四川省Nam Dinh病毒的首次分离鉴定

Isolation and identification of Nam Dinh virus in Sichuan

目的对2015年从四川省西部的甘孜藏族自治州蚊虫中分离到的病毒株SC1502、SC1510进行鉴定并分析其基因特征。方法通过细胞培养的方法对蚊虫标本进行病毒分离,利用Nam Dinh病毒(Nam Dinh virus,NDiV)特异性引物进行逆转录聚合酶链反应(reverse transcription PCR,RT-PCR)鉴定,同时扩增分离株的开放阅读框、RNA依赖的RNA聚合酶(RNA-dependent RNA-polymerase,RdRp)、解旋酶超家族1(superfamily 1 helicase,HEL1)、刺突蛋白(spike protein,S蛋白)基因序列并测序,然后利用Mega Align软件对核苷酸序列和推测的氨基酸序列进行比对分析,利用Mega 7软件绘制系统进化树。结果SC1502、SC1510病毒株能够引起C6/36细胞发生病变,但不能引起BHK-21细胞的病变。同源性分析结果显示,2株四川新分离株与NDiV的RdRp、HEL1、S蛋白的核苷酸和氨基酸同源性最高,分别超过99.4%和97.5%。系统进化分析结果表明,2株四川新分离株与NDiV亲缘关系最近,处于同一进化分支。结论SC1502与SC1510为NDiV,该研究结果丰富了四川省的虫媒病毒病原谱数据库,对相关地区的不明原因疫情分析和调查提供了参考依据。

Objective To understand the genetic characteristics of Nam Dinh virus(NDiV)SC1502 and SC1510 strains which were isolated from mosquitos in Ganzi Tibetan autonomous prefecture in Western Sichuan province in 2015.Methods The virus was isolated from mosquito samples by cell culture,NDiV specific primers were used for the identification of the virus using reserve transcriptase polymerase chain reaction(RT-PCR).The open reading frame,RNA-dependent RNA-polymerase(RdRp)gene,HEL1(superfamily 1 helicase)gene and S gene of the isolates were amplified and sequenced.Mega Align and Mega 7 software were used to analyze the nucleotide and deduced amino acid sequence,and Mega 7 software was used to draw phylogenetic trees.Results SC1502 and SC1510 isolates could grow well in cultured C6/36 cells but not in BHK-21 cells.Sequence alignment showed that the nucleotide and amino acid sequence of RdRp,HEL1 and S gene of SC1502 and SC1510 had 99.4%and 97.5%similarity with that of NDiV.Phylogenetic analysis showed that the two new isolates SC1502 and SC1510 in Sichuan province were very close to NDiV and were in the same evolutionary branch with NDiV.Conclusions SC1502 and SC1510 isolates are NDiV.The results of this study have enriched the pathogen database of arboviruses in Sichuan,and provided a reference for the analysis and investigation of unknown outbreaks in related areas.