云南边境禽流感H5N1亚型病毒血凝素基因分子结构特征分析

Molecular characteristics of the hemagglutinin genes of avian influenza virus subtype H5N1 in the boundary region of Yunnan province

目的分析2003-2008年云南边境禽流感H5N1亚型病毒血凝素(HA)基因分子结构特征。方法对2003-2008年在云南边境采集境外禽类样品420份,做H5/N1亚型特异性RT-PCR及多重RT-PCR检测,对阳性样品中H5N1病毒HA基因进行RT-PCR扩增,克隆至pMD18-T载体测序,并与国内外已知参考毒株序列进行比对及系统发育分析。结果21份代表性病毒样品HA裂解位点序列存在4种不同排列方式,均具有高致病性禽流感病毒分子结构特征;HA受体结合位点、糖基化位点及表位关键性氨基酸位点存在变异;系统发育分析表明可划分为5个不同进化分枝(1、2.4、2.3.2、2.3.4、7)。结论2003-2008年云南边境外H5N1病毒间具有遗传差异,进化分枝2.3.4毒株为当地流行的优势毒株,不同进化分枝或同一进化分枝不同毒株HA基因存在变异。

To elucidate the molecular characteristics of the hemagglutinin （HA） genes of HSN1 subtype of avian influenza viruses in the boundary region of Yunnan province. Of 420 samples were collected from the foreign poultry in boundary re gion of Yunnan province during 2003 to 2008 and these samples were subjected to screening by H5/N1 subtype-specific and multiplex RT PCR. testing. The HA genes of HSN1 viruses from positive samples were amplified by RT PCR and cloned into vector pMD18 T for subsequent sequencing. The alignment with sequences of the known reference strains and phylogenetic analysis were then performed. The genes from 21 representative positive samples with 4 different sequences at the cleavage site were obtained and all of them possessed the molecular characteristic of highly pathogenic avian influenza virus. The mutation of key amino acids had been found among receptor-binding sites, potential glycosylation sites and neutralizing epitopes. Phylogenetic analysis showed those positive samples could be divided into 5 distinct clades, including clade 1, 2.4. 2.3.2, 2.3.4 and 7. It is evident that HSN1 viruses from the foreign boundary region of Yunnan province in 2003 to 2008 show genetic divergence and clade 2,3,4 is the dominant clade in this region.