山东省甲型H1N1流感病毒病原学及基因特性研究

Etiological and Molecular Characteristics of Pandemic Influenza A (H1N1) Virus during 2009-2010 in Shandong Province

在2009-2010年监测年度开展甲型H1N1流感病毒学监测并进行病原学分离鉴定,以及对血凝素基因（HA）和神经氨酸酶基因（NA）特性分析,研究其基因变异情况。采集了17 126份发热患者的鼻、咽拭子标本,采用逆转录实时荧光定量RT-PCR（Real-Time RT-PCR）进行核酸检测,其中甲型H1N1流感病毒核酸检测阳性4004份,总阳性率为23.38%。对阳性标本开展病毒分离,并对分离的甲型H1N1流感病毒的HA、NA基因序列进行测序。利用DNAStar软件对序列进行同源性分析发现与WHO推荐的疫苗株相比,山东省甲型H1N1流感流行株HA、NA基因同源性分别为96.9%~99.3%和99.1%~99.6%;利用Mega 4.0软件进行基因进化分析和氨基酸进化分析发现,与WHO推荐的疫苗株相比,山东省甲型H1N1流感流行株有21个血凝素基因的氨基酸发生替换,其中11个氨基酸位点位于抗原决定簇区,一个糖基化位点发生改变;有16个神经氨酸酶基因的氨基酸发生了替换,一个糖基化位点发生改变;未发生神经氨酸酶蛋白275位H→Y的替换。结果显示山东省甲型H1N1流感暴发流行株HA基因和NA基因均具有高度同源性,HA蛋白和NA蛋白均存在不同程度的氨基酸替换,部分流行株抗原决定簇和糖基化位点发生改变,所有病毒均对达菲类药物敏感。

To isolate and identify the influenza virus in Shandong Province in 2009-2010 and analyze the genetic characteristics of hemagglutinin and neuraminidase gene,further study the variation of gene.A total of 17 126 nasopharyngeal swabs from fever patients were collected and detected by real time quantitative RT-PCR method.The results showed 4004 samples were pandemic influenza A（H1N1） virus positive,with an overall positive rate as 23.38%.The positive samples were incubated and cultured in MDCK cells.The HA and NA genes of isolated pandemic influenza A（H1N1） virus were sequenced,the homology analysis of the HA and NA genes showed an average of 96.9%~99.3% and 99.1%~99.6% sequence identity,respectively,compared with WHO-recommended vaccine strain.The genetic evolution and amino acid substitutions were performed with Mega 4.0 Software.Twenty one amino acids were changed in HA protein,of which 11 were located in the antigenic site;Sixteen amino acids were changed in NA protein,which didn＇t lead to the changes of enzyme sites.Furthermore,one glycosylation site of HA protein and NA protein were changed respectively.No H275Y mutation in NA protein was found.The results showed that the HA and NA genes of the epidemic strains were highly homologous,some mutations in the HA and NA proteins were found,the antigenic site and glycosylation site of some strains were changed during the epidemic process.All the strains were sensitive to oseltamivir.