云南江城蓝舌病病毒16型毒株分离鉴定及其L2基因序列分析

doi:10.16431/j.cnki.1671-7236.2018.11.026

摘要/Abstract

摘要：

为了解近年来云南江城县蓝舌病病毒16型（Bluetongue virus type 16，BTV-16）毒株的流行情况及其L2基因与国外流行株的遗传进化关系，本研究将江城县送检的300份牛肝素钠抗凝血提取红细胞后静脉接种10日龄鸡胚，将收集的鸡胚肝脏捣碎离心，上清液接种于C6/36和BHK21细胞传代。针对出现细胞病变的样品，应用群特异性VP7片段引物进行RT-PCR检测，应用BTV-16 L2基因特异性引物对检测出的BTV核酸阳性样品进行RT-PCR扩增和测序，采用DNAStar和Mega 6.0软件对获得的L2基因编码区序列进行核苷酸、氨基酸同源性比对及遗传进化分析，同时利用BTV-16标准阳性血清对分离到的病毒进行中和试验鉴定。结果显示，江城县发现30个可致细胞病变的样品，其中17个样品经RT-PCR初步确认为BTV；经L2基因序列分析和中和试验鉴定，确定其中6株为BTV-16型毒株；核苷酸、氨基酸同源性比对分析结果显示，6个毒株核苷酸和氨基酸同源性分别在93.4%~98.0%和94.2%~99.1%之间；遗传进化分析发现，其中5株与2001—2008年日本及1982—2011年印度分离的BTV-16毒株亲缘关系较近；1株与1985—1990年日本分离的BTV-16毒株亲缘关系较近。本研究发现，云南江城县BTV-16毒株呈现新旧毒株交叉持续流行态势，但在自然进化中遗传变异不大，有一定的稳定性，本研究在分子水平阐明了云南江城县地方流行BTV-16 L2基因间的遗传和差异，为进一步开展BTV分子流行病学及检测研究提供科学依据。

关键词: 蓝舌病病毒(BTV); 病毒分离; L2基因; 序列分析

Abstract:

In order to understand the prevalence of Bluetongue virus type 16 (BTV-16) strains from Jiangcheng county in Yunnan province in recent years,and the genetic evolution relationship between L2 gene and foreign strains,in this study,300 bovine heparin sodium anticoagulated bloods were taken from Jiangcheng county for extraction of red blood cells,and 10 days old chicken embryos were inoculated intravenously.The collected chicken embryo livers were crushed and centrifuged,and the supernatant was inoculated into C6/36 and BHK21 cells for passage.For the cytopathic samples,the group-specific VP7 fragment primers were used for RT-PCR detection.The BTV-16 L2 gene-specific primers were used to perform RT-PCR amplification and sequencing of the BTV nucleic acid positive samples.The nucleotide and amino acid homology alignments and genetic evolution analysis of the obtained L2 gene coding region sequences were performed using DNAStar and Mega 6.0 softwares.At the same time,the isolated virus was neutralized by BTV-16 standard positive serum.The results showed that 30 cytopathic samples were found in Jiangcheng county,17 of which were initially identified as BTV by RT-PCR;6 of them were identified as BTV-16 strains by L2 gene sequence analysis and neutralization test;The nucleotide and amino acid homology alignment analysis showed that the nucleotide and amino acid homology of the 6 strains were 93.4% to 98.0% and 94.2% to 99.1%,respectively;Genetic evolution analysis found that 5 of them were closely related to the BTV-16 strains isolated in Japan from 2001 to 2008 and in India from 1982 to 2011,one strain was closely related to the BTV-16 strain isolated from Japan in 1985 to 1990.This study found that the BTV-16 strain in Jiangcheng county of Yunnan province showed a cross-continuous epidemic of new and old strains,but the genetic variation in natural evolution was small and had certain stability.This study at the molecular level clarified the local popular BTV from Jiangcheng county in Yunnan province.The genetics and differences between the BTV-16 L2 gene provided a scientific basis for further research of BTV molecular epidemiology and detection.

Key words: Bluetongue virus (BTV); virus isolation; L2 gene; sequence analysis

中图分类号:

S852.65^＋2

寇美玲, 苗海生, 李乐, 李楠, 廖德芳, 李华春. 云南江城蓝舌病病毒16型毒株分离鉴定及其L2基因序列分析[J]. 《中国畜牧兽医》, 2018, 45(11): 3192-3202.

KOU Meiling, MIAO Haisheng, LI Le, LI Nan, LIAO Defang, LI Huachun. Isolation and Identification of Bluetongue Virus Type 16 Strain from Jiangcheng County in Yunnan Province and Sequence Analysis of Its L2 Gene[J]. , 2018, 45(11): 3192-3202.

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