云南省德宏州东方库蠓中西藏环状病毒的分离鉴定

doi:10.11853/j.issn.1003.8280.2022.04.025

中国媒介生物学及控制杂志 ›› 2022, Vol. 33 ›› Issue (4): 580-585.DOI: 10.11853/j.issn.1003.8280.2022.04.025

云南省德宏州东方库蠓中西藏环状病毒的分离鉴定

杨怡铃^1,2, 何于雯¹, 李楠¹, 孟锦昕¹, 殷建忠², 王静林¹

1. 云南省畜牧兽医科学院, 云南省热带亚热带动物病毒病重点实验室, 云南昆明 650224;
2. 昆明医科大学公共卫生学院, 云南昆明 650500

收稿日期:2022-02-26 出版日期:2022-08-20 发布日期:2022-08-12
通讯作者: 王静林,E-mail:wangjl107@163.com
作者简介:杨怡铃,女,在读硕士,主要从事公共卫生研究,E-mail:yylhyj1413@163.com;何于雯,女,硕士,助理研究员,主要从事动物病毒学研究工作,E-mail:heyuwen1117@163.com
基金资助:
云南省科技厅重点基金（2019FA015）；国家自然科学基金（31660714）；云南省程功专家工作站（202005AF150034）；云南省科技人才和平台计划项目（2018HB046）

Isolation and identification of Tibet orbivirus from Culicoides orientalis collected in Dehong prefecture, Yunnan province, China

YANG Yi-ling^1,2, HE Yu-wen¹, LI Nan¹, MENG Jin-xin¹, YIN Jian-zhong², WANG Jing-lin¹

1. Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming, Yunnan 650224, China;
2. School of Public Health, Kunming Medical University, Kunming, Yunnan 650500, China

Received:2022-02-26 Online:2022-08-20 Published:2022-08-12
Supported by:
Basic Research Projects of Yunnan Province (No. 2019FA015); National Natural Science Foundation of China (No. 31660714); Yunnan Chenggong Expert Workstation (No. 202005AF150034); Yunnan Science and Technology Talents and Platform Plan (No. 2018HB046)

摘要/Abstract

摘要： 目的了解云南省德宏傣族景颇族自治州（德宏州）芒市采集库蠓携带西藏环状病毒（TIBOV）情况及其潜在传播媒介，丰富云南边境地区环状病毒的研究。方法 2016年8月在德宏州芒市郊区的牛舍和羊舍用诱蚊灯夜间采集蠓，对采集到的蠓在显微镜下进行初步分类，采用金黄地鼠肾细胞（BHK-21）和白纹伊蚊卵细胞（C6/36）进行病毒分离，阳性分离物分别使用甲病毒属、黄病毒属、TIBOV S7和S10特异引物进行鉴定，对阳性扩增产物测序，采用MEGA Ⅹ等生物信息软件进行序列分析。同时，提取相应库蠓基因组DNA，采用细胞色素C氧化酶亚基Ⅰ（COⅠ）特异引物鉴定库蠓种类。结果将采集到的库蠓分44批研磨，获得1株可引起BHK-21和C6/36细胞病变的毒株（编号：DHC217-11），接种标本研磨的上清液48 h后，BHK-21细胞出现明显病变；接种标本研磨的上清液72 h后，C6/36细胞出现明显病变。甲病毒属、黄病毒属引物扩增均为阴性，TIBOV S7和S10引物扩增均为阳性，测序后分别获得新分离病毒第7节段（S7）1 085 bp和第10节段（S10）723 bp序列。序列分析结果显示，新分离DHC217-11株病毒在S7和S10基因序列构建的遗传进化树上，与6株TIBOV位于同一个进化分支内，它们之间的核苷酸同源性分别为93.4%～99.5%（S7）和81.3%～97.1%（S10），而与蓝舌病毒、鹿流行性出血热病毒等其他环状病毒位于不同进化分支，核苷酸同源性低于62.7%（S7）和58.9%（S10）；进一步分析发现新分离DHC217-11株病毒S7基因序列与2013年芒市库蠓分离TIBOV DH13C120株核苷酸同源性最高，为99.5%，S10基因序列与2017年西双版纳傣族自治州分离的TIBOV SX-2017a株核苷酸同源性最高，为97.1%，提示新分离DHC217-11株病毒为TIBOV。采用库蠓COⅠ基因引物对DHC217-11批库蠓进行分子鉴定，序列分析结果显示DHC217-11批库蠓COⅠ基因序列与东方库蠓遗传进化关系较近，它们之间核苷酸同源性最高为99.8%，提示该批库蠓为东方库蠓。结论从云南省德宏州东方库蠓中分离的DHC217-11为TIBOV，东方库蠓可能是TIBOV潜在的传播媒介。

