1株沼泽绿蛙源蛙病毒的分离鉴定及系统发育分析  

Isolation,identification and phylogenetic analysis of a ranavirus isolated from Rana grylio

作　　者：田思璐 袁羽 徐乐 欧阳萍[1] 陈德芳[2] 黄小丽[2] 耿毅[1] TIAN Silu;YUAN Yu;XU Le;OUYANG Ping;CHEN Defang;HUANG Xiaoli;GENG Yi(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;College of Animal Science and Technology,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]四川农业大学动物科技学院,成都611130

出　　处：《华中农业大学学报》2025年第2期258-264,共7页Journal of Huazhong Agricultural University

基　　金：四川省自然科学基金项目(24NSFSC0858);四川省淡水鱼产业技术体系创新团队建设项目(SCCXTD-25-15);中央引导地方发展科技项目(2024ZYD0317)。

摘　　要：为探究四川某养殖场沼泽绿蛙(Rana grylio)暴发性高死亡率疾病的病因,对病蛙进行病理学检查和病原分离,并结合电镜观察、PCR检测和系统发育分析对分离病原进行鉴定。患病蛙主要病症为皮肤溃疡与四肢肿胀;剖检见肝、脾与肾肿大。组织病理学观察见肝、脾、肾与肠道等组织器官发生变性、坏死与炎症反应。接种病料的鲤鱼上皮瘤细胞25℃培养4 d呈典型细胞病变效应,TCID50为104.12/0.1 mL。电镜观察到大量正六边形、对角线直径约165 nm的病毒粒子呈晶格状排列。对内脏组织及接种细胞进行蛙病毒主要衣壳蛋白(major capsid protein,MCP)基因的特异性PCR检测,结果均为阳性。基于MCP基因全序列的同源性和遗传进化分析显示,分离病毒与蛙病毒属病毒相似性在99%以上,且属于FV3类群。以上结果表明分离病毒(TSL210813)为蛙病毒,是本次沼泽绿蛙疫病的病因。To explore the etiology of a serious infectious disease affecting Rana grylio at a farm in Sichuan Province,pathological examinations and pathogen isolation were conducted on the diseased frogs.The isolated pathogen was identified using transmission electron microscopy,PCR detection,and phylogenetic analysis.External clinical signs included limb swelling and ulcerations on the body surface.The dead frog exhibited an enlarged liver,spleen and kidney.Histopathological observations revealed that the liver,spleen,kidney and other organs exhibited damage characterized by significant degeneration and necrosis foci.The carp epithelioma papulosum cyprini(EPC) cell line,inoculated with diseased samples at 25 ℃,demonstrated typical cytopathic effects(CPE) after four days,including rounding and shedding,with a TCID50 of 104.12/0.1 mL.Transmission electron microscopic observation showed that the virus exhibited a regular hexagonal shape with a crystalline array of its capsid,and the diagonal diameter measured approximately 165nm.PCR by using specific primers for the major capsid protein(MCP) gene was performed on the internal tissues of infected frogs and on EPC cells with CPE,yielding positive results.Phylogenetic analysis based on MCP gene sequences indicated that the sequences of isolated virus shared over 99% identity with ranavirus and was classed as FV3-like.This study confirmed that the isolated virus,designated TSL210813,is a ranavirus,responsible for the disease observed in R.grylio.

关 键 词：沼泽绿蛙 蛙病毒 FV3 蛙病毒主要衣壳蛋白基因 病理损伤 

分 类 号：Q943[生物学—植物学]