A Primary Study On The Pathogenic Characteristics Of The Avian Influenza Virus In Waterfowls And Analysis And Comparison Of It's HA Genes

The thirteen isolates with H5 subtype of avian influenza viruses (ATV) isolated from goose and duck and one isolate isolated from quail were focused in this study.Six isolates from the diseased goose with AI, named WZ2001, SZ2001, JD2001, WJ2001, SH2000 and WJ2000 were inoculated into 35-day-old RongChang-Goose via intravenous respectively. Mortality of goose challenged with these viruses ranges from 20% to 100%, according to the results, six isolates may be divided into three levels of pathogenicity. WZ2002 and SZ2001 are; JD2001 and WJ2001 are moderately pathogenic isolates; SH2000 and WJ2000. Different cytopathic effects (CPE) were observed in cell culture, WJ2001 are highly pathogenic isolates on CEF, SH2000 are low pathogenic isolates.The genomic RNA of the fourteen AIV isolates were extracted and the HA gene of them were successfully amplified by reverse transcription-polymerase chain reaction(RT-PCR). The expected 1.7kb-lengthed product were cloned into pGEMR-T vector and sequenced. The sequencing result showed that the length of the nucleotide sequence of the HA gene amplified was 1732bp containing the whole open reading frame of 1707 nucleotide that encodes a protein of 568 amino acids. The amino acid sequence of the cleavage site has three types: RIRKKR(WZ2001 isolate), RRRKRR(WJ2000, SZ2001and AN2001 three isolates), RRRKKR(other ten isolates), which reveals the molecular basis of the pathogenicity of HPATV. Among these isolates, the homology of HA gene varies from 96.3% to 99.1% in amino acid. The fourteen isolates share 96.1%-97.9% and 95.1%-96.8% homology of amino acid sequence with GD96, HK97 strain respectively, which were isolated inland early; 89.1%-90.9% with IRE83, isolated in Europe; and 84.6%-86.3% with PA83, isolated in North America. It may be concluded that the distribution of strains relies on when and where the virus was isolated.Comparing their important sites, such as proteolytic cleavage site (PCS), receptor binding site (RBS) and potential glycosylation site, we may conclude that it is roughly parallel in their PCS and RBS. There appeared two potential glycosylation sites in 301 and 511 position of WJ2002, one potential glycosylation site in 757 position of AN2001, and one potential glycosylation site in 469 position of JD2001. But it disappeared in 628 position of SH2001 and DT2001.In the phylogenetic tree of HA gene, the fourteen H5 subtype AFV isolates, beside WZ2001 which form branch specialty, are divided into two distinct groups by time, but quail isolate has a higher homology with the lately isolated viruses, rather than its contemporary ones. It is probably suggested that influenza virus transfer their host from goose and duck to non-waterfowl, and then, it varies in non-waterfowl, after which, cause another epidemic in the waterfowl.