一株鲈鲤源鲤春病毒血症病毒的分离鉴定及其病理观察

2016年4月,四川某鲈鲤(Percocypris pingi)养殖场流行一种以鳃、鱼鳔和内脏器官出血为临床特征的传染病。组织病理学观察发现,患病鲈鲤全身多组织器官均发生明显的病理损伤,尤其是肝、脾、肾、鳃和肠表现为明显的出血、变性、坏死以及炎症细胞浸润。取病鱼组织匀浆滤液接种鲤上皮瘤细胞(epithelioma papulosum cyprini, EPC),盲传3代出现典型的细胞病变(cytopathic effects, CPE)。将自然发病鱼组织匀浆滤液和细胞培养病毒液分别接种健康鲈鲤,实验鱼出现与自然发病鱼相同的症状,死亡率分别为60%和50%,而对照组未见异常。对经分离毒株ZLP160415感染出现CPE的EPC细胞制备超薄切片进行电镜观察,发现病毒呈弹状,长90～150 nm,宽40～60 nm;对自然发病鱼、人工感染发病鱼内脏组织和细胞培养病毒液进行鲤春病毒血症病毒(springviremiaofcarpvirus,SVCV)的RT-PCR检测,均扩增出目的条带。基于SVCV糖蛋白基因进行系统发育分析,结果显示分离毒株(ZLP160415)属于Ia型。结合本次疾病的流行病学与病理损伤特点、病毒分离鉴定、人工感染试验结果和透射电镜检查,确定此次流行病的病原为SVCV。

Isolation and identification of a spring viremia of carp virus from Percocypris pingi and resultant pathological lesions

In April 2016, a serious infectious disease characterized by hemorrhage of the gill, swim bladder, and internal organs emerged in a Percocypris pingi farm in Sichuan Province. Histopathological observation showed that the spring viremia of carp virus (SVCV) infection in P. pingi could cause pathological damages in multiple organs, which exhibited hemorrhage, degeneration, necrosis, and infiltration of the inflammation cells, especially in the liver, spleen, kidney, gill, and intestine. The filtrated homogenate was inoculated into the epithelioma papulosum cyprini (EPC) cells, and the typical cytopathic effect (CPE) was formed after three blind passages. Diseased tissue suspension filtered from bacteria and EPC-grown virus were used to inoculate healthy P. pingi. The results showed that the infected P. pingi developed similar clinical symptoms to those described above and suffered 60% and 50% mortality, whereas the control group remained normal. Transmission electron microscopy (TEM) of EPC cells infected with ZLP160415 found that the virus was bullet-shaped, 90-150 nm long, and 40-60 nm wide. RT-PCR of tissue homogenates from the fish naturally infected, the fish artificially infected, and the infected cells were performed, and the results showed that all were SVCV-positive. Based on phylogenetic analysis of glycoprotein genes, the isolate ZLP160415 was classified into the Ia genogroup. From the combination of epidemiology, pathologic lesions, virus isolation and identification, artificial infection, and TEM examination, it was concluded that the SVCV was the pathogen of the disease.