关键词: 西藏环状病毒, 东方库蠓, 分离, 鉴定, 遗传进化分析

Abstract: Objective To investigate the status of Tibet orbivirus (TIBOV) carried by the collected midges and the potential vectors in Mangshi, Dehong Dai and Jingpo Autonomous Prefecture (Dehong prefecture), Yunnan province, China, and to enrich the studies on Orbivirus in the border areas of Yunnan province. Methods Midges were collected with mosquito light trap at night from cattle and sheep houses in the suburbs of Mangshi, Dehong prefecture in August 2016. The collected midges were preliminarily classified under a microscope. The virus was isolated from golden hamster kidney cells (BHK-21) and Aedes albopictus cells (C6/36). The specific primers for Alphavirus, Flavivirus, TIBOV S7 and S10 were used to preliminarily identify the positive isolates. The positive amplified products were sequenced and analyzed by bioinformatics software like MEGA X. Meanwhile, genomic DNA of midges was extracted, and the specific primers for cytochrome c oxidase subunit I (COⅠ) were used to identify midge species. Results The collected midges were divided into 44 batches for grinding. One positive isolate (serial number:DHC217-11) was obtained, which produced obvious cytopathic effect in BHK-21 and C6/36 cells. BHK-21 cells showed obvious cytopathic effect 48 h after inoculation with the supernatant of ground samples. C6/36 cells showed obvious cytopathic effect 72 h after inoculation. The amplification showed negative results using the primers for Alphavirus and Flavivirus, while showed positive results using the primers for S7 and S10 segments of TIBOVs. The sequences of S7 and S10 segments of newly isolated viruses were 1 085 bp and 723 bp after sequencing. The sequence analysis results showed that the newly isolated virus DHC217-11 was in the same evolutionary branch as the six TIBOV strains on the phylogenetic tree constructed by S7 and S10 gene sequences, with a nucleotide homology of 93.4%-99.5% (S7) and 81.3%-97.1% (S10), respectively, and the newly isolated virus DHC217-11 was in the different evolutionary branch from other orbiviruses such as Bluetongue virus and Epizootic hemorrhagic disease virus, with a nucleotide homology of lower than 62.7% (S7) and 58.9% (S10). A further analysis showed that the S7 gene sequence of the newly isolated virus DHC217-11 was 99.5% homologous to the nucleotide sequence of TIBOV DH13C120 isolated from midges in Mangshi in 2013. S10 gene sequence had the highest nucleotide homology of 97.1% with TIBOV SX2017a isolated from Xishuangbanna Dai Autonomous Prefecture in 2017, suggesting that the newly isolated virus DHC217-11 was TIBOV. The COⅠ gene primers were used for molecular identification of midges in batch DHC217-11. The sequence analysis results showed that the COⅠ gene sequence of midges in batch DHC217-11 was closely related to the genetic evolution of Culicoides orientalis, with the highest nucleotide homology of 99.8%, suggesting that midges in batch DHC217-11 were C. orientalis. Conclusion The virus DHC217-11 isolated from C. orientalis collected in Dehong prefecture of Yunnan province is TIBOV. C. orientalis may be a potential vector of TIBOV.

Key words: Tibet orbivirus, Culicoides orientalis, Isolation, Identification, Phylogenetic analysis

中图分类号:

杨怡铃, 何于雯, 李楠, 孟锦昕, 殷建忠, 王静林. 云南省德宏州东方库蠓中西藏环状病毒的分离鉴定[J]. 中国媒介生物学及控制杂志, 2022, 33(4): 580-585.

YANG Yi-ling, HE Yu-wen, LI Nan, MENG Jin-xin, YIN Jian-zhong, WANG Jing-lin. Isolation and identification of Tibet orbivirus from Culicoides orientalis collected in Dehong prefecture, Yunnan province, China[J]. Chinese Journal of Vector Biology and Control, 2022, 33(4): 580-585.

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云南省德宏州东方库蠓中西藏环状病毒的分离鉴定

Isolation and identification of Tibet orbivirus from Culicoides orientalis collected in Dehong prefecture, Yunnan province, China

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