Isolation,Characterization And HA,NA Genetic Analysis Of Goose Avian Influenza Virus

In this study, 1 strain influenza virus identified as H5N1 subtype was isolated from sicked gooses in Jilin. The openning reading frame of its HA gene and NA gene was cloned, sequenced and compared with sequences available in GenBank.According to epidemiologic survey, clinical symptom and dissection, initial diagnosis was made that sicked gooses were infected with high pathogenicity avian influenza. Through viral isolation, blood serologic test, the real-time RT-PCR and subtype identification,it has been proved that the isolated strain from sicked gooses is a H5N1 submodel avian influenza toxicity strain.Assay of the 50%Embryo infective dose (EID50), the intravenous pathogenicity (IVPI) index, intracerebal pathogenicity index ( ICPI) showed that the isotaled strain was highly pathogenic avian influenza virus. A series of biological tests,including heat resistance test, lipid solvent test and acid test, showed that the isotaled strain was sensitive to heat, lipid solvent and acid.Based on HA gene and NA gene of H5N1 avian influenza strains published in Genebank,two specifical primers were designed and synthesized using Primer Primer5.0 . Total RNA of the isotaled strain was isolated by treatment of aliquots of the virus with TRIAZOL. Full-length HA cDNA and NA cDNA were synthized by RT-PCR and respectively cloned into pMDT-18 vectors.The tests of recombinant plasmids digested by enzymes showed that HA gene and NA gene were successfully cloned.Sequences analysis proved that the cloned HA gene of the goose-derived H5N1 Influenza A virus consists of 1707nucleotides and encode a polypeptide of 569 amino acids residues. There were potential glycosylation sites on HA peptide chain, situated in 10, 11, 23, 165, 286 positions of HA1 and 154, 214 positions of HA2.The amino acid sequences of the HA connecting peptide revealed that the isotaled strain had -R-R-R-K-K-R-at the proteolytic cleavage site. The molecular basis of the HA gene was compatible with highly pathogenicity. Sequences analysis proved that the cloned NA gene of the goose-derived H5N1 Influenza A virus consist of 1350 nucleotides and encode a polypeptide of 450 amino acids residues. There were potential glycosylation sites on NA peptide chain, situated in 68, 126, 215 positions of NA.For nucleotide sequence comparison and phyletic evolution analysis, MegAlign of DNAStar software were applied.When compared with HA gene of other influenza a virus(H5N1) from the world...