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目的分析2014年长沙市活禽市场污水中H5N1亚型禽流感病毒(Avian influenza virus,AIV)血凝素(Hemagglutinin,HA)、神经氨酸酶(Neuraminidase,NA)及非结构蛋白(Non-structural,NS)基因进化特征。方法从2014年长沙市活禽市场采集501份环境标本,利用Real-time PCR方法进行A型、H5、H7和H9亚型流感病毒核酸检测,并对单一H5阳性标本进行核苷酸测序,病毒HA、NA和NS基因测序结果进行在线BLAST分析,利用Mega5和Bioedit软件构建核苷酸进化树和氨基酸(Amino acids,aa)比对。结果从501份环境标本中检出A型H5亚型病毒阳性标本177份(检出率35.33%),其中8份标本经核苷酸测序鉴定为H5N1亚型病毒,进化分析表明大部分H5N1病毒HA基因位于2.3.2分支内,聚集形成一个新亚分支,NA及NS基因位于2.3.2.1b亚分支。HA蛋白受体结合位点aa为QSG,表现为禽流感病毒受体特征;NA蛋白未出现H275Y及N295Saa替换,对神经氨酸酶抑制剂敏感;HA、NA及NS蛋白关键分子位点表现为高致病性的分子特征。结论 2014年长沙市活禽市场污水中H5N1亚型禽流感病毒HA、NA和NS基因表现为高致病性的分子特征,但不具有对人易感的受体特征,需要进一步监测。
Objective To analyze the distribution of Hemagglutinin (HA), Neuraminidase (NA) and Non-structural protein (HNN1) of the H5N1 subtype avian influenza virus in live poultry market in 2014 in Changsha City, , NS) gene evolution characteristics. Methods A total of 501 environmental samples were collected from the live poultry market in Changsha City in 2014. Real-time PCR was used to detect influenza A, H5, H7 and H9 subtype influenza viruses. Nucleotide sequencing was performed on single H5 positive samples. BLAST analysis of HA, NA and NS gene sequencing results, using Mega5 and Bioedit software to build nucleotide alignment and amino acid (aa) alignment. Results A total of 177 positive samples of type A H5 subtype (detection rate 35.33%) were detected from 501 environmental samples. Eight of them were identified as H5N1 subtype by nucleotide sequencing. Phylogenetic analysis showed that most of the H5N1 virus The HA gene is located in the 2.3.2 branch, aggregating to form a new sub-branch, and the NA and NS genes are located in the sub-branch 2.3.2.1b. The HA protein receptor binding site aa is QSG, which shows the characteristics of the avian influenza virus receptor. The NA protein does not appear to be replaced by H275Y and N295Saa, and is sensitive to neuraminidase inhibitors. The key molecular sites of HA, NA and NS proteins are Highly pathogenic molecular characteristics. Conclusions HA, NA and NS genes of H5N1 subtype avian influenza virus in live poultry market in Changsha in 2014 show highly pathogenic molecular characteristics but do not have the characteristics of human-susceptible receptors and need further monitoring.