一株似鲇高原鳅源蛙病毒的分离与鉴定

Isolation and identification of a ranavirus from Triplophysa siluroides

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 virus named FYL140220 was isolated from naturally infected Triplophysa siluroides in Leshan, Sichuan Province using epithelima popuasum cuprini （EPC） cells. The infected EPC cells showed circular shrinkage, necrosis, and desquamation, which formed significant plaque-lesion characteristics. Diseased tissue suspension filtered from bac- teria and EPC-grown virus were used to inoculate healthy Triplophysa siluroides. As a result, the infected T. siluroides developed similar clinical symptom to those described above and suffered 30% and 40% mortality, whereas the uninfected control EPC cells remained normal. Electron microscopy revealed icosahedral viral particles in the cells. The FYL140220 virus had an average diameter of （103±7） nm, and the hexagonal shape was highly similar to that of other Iridoviridae viruses. DNA was extracted from the EPC-grown virus and naturally and artificially infected internal T. siluroides tissues for polymerase chain reaction （PCR） amplification of the conserved region of the ranavirus major capsid protein （MCP） gene, revealing a 500obp fragment. The MCP homology and genetic evolution analysis showed that FYL140220 and other ranavirus strains formed a tight cluster and shared 99.8% identity with CGSV-G and 99.6% with RGV. Taken together, these results confirm that FYL140220 is a ranavirus. This is the first report on a natural ranavirus infection and mortality caused by this pathogen in cultured Triplophysa.