摘要
采用细胞培养、超薄切片电镜观察、巢式PCR检测以及病毒DNA克隆与测序分析等技术。从湖北武汉一循环水养殖系统中患出血病异育银鲫（Carassius auratusgibelio）体内分离鉴定了一株鲤疱疹病毒2型（Cyprinid herpesvirus2，CyHV-2）病毒，命名为鲤疱疹病毒2型武汉株（CyHV-2．WH）。患病鱼组织匀浆液接种锦鲤鳍条细胞系（Koi．Fin）可产生典型的细胞病变效应。用患病鱼组织匀浆液与细胞培养物分别人工感染异育银鲫，均可重复出与自然发病相同的症状，死亡率达100％。患病鱼脾与肾脏组织超薄切片电镜观察结果显示，病毒为具囊膜的球形病毒，囊膜直径为170-200am，病毒衣壳直径为110～120nm，病毒在细胞核内复制、组装。采用CyHV-2的特异引物对病鱼样本和感染细胞培养物样本进行巢式PCR检测，均能扩增出357bp的目的条带，将该扩增产物进行测序与比对分析后发现，其与GenBank中已报道的CyHV-2毒株的DNA解旋酶基因高度同源（99．44％以上）。与鲤疱疹病毒3型（CyHV-3）也有较高的同源性（81．55％）。系统进化分析结果显示，CyHV-2．WH株与JS2012、H．Fukuda、YCll0907等CyHV-2病毒株属同一分支。
By using cell culture and virus infection methods, a virus had been isolated from gibel carp （Caras- siusauratus gibelio） suffering with severe hemorrhage, which were cultured in a circulation aquaculture system in Wuhan,and then identified as Cyprinid herpesvirus 2（CyHV-2）, designated as CyHV-2 WH strain, by electron microscopy observation, nested-PCR and viral DNA sequencing and sequence alignment analysis. The tissue ho- mogenate of diseased fish could cause typical cytopathic effect in Koi-Fin cells. In artificial infection test, the typical symptoms of hemorrhage could be reproduced as naturally infected fish by infecting both tissue homoge- nate and cell cultured virus, and the death rate reached 100%. Electron microscopy observation of ultra-thin sec- tion samples of spleen and kidney tissues revealed that the viral nucleocapsid was spherical in shape measuring 110-120 nm in diameter with a 170-200 nm envelope. The virions replicated and assembled in nucleus. A 357 bp DNA fragment obtained by the nested-PCR assay of both tissue homogenate and cell culture. Sequence alignment analysis of the DNA fragment showed that WH shared so high identity （〉99.44%） with the published DNA heli- case gene sequence of CyHV-2 stains in GenBank, meanwhile, it shared 81.55% identity with CyHV-3 stains. Phylogenetic analysis showed that WH strain formed an independent branch on the phylogenetic tree with other CyHV-2 strains such as JS2012, H.Fukuda and YC110907.
出处
《中国水产科学》
CAS
CSCD
北大核心
2013年第6期1303-1309,共7页
Journal of Fishery Sciences of China
基金
现代农业产业技术体系建设专项资金资助(CARS-46-11)
中国水产科学研究院基本科研业务费资助项目(2013A0606)
关键词
鲤疱疹病毒2型
异育银鲫
病毒分离
病毒鉴定
潜伏感染
Cyprinid herpesvirus 2（CyHV-2）
gibel carp
virus isolation
virus identification
latency
