虹鳟传染性胰脏坏死病毒的分离鉴定及聚类分析

Isolation,identification and cluster analysis of an infectious pancreatic necrosis virus

为研究传染性胰脏坏死病毒(IPNV)感染虹鳟Oncorhynchus mykiss的病理特性,取云南省某养殖场大规模死亡的虹鳟稚鱼进行了病原的分离与鉴定,试验中利用鲑鳟鱼常见病毒敏感细胞大鳞大麻哈鱼胚胎细胞(Chinook salmon embryo cells,CHSE-214)、鲤上皮瘤细胞(Epitheliaoma papulosum cyprinid,EPC)和虹鳟性腺细胞(Rainbow trout gonad cell line,RTG-2)对虹鳟稚鱼无菌组织悬液进行病毒培养,并对产生细胞病变的上清进行病毒RNA提取及鲑鳟鱼常见病毒的RT-PCR检测,利用敏感细胞进行病毒分离株的噬斑培养及滴度测定,并对病毒形态进行透射电镜观察。结果表明:细胞培养显示,该组织悬液能使CHSE-214和RTG-2细胞产生明显的细胞病变;RT-PCR结果显示,检测样本呈IPNV阳性,传染性造血器官坏死病毒(IHNV)呈阴性;该IPNV分离株(命名为Ch Rtm213)在CHSE-214、RTG-2和EPC细胞上的病毒滴度分别为10^(5.2)、10^(3.2)、10^(2.3) TCID_(50)/m L,在CHSE-214细胞上培养4 d即可形成肉眼可见的病毒噬斑;在透射电镜下清晰可见大量病毒粒子呈晶格状排列于细胞质内;Ch Rtm213分离株与西班牙毒株2310(AY489225)VP2相似度较高,核苷酸序列一致性为96.4%;聚类分析结果显示,Ch Rtm213分离株与参考毒株加拿大Jasper(ATCC:VR-1325)聚为一簇,属于Gengroup 5基因型。研究表明,Ch Rtm213分离株异于已报道的IPNV分离株,具有不同的血清型及病毒毒力,可为传染性胰脏坏死病毒的检测及防控提供参考。

A pathogen of an infectious pancreatic necrosis （IPN ） was isolated and cultured in rainbow trout On-corhynchus mykiss collected from Yunnan Province by chinook salmon embryo cells （ CHSE -214） , epitheliaoma papulosum cyprinid （ EPC ） and rainbow trout gonad cell line （ RTG - 2 ） inoculated with sterile tissue suspension. The RNA was extracted from cells with cytopathic effect, and identified by RT - PCR assays used for common salm-on virus detection. The isolate was further analyzed with plaque assay, virus titer detection, and electron microsco-py observation. The cell culture showed that the tissue suspension led to produce typical cytopathic effect （ CPE ） on CHSE -214 and RTG -2 instead of EPC . T he RT-PCR showed that the sample was positive to infectious pancre-atic necrosis virus （IPNV ） and negative to infectious hematopoietic necrosis virus （IHNV ）. The IPNV isolate was named as ChRtm213, with titer of 10^5.2 TCID50/mL on C H S E -214, 10^3.2 TCID50/ mL on RTG -2 and 10^2.3 TCID50/mL on EPC . ChRtm213 produced plagues in CHSE -214, and plenty of virons in crystalline arrangement was observed in cytoplasm. Th e ChRtm213 had the maximal homology with Spain IPNV -2310 isolate, with nucleo-tide identity of 96. 4%. The cluster analysis revealed that ChRtm213 , in the same cluster with reference strain Canada isolate Jasper （ATCC： VR -1325） , belonged to Gengroup 5. A novel IPNV was isolated and identified from Yunnan Province, and the findings provide reference for the monitoring and vaccine development of IPNV in China.