2014年长沙市活禽市场污水中H5N1亚型禽流感病毒HA、NA及NS基因进化分析

Evolution of the HA,NA and NS genes of H5N1 avian influenza viruses from sewage in live bird markets in Changsha,2014

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。

The aim of this study is to analyze the evolutional and molecular characteristics of Hemagglutinin （HA）, Neura- minidase（NA）and non-structural （NS） genes of H5N1 avian influenza virus （AIV） from sewage in live bird markets （LBMs） in Changsha, 2014. Five hundred and one specimens were collected from environment in LBMs in Changsha, 2014,and real-time RT-PCR was used for influenza A typing and subtyping （HS, H7 and H9） detection. Sequencing were used for the positive of single H5. The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Align- ment Search Tool （BLAST）. The phylogenetic trees for HA,NA and NS genes and the ClustalW Multiple alignments of amino acids were constructed using MEGA 5 and BioEdit software, respectively. Results showed that of 501 environmental samples, 177 （35.33%） samples were positive for influenza A viruses and H5 subtype. Eight H5N1 subtype AIV were confirmed by se- quencing from the samples of the positive of single H5. Phylogenetic analysis indicated that most of HA genes of the HSN1 subtype AIV strains isolated in Changsha city were located in 2.3.2 and clustered into new subclade, and the most of NA and NS genes in this study were clustered into subclade 2.3.2. 1b. QSG of the HA protein of the receptor binding site were found in these H5N1 viruses, and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of AIV. In strains from this study, we did not found amino acid substitutions of the NA protein at H275Y and N295S, and sensitive to neuraminidases, and the high pathogenicity molecular characteristics of HA, NA and NS genes were showed in these viruses. In conclusion, molecular characteristics of the HA, NA and NS of these H5N1 subtype viruses in this studyshowed high pathogenicity, but that may not facilitate human infection. So, the prevalence and genetic evolution of this virus should be closely monitored.