草鱼呼肠孤病毒JX-0902株的分离和鉴定  

Isolation and identification of grass carp reovirus strain JX-0902

作　　者：刘永奎[1,2] 王庆[1] 曾伟伟[1] 石存斌[1] 张超 陈道印 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,广东广州510380 [2]上海海洋大学,上海201306 [3]济宁市渔业监测站,山东济宁272019 [4]南昌县莲塘鱼病防治所,江西南昌330200

出　　处：《中国水产科学》2011年第5期1077-1083,共7页Journal of Fishery Sciences of China

基　　金：农业部公益性行业科研专项(200803013);现代农业产业技术体系建设专项资金(nycytx-49-14)

摘　　要：收集江西南昌地区患典型草鱼出血病的病鱼组织,进行超薄切片电镜观察,结果显示,在脾、肾样品中发现大量病毒颗粒,病毒无囊膜,近球形,直径约70 nm,形态与已报道的草鱼呼肠孤病毒(grass carp reovirus,GCRV)相似。取病鱼肌肉、内脏研磨,组织液经离心、过滤除菌后进行鱼体人工感染试验和细胞感染实验,结果发现,人工感染试验鱼出现死亡,且具有典型出血症状;组织滤液感染草鱼肾细胞系(CIK)细胞后,盲传6代未观察到典型细胞病变效应,但感染细胞固定后经超薄切片电镜观察,细胞质内有大量病毒存在,与病鱼组织切片观察到的病毒形态一致。SDS-PAGE结果进一步揭示,新分离病毒株(暂命名JX-0902)基因组由11条dsRNA组成,呈现水生呼肠孤病毒基因组典型特征,但基因组带型与GCRV 873株存在差异,而与HZ08株接近。根据GenBank上已提交的草鱼呼肠孤病毒S6序列(GQ896337)设计特异引物,以JX-0902株的cDNA作为模板,可扩增出特异性条带。基于S6序列的聚类分析结果显示,JX-0902与GCRV HZ08株、GD108株S6同源性高达98%、99%,该分离株应为草鱼呼肠孤病毒。We identified a new strain of reovirus in grass carp from Nanchang city,Jiangxi Province.We collected diseased yearling grass carp,Ctenopharyngodon idellus exhibiting typical hemorrhage symptoms from Nanchang city and observed large numbers of viral cells in ultra-thin sections of the spleen and kidney.The virus had a non-enveloped structure with a diameter of approximately 70 nm,very similar to the characteristics of the grass carp reovirus（GCRV）.We isolated samples of the virus by homogenizing the tissue,centrifuging the homogenate,then conducting sterilizing filtration.We were able to replicate the hemorrhaging by artificially infecting grass carp with the purified sample.Artificial infection of grass carp kidney cell line（CIK） revealed no obvious cyto-pathic effects（CPE） after six passages.However,observations under the electron microscope revealed that the virus was propagated in the cytoplasm,and had the same shape as the virus observed in the tissue sections.The newly isolated virus（temporarily named GCRV JX-0902） consisted of 11 segments of dsRNA,a typical charac-teristic of aquareovirus.There were significant differences in the genome banding pattern of GCRV873 and JX-0902,but no difference between HZ08 and JX-0902.We designed PCR primers based on sequence segment 6（GQ896337） of GCRV in GenBank to amplify specific bands using JX-0902 cDNA as the template.The amino acid sequence of JX-0902 S6 had 98% and 99% identity with GCRV-HZ08 and GD108,respectively.Thus,we conclude that the new isolate is a member of the genus of aquareovirus.

关 键 词：草鱼出血病 呼肠孤病毒 江西分离株 分离 鉴定 

分 类 号：S941[农业科学—水产养殖]