摘要
目的调查和监测云南省库蠓中携带的新发或未被重视的虫媒病毒,为人和动物健康保障提供依据。方法于2021年7月在云南勐腊某猪舍采用紫外诱捕灯采集库蠓,低温环境中带回实验室。约200只库蠓分装到1个离心管,研磨后取上清接种到C6/36细胞,连续盲传数代;出现细胞病变效应(cytopathic effect,CPE)细胞,通过RNAiso Plus提取总RNA,用1%琼脂糖凝胶进行电泳;根据电泳带型,选用病毒特异引物,通过qRT-PCR检测鉴定阳性分离物种类。测定病毒种类后,设计引物扩增病毒的基因组,测序所得序列用Lasergene SeqMan拼接,通过NCBI的开放阅读框(open reading frame,ORF)预测工具分析编码区,从GenBank下载基因序列并通过MEGA 6.0构建系统发育树,分析遗传进化关系。结果1株阳性分离物(ML38-1)含Oya病毒(Oya virus,OYAV);拼接得到的基因组序列含3个片段,S片段长986 nt,编码233 aa;M片段长4481 nt,编码1433 aa;L片段长6887 nt,编码2261 aa。毒株ML38-1与2013年云南师宗采集库蠓中分离的OYAV(SZC50)同源性最高,S、M、L片段的相似性为99.8%~99.9%。系统发育树上,ML38-1的S片段与云南师宗库蠓、中国蚊子和越南库蚊中分离的OYAV(序列号:OP672242.1、JX983192.1、MH484317.1)、越南库蚊中分离的Cat Que virus(序列号:NC 024075.1)处于同一进化分支;M片段与云南师宗库蠓和中国蚊子中分离的OYAV(序列号:OP672243.1、JX983193.1)处于同一进化分支;L片段与中国分离的OYAV(序列号:P672244.1、JX983194.1、PQ463773.1、MK609491.1)和Cat Que病毒(序列号:OP129710.1)处于同一进化分支。结论从2021年在云南勐腊猪舍采集的库蠓中分离到1株OYAV,该毒株与云南师宗库蠓中分离的OYAV高度同源。OYAV在云南省的库蠓中存在扩散,应持续加强监测。
Objective To investigate newly identify or overlooked arboviruses carried by Culicoides in Yunnan Province,providing a basis for human and animal health protection.Methods In July 2021,Culicoides were collected using UV light traps at a pigsty in Mengla,Yunnan and taken back to the laboratory in a low-temperature environment.Every 200 Culicoides was packed into one centrifuge tube,homogenized and the supernatants were inoculated into C6/36 cells to passage several generations for CPE.Total RNA of cells with CPE was extracted using TaKaRa RNAiso Plus,and electrophoresis was performed on 1%agarose gel.According to the electrophoresis band type,the isolates was identified by qRT-PCR using specific primers,and the type of positive isolates was identified.After determining the type of virus,primers were designed to amplify the viral genome.The obtained sequence was concatenated using Lasergene SeqMan,and the open reading frame was analyzed using NCBI ORF Finder.The gene sequence was downloaded from GenBank and a phylogenetic tree was constructed using MEGA 6.0 to analyze genetic evolutionary relationships.Results One isolate(ML38-1)was found to contain Oya virus(OYAV).The genome obtained sequence contains three fragments:the length is 986 nt for S fragment encoding 233 aa,4481 nt for M fragment encoding 1433 aa,and 6887 nt for L fragment encoding 2261 aa.The strain of ML38-1 share the highest homology with OYAV(SZC50)isolated from Culicoides collected in Shizong,Yunnan in 2013,with similarities of 99.8%to 99.9%among the S,M,and L fragments.On the phylogenetic tree,the S fragment of ML38-1 belongs to the same evolutionary branch as OYAV isolated from Culicoides collected in Shizong,Yunnan,mosquitoes collected in China,and Culex spp.collected in Vietnam(GenBank accession numbers:OP672242.1,JX983192.1,MH484317.1),as well as Cat Que virus isolated from Culex collected in Vietnam(GenBank accession number:NC 024075.1);The M fragment belongs to the same evolutionary branch as OYAV isolated from Culicoides collected in Shizong,Yunnan and mosquitoes collected in China(GenBank accession numbers:OP672243.1,JX983192.1);The L fragment belongs to the same evolutionary branch as the Oya virus(GenBank accession numbers:P672244.1,JX983194.1,PQ463773.1,MK609491.1)and Cat Que virus(GenBank accession number:OP12970.1)isolated from China.Conclusion One strain of OYAV was isolated from Culicoides collected in Mengla,Yunnan in 2021,which is highly homologous to the OYAV isolated from Culicoides in Shizong,Yunnan.OYAV has spread among Culicoides in Yunnan Province,and monitoring should be continuously strengthened.
作者
李苏胜
何于雯
杨振兴
孟锦昕
李楠
王静林
LI Susheng;HE Yuwen;YANG Zhenxing;MENG Jinxin;LI Nan;WANG Jinglin(Yunnan Tropical and Subtropical Animal Viral Disease Laboratory,Key Laboratory of Transboundary Animal Diseases Prevention and Control(Co-construction by Ministry and Province),Yunnan Animal Science and Veterinary Institute,Kunming,Yunnan 650224,China;Yunnan Provincial Key Laboratory of Public Health and Biosafety&Yunnan Key Laboratory of Cross-Border Infectious Disease Prevention and New Drug Development,School of Public Health,Kunming Medical University,Kunming,Yunnan 650500,China)
出处
《中国热带医学》
北大核心
2025年第8期951-957,共7页
China Tropical Medicine
基金
国家重点研发计划项目(No.2022YFC2601603,No.2024YFD1800101)
国家自然科学基金项目(No.32260896)。
关键词
库蠓
Oya病毒
分离与鉴定
基因序列分析
Culicoides
Oya virus
isolation and identification
gene sequences analysis
