Isolation, Identification And Pathogenicity Of Avian Influenza Virus From Goose

Avian influenza(AI) is a number of influenza A virus and can cause fatal disease for avian specises. HPAIV was placed the class A infectious disease by OIE.Since,It was reported in 1878 and has beening happened all over the world.It has not only led to the seriously loss of economy of the poultry enterprise all over the world, but also threated human health seriously. Avian Influenza Virus(AIV)H5N1 in 1997,H9N2 in 1999,H7N7 in 2003 and H5N1 in 2004 infected human beings successively so that people understand the public sanitation significance of avian influenza.In this study, 1 strain of H5 subtype avian influenza virus(AIV)was isolated from gooses. This was found as globular enveloped virion under TEM.The hemagglution inhibition(HI), Neuraminidase inhibition(NI) test and fluorescence RT-PCR indicated that this isolate belong to H5 subtype of influenza A virus.The results of the 50 percent embryo Lethal doses(ELD50),Intravenous pathogenicity index(IVPI), intracerebral pathogenicity index(ICPI) showed that 1(from gooses) of the H5N1 AIV isolate is highly pathogenic AIV unstable to heat, and are inactivated under 56℃for 30min,60℃for 10min and 65℃～70°for 3min. G7 is sensitive to ether, chloroform and acid.Based on published gene sequences in Genebank, 1 pairs of specific primers which is used to amplify HA gene of H5N1 AIV isolates were designed and synthesised. First the viral RNAs of all the isolate was extracted and used in reverse transcription-polymerase chain reaction(RT-PCR)for amplifying the full-length cDNAs of HA genes of the isolate. The cDNAs were then cloned to vector pMD18-T, the gained recombinants were transferred into JM109 bacteria and identified by enzyme digestion, PCR confirmation and then sequenced. The results of sequence analysis showed that the cloned HA genes of H5 subtype AIV isolate from gooses contain whole open reading frame, consist of 1707 bp and encode 568 amino acids residues. There are 6 potential glycosylation sites and 6 basic amino acids(R-R-R-K-K-R) insert at the cleavage sites in HA genes of the AIV isolates which is a symbol of highly pathogenic avian influenza. Amino acids of receptor-binding sites are YWIHELY, amino acids of left edge of receptor-binding sites are SGVSS, amino acids of right edge of receptor-binding sites are NGQSG; amino acids of receptor-binding sites 226 and 228 were of binding to Avian.HA genes of the isolates and reference isolates are analysised by software.The results of phylogenetic analysis showed homology of nucleotides and amino acids of HA genes is 96.3%～99.1% and 94.6%～98.8% among all the isolates; The results of phylogenetic analysis showed homology is high in same subtype and doesn't have obvious difference because of hosts, and genes of the isolates come from different source and differrent areas.Virus isolation and viral antigen distribution of a goose AIV isolate in different tissues of infected chickens by nose and eye drop application, intravenous injection were studied.The results showed that the AIV can be isolated from trachea, cloacal swabs at postinnoculation 12h-192h by virus isolation, the AIV can be detected from heart, liver, spleen, lung, kidney, brain and muscle at postinnoculation 24h-216h by fluorescence RT-PCR and inmunofluorecence. The results show that the isolate is highly pathogenic to chicken, and the time of antigen distribution is different with different tissues.To draw assistance from histopathology, G7 was observed by histopathologic method. It was considered that the avian influenza virus was highly pathogenic to chickens. The histologic changes were hyperemia, hemorrhage, degeneration and necrosis in myocardium, lung, kidney, liver, pancreas, spleen and thymus. Especially in the fat of coronary groove, the multifocal haemorrhages was more serious.