The Evolution Of H5N1 Influenza Viruses Among Wild Birds At Qinghai Lake In China

In 2005,highly pathogenic H5N1 influenza viruses firstly caused a massive outbreak of lethal disease among wild birds at Qinghai Lake in Western China. To understand the effect of wild birds during the transmission of the AIV, we sequenced and analysed the entire genome of 15 H5N1 viruses isolated from 6 different avian species at Qinghai Lake in China. Viral RNAs were extracted from infectious allantoic fluid by using LS Trizol RNA extraction kit. RT-PCR for the amplification of all the gene fragments was performed by using fragment-specific primers designed and determined with OLIGO4.0 and Gendoc sequence analysis software. The PCR products were purified with huashun gel extraction purification kit and sequenced by using CEQ DTCS-Quick Start Kit on a CEQ 8000 DNA sequencer. Sequence data were compiled and analyzed by using the sequence analysis software package (DNASTAR). The Megalign program of DNASTAR was used for multiple sequence alignment and generation of phylogenetic trees.The result of the sequence analysis revealed 4 different H5N1 genotypes in this study. In 15 virus, difference were seen in PB2, PA and NS genes less than 95% identity and other genes were similar.All of these viruses had a series of basic amino acids at the HA cleavage site (RRRKKR), characteristic of other influenza viruses highly pathogenic in chickens.They also had a 20-amino-acid deletion in the NA stalk (residues 49-68) as compared with the NA of the Goose/Guangdong/1/96 (GSGD/1/96) virus. In 15 viruses, NA protein had 5 additional glycosylation sites. SNT is at position 70 and 386 is at position SFS in the NA protein.Among these isolates, the sequences of PB2 gene segments were K at position 627 except BHGs/QH/1/05,BHGs/QH/2/05 and RS/QH/I/05.The PB1 genes of 15 viruses divided into 3 subgroups: BHGs/QH/2/05 belonged to one subgroup. Its PB1 gene homology was 96.6%-97.8% and PB1 amino acid homology was 97.8%-99.2% with the test 14 viruses; WS/QH/1/05, BHGs/QH/1/05 and RS/QH/1/05 were in the second subgroup with the same homology in genes and amino acids. The PB1 genes homology of this 3 viruses were 98.5%-99.0% with viruses isolated from wild birds and...