2015-2016年深圳市登革Ⅲ、Ⅳ型分离病毒株E/NS1基因序列特征

Isolation and identification of dengue type Ⅲ and type Ⅳ viruses and sequence analysis of E/NS1 gene in Shenzhen City during 2015 and 2016

目的了解2015-2016年深圳市分离的登革Ⅲ、Ⅳ型病毒E/NS1基因序列特征及登革Ⅲ、Ⅳ型病毒流行规律,探究其可能的传播来源。方法收集2015-2016年深圳市登革热患者病例资料及急性期血清,用C6/36细胞培养分离登革病毒,用FQ-PCR对其进行血清分型,分离成功的病毒株用反转录-聚合酶链反应(PCR)方法扩增E基因和NS1基因,进行序列分析,绘制系统进化树,氨基酸比对分析4个不同血清型NS1蛋白。结果从5份Ⅲ型的登革病毒标本中成功分离4份病毒株,3份Ⅳ型登革病毒标本中成功分离2份登革病毒;BLAST分析保守E基因结果表明,与DENV-3分离株同源性最高(99%)的毒株主要是印度尼西亚2010和2015年分离株、菲律宾2015年分离株。与DENV-4分离株同源性最高(99%)的毒株主要是菲律宾2013年分离株、印度尼西亚2010分离株、意大利2009年分离株。NS1基因序列分析显示所选4株不同血清型的病毒株氨基酸相似性为77.69%,4个不同血清型的登革热NS1抗原存在5-7个氨基酸保守区域。结论2015-2016年深圳市输入的登革Ⅲ、Ⅳ型病毒可能来自印度尼西亚和菲律宾,应加强出入境人员的监测工作,避免输入引起本地感染的风险。

We aimed to determine the envelope（E） gene sequence and the NS1 gene sequence of type Ⅲ and Ⅳ dengue virus（DENV3-4）isolated in Shenzhen during 2015 and 2016,and to understand the molecular virology characteristics of these strains as well as to explore their possible origin. The medical record of dengue fever patients and acute phase serum in Shenzhen during 2015 and 2016 were collected. The dengue virus was isolated and cultured by use C6 / 36 cells. Serum was obtained by FQ-PCR. The E gene and NS1 gene were amplified by reverse transcriptase-polymerase chain reaction （PCR） and draw the phylogenetic tree for sequence analysis. Amino acids sequence of NS1 proteins were used to analyze the common conservative regions of four different serotype. There were four strains of type 3 dengue virus were successfully isolated, and two strains of type 4 dengue virus were successfully isolated. The results of E gene BLAST analysis showed that strains with the highest homology （99%） to DENV-3 isolates were mainly isolates from Indonesia in 2010 and 2015 and from Philippines in 2015. Strains with the highest homology （99%） to DENV-4 isolates were the Philippines 2013 isolate, Indonesia 2010 isolate and Italy 2009 isolates. The genetic sequence analysis of NS1 gene showed that the amino acid similarity of the four virus strains with different serotype was 77.69%. Four different serotype dengue NS1 antigens had 5 to 7 amino acid conservative areas. Shenzhen imported type of dengue Ⅲ, Ⅳ dengue virus may come from Indonesia and the Philippines in 2015-2016. Entry-exit personnel should step up their monitoring efforts to avoid the risk of entering local-caused infections.