库蠓中盖塔病毒的分离及其分子鉴定

Isolation and identification of Getah virus from Culicoides in Yunnan

目的 探讨1株（SZC30）云南省库蠓分离病毒的分类及分子特征.方法 2013年7月采用灯诱的方法在云南省曲靖市师宗县五龙乡采集库蠓,采用BHK-21和C6/36细胞进行病毒分离,阳性分离物脑内接种1日龄乳鼠;分别用甲病毒属和盖塔病毒特异性引物对阳性分离物进行RT-PCR扩增、测序,用Clustal X1.83、BioEdit、DNAStar、Mega5.1等生物信息学软件进行序列分析.结果 采集到库蠓共3 500只,库蠓分为41批进行病毒分离,其中分离到一株病毒（SZC30）对BHK-21与C6/36细胞产生明显病变;分别用甲病毒属和盖塔病毒NS1特异性引物进行RT-P C R扩增结果为阳性;NS1基因序列分析结果显示SZC30株病毒与YN0540及SC1210两株中国分离的盖塔病毒位于同一进化分支内,核苷酸同源性在97％～100％之间,氨基酸同源性在97％ ～100％之间.结论 从云南省采集库蠓中分离到的SZC30株病毒为盖塔病毒.

Objective To discuss the taxonomy and molecular characteristics of one virus strain （SZC30） isolated from Culicoides in Yunnan.Methods Culicoides were collected with light trap method in the Wulong Village of Shizong County of Yunnan Province in July,2013.BHK-21 and C6/36 cells were used for virus isolation.The positive isolates were inoculated into brain of one-day suckling mice.Alphavirus and Getah virus specific primers were used to amplify the genome of the virus isolation by RT-PCR.The products of RT-PCR were sequenced.Clustal X1.83,DNAStar,Mega5.1 were used for bioinformatics analysis.Results Totally 3 500 culicoides were collected and divided into 41 batches for virus isolation.One isolate （SZC30） produced cytopathic effect （CPE） on BHK-21 and C6/36 cells;the result of RT-PCR with Alphavirus and Getah virus NS1 specific primer were positive;the sequence analysis of NS1 gene suggested that SZC30 and two Getah virus strains （YN0540,SC1210） from China were in the same evolutionary branching,the nucleotide homology were 97%-100%,and the amino acid was 97%-100%.Conclusions SZC30 isolated in Yunnan province was identified as Getah virus.