2014年云南地区禽流感H5N1亚型病毒全基因进化及分子结构特征分析

Evolutionary and molecular characteristics of full-length genes of H5N1 subtype avian influenza virus from Yunnan region in 2014

2014年3月云南地区发生H5N1亚型禽流感疫情。为了解引起该疫情的病毒全基因变异特征及遗传进化关系，我们在云南通海、安宁、大理采集死亡鸡喉气管40份，经H5N1亚型禽流感病毒（AIV）特异性多重RT-PCR检测。从检测结果呈阳性的26份样品中每个地区挑选2份病毒阳性样品进行全基因测序分析，结果显示6株AIVs均属于2．3．4．4进化小分支。同一病毒株不同基因节段呈现不同进化关系。蛋白序列的变化主要为：HA的受体结合位点和表位关键性氨基酸位点存在变异，新增加2个糖基化位点；NA缺失4个糖基化位点；PB2的aa627为谷氨酸（E）；NS在aa80-aa84发生缺失；PB1、PA、NP、M关键性氨基酸位点均存在部分替代或突变。本研究测序的6个H5N1亚型AIVs与之前的AIVs间具有遗传差异，这种进化小分支2．3．4．4的AIVs已成为当地流行的优势病毒株。

To investigate the variation and evolution characterizations of the H5N1 subtype avian influenza virus in Yunnan area, a total of the clinical samples were collected from dead chicken in Tonghai, Arming, Dali of Yunnan region during the prevalence of the highly pathogenic avian influenza in chickens in March 2014, and the viruses were detected by H5/N1 subtype-specific multiplex RT-PCR. The full-length genes of 6 H5N1 subtype A1Vs from the positive samples were amplified and sequenced. The alignment and phylogenetic analysis showed that the 6 full-length sequences of the H5N1 subtype AIV were from the positive samples were belonged to Clade 2.3.4.4. The different genes of same isolate presented different evolutionary relationships. The mutation of key amino acids were found among receptor binding sites and the epitopics in HA, which resulted in two new additional potential glycosylation sites lossing 4 potential glycosylation sites in NA. The aa627 in PB2 still was Glu. However, the aa80-aa84 were deleted in NS and same substitutions and inserts/deletions of amino acids were also found in PB 1, PA, NP and M. It was evident that H5N1 subtype AIVs showed genetic divergence with the heretofore AIVs and the virus of Clade 2.3.4.4 had become dominant epidemic strains in the region.