1株牛蛙虹彩病毒的分离鉴定及其致病性

Isolation, identification and pathogenicity of a Rana grylio virus

为了明确一种牛蛙(Rana catesbiana Shaw)传染性疾病的致病病原,本研究利用收集的病料进行病毒分离培养、鉴定和致病性分析。结果显示,接种病料的鲤鱼表皮瘤细胞(epithelioma papulosum cyprini cell line, EPC)出现变圆、脱落等典型细胞病变效应(cytopathic effect, CPE),病毒粒子的电镜观察表明其可能为虹彩病毒,进一步的基因组PCR鉴定和序列的同源性分析证实分离的病毒为一株蛙虹彩病毒(Rana grylio virus, RGV),称之为RGV-FJ。攻毒试验表明,感染RGV-FJ的牛蛙出现腹部及大腿出血,肝脏变白肿大,脾脏、肾脏及肺部出血等症状。组织病理学观察发现,攻毒组牛蛙皮肤的黏液层细胞肿胀、排列紊乱,细胞间红细胞浸润,上皮细胞肿胀、坏死;肝细胞肿胀或萎缩、坏死、排列紊乱,门静脉与肝组织剥离,红细胞渗出且肿胀成不规则形态;肾脏的肾小管管腔狭窄,细胞界限模糊,大量红细胞和单核细胞浸润;肺部囊泡细胞排列紊乱,有大量红细胞浸润、淤积。同时利用透射电镜在蛙的肺部观察到病毒粒子。PCR检测显示,攻毒组牛蛙肝脏、脾脏、肾脏、肠道、肌肉、皮肤和肺中均可检测到RGV-FJ,这表明牛蛙发生了RGV-FJ的系统侵染;攻毒组牛蛙从攻毒第2天开始出现死亡,第4天的累计死亡率达到94.12%;并从病死牛蛙的主要内脏器官中重新分离出了RGV-FJ。上述结果表明,分离的RGV-FJ具有较强致病性,这为进一步开展分离病毒的致病机理研究奠定基础。

In order to identify the pathogen agent of an infectious disease of bullfrog(Rana catesbiana Shaw),the collected disease samples were used for virus isolation, identification and pathogenicity analysis.The results showed that the carp epithelioma papulosum cyprini cell line(EPC) inoculated with the disease samples exhibited typical cytopathic effects(CPE),such as rounding and shedding.Electron microscope observation on the virions indicated that it might be an iridovirus.Further PCR identification and sequence homology analysis confirmed that the isolated virus was a Rana grylio virus(RGV),named RGV-FJ.The viral challenge experiment showed that the bullfrog infected with RGV-FJ had typical symptoms of abdominal and thigh hemorrhage, white and swollen liver, spleen, kidney and lung bleeding.Histopathological observation showed that the mucous layer cells of the skin in the challenge group were swollen and disordered, intercellular red blood cell infiltration, epithelial cells swelling and necrosis.Hepatocytes swelling or atrophy, necrosis, disordered arrangement, peeling of portal vein and liver tissue, exudation and swelling of red blood cells into irregular shape.The renal tubular lumen of the kidney was narrowed, the cell boundary was blurred and massive erythrocytes and monocytes was infiltrated.The arrangement of pulmonary vesicle cells was disordered, with a large number of red blood cells infiltrating and silting.Viral particles were observed in the lungs of the bullfrog by transmission electron microscope.PCR detection showed that RGV-FJ could be detected in the liver,spleen,kidney,intestine,muscle,skin and lung of bullfrog in the challenge group,which indicated that the bullfrog had been systematically infected with RGV-FJ.The fatality rate analysis showed that the bullfrog in the challenge group died from the second day after infection,and the cumulative mortality rate reached 94.12% on the fourth day.RGV-FJ was re-isolated from the main internal organs of the challenge bullfrog.The above results show that the isolated RGV-FJ has high pathogenicity,which provides a research basis for further research on the pathogenic mechanism of the isolated virus.