Isolation, Identification And Molecular Characterization Research Of The H5N2 Canine Influenza Virus

56 nasal swabs and serum samples from 56 dogs exhibiting the respiratory disease signs, including coughing, sneezing, sniffling and heightened temperature were collected in this study. The serum samples were detected with the HI assay,NI assay and BLAST analysis and were identified as the H5N2 subtype ultimately. Two pairs of primers were designed based on corresponding genes subtype of H5 subtype of influenza virus according to GenBank. The canine virus isolated (CA/SD/JT01/09) as a template, its 8 genes were amplified by RT-PCR method. Then, the RT-PCR products were cloned into pMD18-T vector and sequenced. For the sake of researching the prevalence condition of H5N2 virus between dogs, we collected and conducted a large number of the canine serum samples for the antibody analysis. Finally, we have successfully isolated the anti-H5N2 antibodies from Small part of the samples. It indicates that the H5N2 influenza virus can spread between dogs.As a result, the ORF length of the HA gene of CA/SD/JT01/09 was 1467 bp, encoding 466 amino acids. The amino acid sequence at HA0 cleavage site in CA/SD/JT01/09 was PENQTR↓G, which is representative of a non-highly pathogenic.There are 4 glycosylation sites in the isolated segment of the HA proteins.They were 181th-NNTD-,209th-NPTT-,302th-NSSM- , 500th -NGTY- respectively, moreover, three of which in the HA1 protein,while one in the HA2 protein,and the number of the isolated virus was different from the individual reference strains. CA/SD/JT01/09 HA protein amino acids occurred some bit mutations respective in the 45th,184th,444th amino acid site. Due to not be at the important receptor binding site of HA protein, its antigenicity and biological properties have not been changed. The ORF length of the NA gene of CA/SD/JT01/09 was 1172 bp, encoding 388 amino acids. There are 4 glycosylation sites in the isolated segment of the HA proteins.At the 50th and 338th amino acid of the NA, the mutations had occurred.In addition, the 50th amino acid V that located in NA stalk was replaced by A. It can be speculated that the mutation will produce some impact on the virus host range and replication capacity. The ORF length of the NP gene of CA/SD/JT01/09 was 1594 bp, encoding 499 amino acids. The NP gene sequence of the isolated virus has the highest nucleotide identity with A/mallard/Italy/36/2002(H5N3) (97%). The 339th, 376th, 461th amino acid residue respective was R, N, A in the NP protein of the CA/SD/JT01/09 virus, while was C, D, D in the referenced viruses. The gene length of other gene segments PB2, PB1,PA,M, NS of CA/SD/JT01/09 were 2174bp, 2134bp,2062bp, 1027bp, 890bp respectively, encoding 724, 711, 687, 252, 226 amino acids. The nucleotide homologies of the each gene between SW/SD/JT/07 and the referenced strains were greater than 90%.Phylogenetic analysis showed that the gene sequence homologies between the isolated influenza virus CA/SD/JT01/09 and referenced sequences were greater than the maximum 93.3%. The HA gene have the highest homology with virus SW/FJ/F1/01 (H5N1). It belongs to Asian poultry lineage. The N2 gene had the closest relationship with the H9N2 influenza virus isolated from turkey at 1966, Wisconsin. The analysis showed that the NA gene belonging to recent the North of American Avian lineage. NP gene has the closest relationship with SW/FJ/F1/01 (H5N1). It belong to the the European lineage. The remanent fragments have the highest nucleotide identity with virus SW/FJ/F1/01 (H5N1),also have the closest relationship with SW/FJ/F1/01 (H5N1).This showed that the source of the isolated virus gene fragments was very complex. We conjecture that the isolated virus CA/SD/JT01/09 was a new recombinant that was recombinated by H9N2 influenza virus and H5N1 influenza virus.This is the first report that the genotype H5N2 virus were found to be associated with canine worldwide. Because of the often contact between dog and humans, maybe the CIV can give rise to threat on human health, so the question becomes important about the zoonotic potential. We successfully isolated the H5N2 influenza virus and obtained its 8 gene sequences. Moreover, the gene sequences, amino acid sequences and homology analysis had been performed in this reasearch.These results will provide important molecular epidemiology information for the prophylaxis and treatment of CIV.