H9亚型禽流感病毒钦州株HA基因的克隆与遗传进化

采集钦州活禽交易市场的鸡气管和泄殖腔的棉拭予样品，用H9亚型分型引物进行PCR初步筛选，阳性样品经SPF鸡胚尿囊腔接种分离病毒，通过RT-PCR方法扩增HA基因，并将其克隆到pMD—18T载体后进行序列测定和分析。结果表明，获得1株H9亚型禽流感病毒命名为：A／Chicken／Guangxi／qz40／2009（简称qz40）。测序结果表明qz40的HA基因片段全长1683bp，编码560个氨基酸；序列同源性比较结果表明，该毒株与参考毒株的核苷酸序列同源性为82．8％．99．9％，推导氨基酸同源性为87．5％．99．6％；HA基因的裂解位点氨基酸顺序为RSSRIGLF，为低致病性毒株；含有7个潜在的N-糖基化位点，其中5个位于HAl部分、2个位于HA2部分；基于H9亚型HA基因的进化树分析表明，qz40株属于欧亚种系的A／Chicken／Beijing／1／94（Ck／Bei—like）群系。

Cloning and phylogenetic analysis of HA gene of an avian influenza virus H9 subtype isolate from Qinzhou of Guangxi

In order to explore the epidemic rule of H9 subtype avian influenza virus in Qinzhou area, specimens of paired tracheal and cloacal swabs were collected from live poultry markets in Qinzhou and screened by PCR with H9 subtyping primers, then the positive specimens were subjected to virus isolation by using SPF chicken embryos. One influenza A virus was determined as H9 subtype by RT-PCR, named A/Chicken/Guangxi/qz40/2009（qz40 in short）. The cDNA of the qz40 HA gene was amplified by RT-PCR and sequenced. The results showed that the full length of HA gene consisted of 1683 nucleotides（nt） encoding 560 amino acid, nucleotide and amino acid sequence homology of HA gene was 82.8%-99.9% and 87.5%-99.6%, respectively. Genetic analysis revealed that the qz40 had an RSSR ↓GLF motif at the cleavage site of HA, rep- resenting low pathogenicity in chicken. In addition, this strain had 7 potential glycosylation sites. Phylogenetic analysis of the H9 HA gene showed that the qz40 strain was classified into the lineage represented by A/Chicken/Beijing/1/94（Ck/Bei-like）.