2012年-2013年云南边境禽流感病毒 H5N1亚型血凝素基因分析

Molecular Analysis of Hemagglutinin Genes of Avian Influenza Virus H5N1 Subtype in the Border of Yunnan Province During 2012 to 2013

为了解2012年-2013年云南边境禽流感病毒 H5N1亚型血凝素（HA）基因变异及进化特征，在云南边境采集境外家禽和野生鸟类棉拭子样品1500份，经禽流感病毒 H5N1亚型特异性多重 RT-PCR 检测，选择代表性阳性样品，进行病毒 HA 基因扩增、纯化、克隆和测序，并与已知毒株和参考毒株序列进行序列比对及系统遗传进化分析。结果自1500份样品中检出 H5N1亚型阳性样品60份，60份阳性样品病毒 HA基因测序获得23种 HA 序列，可划分为2个不同进化分支（2．3．2，2．3．4），2．3．2进一步可划分为3个进化小分支（Ⅱ-1～Ⅱ-3），在23份代表性阳性样品中，17份样品属于进化小分支2．3．2Ⅱ-1，3份属于2．3．2Ⅱ-2，其余3份属于2．3．4；阳性样品中病毒 HA 裂解位点序列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、表位关键性氨基酸位点及其他氨基酸位点存在变异，部分位点的变异具有分支或亚（小）分支特异性。2012年-2013年期间云南边境禽流感病毒 H5N1亚型间具有遗传差异，进化分支2．3．2Ⅱ-1成为当地流行的优势毒株。

To elucidate the molecular variation and evolution characteristecs of the hemagglutinin （HA） genes of avian influenza virus H5N1 subtype in the border of Yunnan province during 2012 to 2013,swab samples were collected from foreign poultry and wild birds in the region and screened by H5N1 subtype-specific multiplex RT-PCR.The HA genes of H5N1 viruses from the positive samples were amplified,pu-rified,and cloned into pMD18-T vectors for sequencing.The alignment and phylogenetic analysis were performed with sequences of the known reference strains.60 positive samples were found out form 1 500 samples.23 different HA sequences were obtained from the 60 positive samples and can be divided into 2 distinct clades （2.3.2 ,2.3.4）,and the clade 2.3.2 can be further divided into 3 smaller clades （Ⅱ-1~Ⅱ-3）by phylogenetic analysis.Among 23 representative samples,17,3 and 3 belonge to clad or sub-clade 2.3.2Ⅱ-1,2.3.2Ⅱ-2 and 2.3.4 respectivly.The cleavage site of HA from positive samples were all pos-sessed molecular characteristics of highly pathogenic avian influenza virus.The mutation of key amino acids was found among receptor binding sites,potential glycosylation sites,neutralizing epitopes and oth-ers.Some mutation sites show calde or sub-clade specific.H5N1 viruses from the border of Yunnan prov-ince during 2012 to 2013 have genetic divergence,and clade 2.3.2 Ⅱ-1 viral strains become the dominant strains in the region.