| 虹鳟传染性胰脏坏死病毒的分离鉴定及聚类分析 |
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| 引用本文： | 刘淼,徐黎明,赵景壮,曹永生,刘红柏,尹家胜,卢彤岩.虹鳟传染性胰脏坏死病毒的分离鉴定及聚类分析[J].大连海洋大学学报,2017,32(1). |
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| 作者姓名： | 刘淼 徐黎明 赵景壮 曹永生 刘红柏 尹家胜 卢彤岩 |
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| 作者单位： | 中国水产科学研究院黑龙江水产研究所,黑龙江 哈尔滨,150070 |
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| 基金项目： | 黑龙江省应用技术研究与开发计划项目，黑龙江省自然科学基金资助项目，中央级公益性科研院所基本科研业务费专项 |
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| 摘 要： | 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。
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| 关 键 词： | 虹鳟 传染性胰脏坏死病毒(IPNV) CHSE-214 基因型 |
Isolation,identification and cluster analysis of an infectious pancreatic necrosis virus |
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| Authors: | LIU Miao XU Li-ming ZHAO Jing-zhuang CAO Yong-sheng LIU Hong-bai YIN Jia-sheng LU Tong-yan |
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| Abstract: | 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. The 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 105. 2 TCID50/mL on CHSE-214, 103. 2 TCID50/mL on RTG-2 and 102. 3 TCID50/mL on EPC. ChRtm213 produced plagues in CHSE-214, and plenty of virons in crystalline arrangement was observed in cytoplasm. The 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. |
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| Keywords: | Oncorhynchus mykiss infectious pancreatic necrosis virus CHSE-214 gene type |
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