| During the period from May to July in 2005, a large quantity of migratory birds in Qinghai Lake and surrounding areas died due to H5N1 highly pathogenic avian influenza virus. Because of living in water of migratory birds, the water is a important kind of dangerous vector of avian influenza virus. For understanding the occurrence and transmission model of avian influenza, we established a method to concentrate avian influenza viruses in water samples using nitrocellulose membrane. H5N1 highly pathogenic influenza virus was isolated from water sampled around Qinghai Lake. We investigated its pathogenicity, biological characteristics, genetic variation etc, and this will provide scientific references for researches on monitoring and early warning system.Avian influenza viruses in tap water and sewage were concentrated by nitrocellulose membrane filter, and the recovery rate were 96.14% and 93.44% respectively, no obvious difference. Avian influenza viruses were carried out 10-fold serial dilutions and then recovered, and no obvious difference of recovery rates was observed among different concentrations. When the concentration of virus was 96PFU/L, the recovery rated in tap water was above 78.13%, and in sewage above 47.92%. In this study, we compared the best elution efficiency among glycine buffer, beef extract solution and phosphate buffer in different pH (7, 9.5, and 11). The result shown that 0.1mol/L glycine buffer at pH 9.5 had the highest recovery rate. In this study, H5N1 highly pathogenic influenza was isolated by established method.The virus was intranasal inoculated in duck, and it can be detected in lung, kidney, liver, spleen, brain and breast muscle after 24h from pathogenicity experiment, and the highest titer appeared 36h post inoculation. The virus showed the descent tendency 60h post inoculation and can not be detected in liver, spleen and kidney, while it maintained highest titer in lung, brain and breast muscle. The same trend which mice was infected by influenza viruses, but the difference is that isn't detected viruses in muscle after 60h.These results showed that influenza virus has tropism for organization. The virions were replicated in specific pathogen free(SPF) chicken embryo and total RNA were extracted from allantoic fluid of chick embryo. Using usual primers of avian influenza virus, full-length cDNAs of eight genes of isolate Qinghai/2007(H5N1) were amplified by RT-PCR, cloned into vector pMD18-T and sequenced. Compared with published sequences of avian influenza viruses on Genbank, we identified this isolate as H5N1 subtype avian influenza virus and that all eight cDNA fragments included the complete open reading frame. Sequences of 8 genes of this isolate were analyzed using DNAstar software together with a total of 11 strains avian influenza virus. The homology of HA gene between this isolate and strain Shandong/2003, which belonges to Eurasian 1ineage and was in the same evolution branch with this isolate, was 98.6%, while it was 86% between this isolate and strain Ireland/1983(H5N8) in America in 2004, which located in a different evolution branch. It suggested that this isolate originated from Eurasian lineage. The amino acid sequence of the cleavage site on HA is PQRERRRKKR, with more than 5 basic amino acids, which shows that the strain is high pathogenic influenza virus. Homologies of the other seven genes with Shandong/03 were all above 95.7%, implied that these two isolates have very intimate relationship. Therefore, we suppose that this isolate possibly came from Shandong/03. |
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