study may be derived from different sublines.Therefore,the isolation of influenza virus is toxic.The strain may be a recombinant virus.The molecular biological characteristics of the eight isolated strains were further analyzed.The amino acid sequences of the isolates were deduced by DNAStar software and compared with reference strains.The results of amino acid sequence analysis showed that the amino acid sequence homology of the 8 isolates was 99.3% to 100%;the amino acid at position 234 of the 8 isolates HA protein was leucine(Leu)and had SA ?-2,6-Receptor binding properties of-Gal;The amino acid sequence at the cleavage site of the HA protein is PSRSSR?GL,which is consistent with the characteristics of the low pathogenic influenza virus;HA protein contains 8 potential glycosylation sites,and the reference strain In comparison,8 strains of isolates all had a potential glycosylation site at position 218,a potential glycosylation site at amino acid 313,and 627 amino acid residues of the 8 isolates of PB2 protein.The point is lysine(Lys),indicating that the 8 strains have mammalian receptor binding properties.This study confirmed that crickets and foxes can infect H9N2 subtype influenza virus.This may not only cause harm to the fur animal husbandry industry,it may lead to infections by foxes and fleas,thus causing certain safety hazards.In order to understand the prevalence of H9N2 subtype influenza virus and the evolution of genes in the foxes and centipedes,the whole genome sequencing and analysis of H9N2 subtype influenza virus isolates was conducted in this study to explore the origin and genetics of each gene fragment of the isolates.The law of change has enriched the epidemic data of the H9N2 subtype influenza virus.