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从四川乐山某养殖场自然发病的似鲇高原鳅(Triplophysa siluroides)体内分离到一株病毒FYL140220。病毒感染鲤上皮瘤细胞系(epithelima popuasum cuprini,EPC)后,细胞呈现圆缩、坏死、脱落、明显空斑的病变特征。将自然发病鱼组织过滤除菌液和细胞培养病毒液分别接种健康似鲇高原鳅,试验鱼出现与自然发病鱼相同的症状,死亡率分别为30%和40%,而对照组无异常。对经FYL140220感染出现典型细胞病变(CPE)的EPC细胞制备超薄切片进行电镜观察,发现病毒呈正六边形,对称20面体,对角线直径约(103±7)nm;提取细胞培养病毒液、自然发病鱼和人工感染发病鱼的内脏器官总DNA进行蛙病毒主要衣壳蛋白(maior capsid protein,MCP)基因保守区域的PCR扩增,均能扩增出预期大小约500 bp的特异性条带。MCP基因同源性与遗传进化分析表明,分离株FYL140220与蛙病毒属成员聚为一支,尤其与大鲵蛙病毒与沼泽绿蛙病毒同源性最高,分别达99.8%和99.6%。结合PCR检测、电镜观察和系统发育分析,确认分离病毒FYL140220为蛙病毒,这是首次报道蛙病毒可自然感染似鲇高原鳅并致死。
A strain of virus FYL140220 was isolated from the naturally occurring Triplophysa siluroides in a farm in Leshan, Sichuan Province. After the virus was infected with epithelium popuasum cuprini (EPC), the cells showed rounding, necrosis, shedding and obvious plaque lesions. The naturally occurring fish tissue filtration and cell culture virus solution were inoculated with healthy loach highland loach, the test fish with the same symptoms of naturally occurring fish, the mortality rate was 30% and 40%, while the control group was normal. The ultrathin section of EPC cells infected with typical cytopathic effect (CPE) infected by FYL140220 was observed under electron microscope. The virus was found to be regular hexagonal and symmetric 20-sided with a diagonal diameter of about (103 ± 7) nm. The cell culture virus solution , The naturally occurring fish and the total DNA of the internal organs of the fish infected by artificial infection were amplified by polymerase chain reaction (PCR) from the conserved region of main frog virus maior capsid protein (MCP) band. The homology and genetic evolution analysis of MCP gene showed that the isolates FYL140220 and Frogivirus belonged to one member, especially those homologous to the frog Rana virus and the marsh frog virus, which reached 99.8% and 99.6% respectively. Combined with PCR detection, electron microscopy and phylogenetic analysis, the isolated virus FYL140220 was identified as frog virus, which is the first report that frog virus can spontaneously infect and cause death of the highland loach.