2009年山东省四起甲型H1N1流行性感冒样病例暴发疫情病原学分析

目的 通过对2009年山东省济宁市4起流行性感冒(简称流感)样病例暴发疫情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.方法 采集4起流感样病例暴发疫情中发热患者的鼻咽拭子标本34份,采用逆转录实时PCR(realtime RT-PCR)方法进行核酸检测,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,利用Mega 4.0软件进行基因进化分析和氨基酸进化分析.与WHO推荐的疫苗株及国内代表株进行对比.结果 在34份鼻咽拭子标本中,17份甲型H1N1流感病毒阳性,11份标本分离培养出了甲型H1N1流感病毒.将其中的7株进行HA基因和NA基因测序,HA、NA基因的同源性分别为98.4%～99.6%、99.2%～100.0%.与WHO推荐的疫苗株及国内代表株相比,有11个HA基因的氨基酸发生替换,分别为38、40、56、90、100、145、172、173、220、303及338位,其中38、40和303位位于抗原决定簇C区,172和173位位于抗原决定簇D区,56位位于抗原决定簇E区,同时40位为糖基化位点;有7个NA基因的氨基酸发生了替换,为80、106、241、248、351、369和386位,386位为糖基化位点;未发生神经氨酸酶蛋白275位H-Y的替换.结论 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.

Analysis of etiology of four pandemic influenza A (H1N1) virus outbreaks in Shandong province, in 2009

Objective To isolate and identify the influenza virus that caused four influenza-likeillness outbreaks in Jining city of Shandong Province in 2009 and analyze the genetic characteristics of hemagglutinin (HA) and neuraminidase (NA) gene,the variation of these genes were studied. Methods 34 nasopharyngeal swabs from fever patients of four influenza-like-illness outbreaks were collected and diagnosed by real time quantitative RT-PCR method. The positive samples were incubated and cultured for virus. HA and NA genes of isolated pandemic influenza A (H1N1) virus were sequenced, the homology analysis was done with DNAStar software and the genetic evolution and amino acid substiutions were performed with Mega 4. 0 software. The squences were compared with WHO recommended vaccine virus,native reference virus. Results Seventeen of 34 nasopharyngeal swabs were positive, 11 pandemic influenza A (H1N1) viruses were isolated and HA and NA genes of 7 strains were sequenced. Phylogenetic analysis for hemagglutinin and neuraminidase gene of Shandong outbreak strains showed that there were 98. 4% -99. 6% and 99. 2% - 100. 0% sequence identity. Compared with WHO-recommended vaccine strain, the reference virus in mainland China strain, eleven amino acids were changed for HA protein, including position 38,40,56,90,100,145,172,173,220,303 and 338, and 38,40,303 of HA protein were located in the antigenic determination C cluster, 172,173 in the D cluster,56 in the E cluster, site 40 of HA protein were glycosylated. In NA protein, seven amino acids were changed, including position 80,106,241,248,351,369and 386,site 40 of NA protein were glycosylated. No mutations of 275 in NA protein were found. Conclusion The HA and NA genes of the epidemic strains showed high homology, some mutstions in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process.