CAJ | 학술논문

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 herpesvirus 2, 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170~200 nm,病毒衣壳直径为110~120 nm,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357 bp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、YC110907等CyHV-2病毒株属同一分支。
채용세포배양、초박절편전경관찰、소식PCR검측이급병독DNA극륭여측서분석등기술,종호북무한일순배수양식계통중환출혈병이육은즉(Carassius auratusgibelio)체내분리감정료일주리포진병독2형(Cyprinid herpesvirus 2, CyHV-2)병독,명명위리포진병독2형무한주(CyHV-2-WH)。환병어조직균장액접충금리기조세포계(Koi-Fin)가산생전형적세포병변효응。용환병어조직균장액여세포배양물분별인공감염이육은즉,균가중복출여자연발병상동적증상,사망솔체100%。환병어비여신장조직초박절편전경관찰결과현시,병독위구낭막적구형병독,낭막직경위170~200 nm,병독의각직경위110~120 nm,병독재세포핵내복제、조장。채용CyHV-2적특이인물대병어양본화감염세포배양물양본진행소식PCR검측,균능확증출357 bp적목적조대,장해확증산물진행측서여비대분석후발현,기여GenBank중이보도적CyHV-2독주적DNA해선매기인고도동원(99.44%이상),여리포진병독3형(CyHV-3)야유교고적동원성(81.55%)。계통진화분석결과현시, CyHV-2-WH주여JS2012、H.Fukuda、YC110907등CyHV-2병독주속동일분지。
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.