两株Ⅱ型草鱼呼肠孤病毒的分离、鉴定及生物学特性比较  

Isolation and identification of two new isolates of genotypeⅡgrass carp reovirus and comparision of their biological characteristics

作　　者：高彩霞 曾伟伟 汤亚方 王英英[2] 王庆[2] 周文礼 吴杰兴 刘世旭 尹纪元 李莹莹[2] GAO Caixia;ZENG Weiwei;TANG Yafang;WANG Yingying;WANG Qing;ZHOU Wenli;WU Jiexing;LIU Shixu;YIN Jiyuan;LI Yingying(Department of Aquaticture,Tianjin Agricultural University,Tianjin 300384,China;Key Lab of Fishery Drug Creation of Ministry of Agriculture and Rural Affairs/Key Lab of Aquatic Animal Immune Technology of Guangdong Province/Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China;Key Lab of Animal Molecular Design and Precision Breeding of Guangdong Province of Foshan University/School of Life Science and Engineering,Foshan University,Foshan 440605,China)

机构地区：[1]天津农学院水产学院,天津300384 [2]中国水产科学研究院珠江水产研究所,农业农村部渔药创制重点实验室,广东省水产动物免疫技术重点实验室,广东广州510380 [3]佛山科学技术学院广东省动物分子设计与精准育种重点实验室,佛山科学技术学院生命科学与工程学院,广东佛山440605

出　　处：《水产学报》2020年第6期1013-1024,共12页Journal of Fisheries of China

基　　金：广东省自然科学基金(2017B030311018);广东省现代农业产业技术体系创新团队建设专项资金(2019KJ119,2019KJ150);大宗淡水鱼产业技术体系(CARS-45-17);Ⅱ型草鱼呼肠孤病毒灭活疫苗规模化生产关键技术研究(2019ZD0703)。

摘　　要：为研究Ⅱ型草鱼呼肠孤病毒(grass carp reovirus,GCRV)不同分离株毒力强弱及生物学特性的差异,该研究从患病和健康草鱼体内分离到Ⅱ型GCRV各一株,分别命名为ZH180804和CQ180701,并从细胞培养特性、致病性、基因组带型、基因序列差异、遗传进化关系等方面进行比较分析。结果显示,ZH180804对草鱼和稀有鲫的致死率分别为80%和100%,CQ180701对草鱼和稀有鲫的致死率分别为0和10%,初步表明ZH180804为强毒株,CQ180701为弱毒株,且弱毒株感染过的草鱼对强毒株的攻击感染具有较好的免疫保护;2株分离株在草鱼鳔细胞(GSB)、稀有鲫卵细胞(GRE)及稀有鲫尾鳍细胞(GRF)中均能增殖但不产生致细胞病变效应(CPE),且ZH180804株的增殖量是CQ180701株的1000倍以上;十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示,2株分离株基因组带型相似,但S7、S8、S9、S10与S11节段的分布存在一定的差异;2株病毒的S7与S11节段的基因序列同源性分别为98%和99%。基于S7与S11节段编码的氨基酸序列构建的遗传进化树显示,2株分离株聚在同一个分支上,具有较近的亲缘关系。研究表明,从患病和健康的草鱼中分离的2株Ⅱ型GCRV有较多共性,但其复制能力、致病性等方面存在较大差异。In order to study the virulence and biological characteristics of different isolates of grass carp reovirus(GCRV)typeⅡ,we researched two new isolates of genotypeⅡGCRV,ZH180804 and CQ180701,which were isolated from diseased and healthy Ctenopharyngodon idella,respectively.The characteristics of cell culture,pathogenicity,genomic segment,gene sequence and phylogenetic differences of the two strains were compared and analyzed.The results showed that mortality rate of ZH180804 to C.idella and Gobiocypris rarus were 80%,100%,and those of CQ180701 were 10%,0%,respectively.The data of mortality rate showed that ZH180804 is a virulent strain,and CQ180701 is a low virulent strain.Cell infection experiment showed the two isolates could proliferate in grass carp swim bladder cells(GSB),Gobiocypris rarus egg cells(GRE)and G.rarus fin(GRF)cells,but did not cause cytopathic effect(CPE),and viral multiplication of ZH180804 strains was 1000 times higher than that of CQ180701 strains.The banding patterns of two isolates in sodium dialkyl sulfate-polyacrylamide gelelectrophoresis(SDS-PAGE)were partially identical,but there were some differences between the segments of S7,S8,S9,S10 and S11.The sequence homology of the S7 and S11 genes between two viruses are 98%and 99%,respectively.Phylogenetic analysis indicated that isolates ZH180804 and CQ180701 belong to the same branch,and it means that they are very closely related.All results above indicate that two strains of GCRV typeⅡisolated from diseased and healthy grass carp have more common characteristics,but their replication ability and pathogenicity are different.

关 键 词：草鱼 稀有鲫 草鱼出血病 草鱼呼肠孤病毒 基因Ⅱ型 强弱毒株 生物学特性 

分 类 号：S941.41[农业科学—水产养殖]