Isolation And Whole Genome Sequence Analysis Of Equine H9N2Influenza Virus In Guang Xi

Using RT-PCR and serology, H9N2subtype influenza virus was tested in samples of horse tissue and serum collected from four cities in Guangxi. The results showed that among the284tissue samples inoculated from chick embryo, one was positive with the positive rate of0.35%and7were positive with the positive rate of0.54%amongst the1110serums. An isolate of virus was identified as H9N2subtype influenza virus by hemagglutination, hemagglutination-inhibiton, and named A/equine/Guangxi/3/2011(H9N2)(abbreviated to GX3virus). Complete genome was sequenced and analyzed. The pathogenicity of the virus was determined. The ELD50and ICPI value were10-6.43/0.2mL and0.09, respectively. Animal experiment demonstrated that GX3virus was a low pathogenic influenza virus and possessed good immunogenicity. Analysis of key sites in GX3virus indicated that amino acids in183,225,227and228sites were very conservative. The226th amino acid for leucine (Leu) had the receptor SAa-2,6-Gal specificity. The amino acid sequence at HA1and HA2amino acids cracking site in GX3virus was RSSRGLF, which was a low pathogenic influenza virus. The627th,701st,714th amino acid in PB2gene was Glu, Asp and Ser, respectively; the66th amino acid in PB1-F2gene was Asn, respectively; the319th in NP gene and92nd in NS1protein was Asn and Asp, respectively. Both proved that GX3was a low pathogenic virus. Through glycosylation analysis, it found that both HA and NA gene had eight glycosylation sites. HA gene had one more glycosylation site in the313th amino acid, and NA gene had one more glycosylation site in the44th amino acid than the reference virus. Other glycosylation sites were relatively conservative with no evident change. The finding of119E,151D,276E,292R and294N in NA gene implied GX3virus was sensitive to neuraminidase, while S3IN in M2gene exhibited GX3was resistant to amantadine Phylogenetic tree and nucleotide homology analysis indicated that HA and NS genes of GX3virus derived from Beijing, NA gene from G1-like, PB2and M gene from G1-like, PA, NP and PB1from SH/F/98-like. Genes of GX3derived from4strains of classical H9N2influenza virus and reassorted a new genotype virus.