摘要目的 了解云南省边境地区禽流感H5N1亚型病毒遗传多样性.方法 2009-2011年7月在云南省边境地区采集境外家禽和野生鸟类棉拭子样品,经H5/Nl亚型特异性多重RT-PCR检测,阳性样品进行病毒HA基因扩增,克隆至pMD 18-T载体测序,并与已知参考毒株进行序列比对及系统发育分析.结果 36份阳性样品病毒HA基因测序获得15种HA序列,存在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),2.3.4进一步可划分为2个进化小分支(Ⅰ-1和Ⅰ-2).2.3.2Ⅱ-1、Ⅱ-2毒株是新出现的H5N1亚型病毒变异株.结论 2009- 2011年7月云南省边境地区H5N1亚型病毒具有遗传多样性,病毒经历了多分支(2.3.2、2.3.4)至单一支(2.3.2)的进化过程.

abstractsObjective To elucidate the genetic diversifications of avian influenza subtype H5N1 viruses in the boundary regions of Yunnan province during 2009 to July,2011.Methods Swab samples were collected from foreign poultry and wild birds in boundary regions of Yunnan province during 2009 to July,2011 and tested by H5/N1 subtype-specific multiplex RT-PCR.The HA genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD 18-T vectors for sequencing.Both alignment and phylogenetic analysis were performed with sequences of the known reference strains.Results Fifteen different HA sequences were obtained from 36 representative positive samples and could be divided into 2 distinct Clades (2.3.2 and 2.3.4).Through phylogenetic analysis,Clade 2.3.2 and 2.3.4 could then be further divided into 3 ( Ⅱ -1 to Ⅱ -3 ) and 2 smaller clades ( Ⅰ -1 and Ⅰ -2),respectively.The viruses of Clade 2.3.2 Ⅱ -1 and Ⅱ-2 were new variant strains of H5N 1 virus.The cleavage sites of HA from positive samples all possessed molecular characterization of highly pathogenic avian influenza virus.Mutation of key amino acids had been found among receptor binding sites,potential glycosylation sites,neutralizing epitopes and others.Conclusion It seemed evident that the H5N1 subtype viruses showed genetic diversifications and had undergone the evolution progress of multi-clade (2.3.2,2.3.4) to single caide (2.3.2) in the boundary regions of Yunnan province,during 2009 to July,2011.