鲤病毒病原的感染性测定

为查明养殖鲤（Cyprinus carpio）突然大批发病死亡的原因，对鲤病样品进行了细胞攻毒、空斑测定、电镜观察，以及鱼体感染等实验。先以患病鲤的组织匀浆液，经过滤后，分别接种到草鱼鳍条细胞（GCF）、鲤上皮瘤细胞（EPC）等14种培养细胞中。利用倒置显微镜观察显示，在1-2d内，该病鱼组织匀浆液可使其中9种细胞出现典型的细胞病变。收集出现病变的细胞液（即病毒悬液），进一步进行病毒滴度检测、空斑测定和鱼体感染实验。结果显示，在GCF细胞上的病毒滴度为10^7.3TCID50／mL；在FHM，TSB和GCO等细胞中可产生直径1～4mm的圆形空斑，空斑的大小与宿主细胞的种类和接种的病毒浓度有关。通过对感染细胞制备的超薄切片和病毒负染样品进行电镜观察，显示这是一类呈典型子弹头样的弹状病毒颗粒。感染了病毒悬液的鲫和鲤先后在第2天和第3天开始出现病症，间隔1～2d后发病的鱼开始死亡，至第14天，两种感染鱼的死亡率均达到83．3％。收集人工感染后濒死的鲫和鲤，分别制备组织匀浆液，回接感染鱼类培养细胞，24h内能使其出现与原发病鲤组织匀浆液所引起的类似的细胞病变。因此证实患病鲤是由病毒病原感染所致。中国水产科学，2006，13（4）：617—623]

Detection of viral pathogen from diseased common carp (Cyprinus carpio) by infectious tests

The viral pathogens are the most important epidemic disease affecting fish,which cause significant morbidity and mortality in fish.It has substantial impact on the production of fish and can not be prevented effectively.In order to determinate which pathogen caused the mass mortality cultured common carp(Cyprinus carpio),a series of experiments about cell lines challenged with virus supernatants from diseased fish homogenate,plaque assays,observed with electron microscope and artificial infection in fish were done.Diseased fish samples were collected,homogenized,filtered,then inoculated onto monolayer cultures of 14 cell lines including Ctenopharyngodon idellus ovaries(GCO),Ctenopharyngodon idellus fins(GCF),Channel catfish ovary(CCO), Epithelioma papulosum cyprini(EPC), Pimephales promelas(FHM),Cyprinus carpio leucocyte cell(CLC),Trionyx sinensis blood(TSB), Carassiu auratus gibelio heart(GCH), Ameiurus nebulosus(BB), Carassius auratus blastula(CAB),Carassius auratus vertebral column bone(CAVB), Ctenopharngodon idellus kidney(CIK), Gobiocypris rarus ovary(GRO) and Hypophthalmichthys molitrix ovary(HMO).The results show that observation of the cytopathogenic effect(CPE) appeared in 9 of these cell lines,GCO,GCF,CCO,EPC,FHM,CLC,TSB,GCH and BB.The CPE involved cell rounding,detachment from the monolayer, and subsequent destruction of monolayer.Virus titers of about 10~(7.3) TCID_(50)/mL were obtained by infecting the GCF cells.Viral plaques were formed after virus supernatants attachment to three cell lines FHM,TSB,and GCO,and the plaques of 1 to 4 mm in diameter is probably dependent on host cell lines and viral diluents.Electron microscopy observation revealed that the viral particles of ultrathin sections and negatively stained had a typical bullet-shape morphology.Initial symptoms appear in crucian carp(Carassius auratus) and common carp(Cyprinus carpio) in 2 and 3 days post-infection(p.i.) respectively.The diseased fish died in 1 to 2 days after the symptoms appearing.The late mortality rate of infected fish was 83.3% until the 14 days p.i.The dying fish were homogenized,filtered,then inoculated onto fish cell lines,the CPE were observed obviously in 24 hours p.i.and similar to those induced by the original homogenate of the diseased common carp.It is supported that the diseased fish were infected by the viral pathogen.Also the viral pathogen has strong infectivity to the crucian carp(Carassius auratus) and common carp(Cyprinus carpio).The detection of viral pathogen from diseased common carp(Cyprinus carpio) by infectious tests in our researches is a base to the isolation and indentification of the viral pathogen,also to the diagnosis and prophylaxis of it.