一种新型草鱼呼肠孤病毒人工感染方法  

PRELIMINARY STUDY ON A NEW METHOD OF GCRV ARTIFICIAL INFECTION

作　　者：褚鹏飞[1,2] 何利波[1] 汪浩[1,2] 陈庚[1,2] 李勇明[1] 黄容[1] 廖兰杰[1] 朱作言[1] 汪亚平[1] CHU Peng-Fei HE Li-Bo WANG Hao CHEN Geng LI Yong-Ming HUANG Rong LIAO Lan-Jie ZHU Zuo-Yan WANG Ya-Ping(State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China University of Chinese Academy of Sciences, Beijing 100049, China)

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院大学,北京100049

出　　处：《水生生物学报》2016年第6期1166-1171,共6页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金重点项目(No.31130055);淡水生态与生物技术国家重点实验室自主课题(No.2014FBZ040)资助~~

摘　　要：研究从患病草鱼中新分离到一株草鱼呼肠孤病毒(Grass carp reovirus,GCRV),对其进行了病毒纯化与电镜观察、基因组RT-PCR分型以及病毒量定量分析等,并在此基础上探索了一种新的病毒人工感染方法。取病鱼肌肉组织进行病毒纯化与电镜观察,观察到大量病毒粒子,直径在70—80 nm。病毒基因组RT-PCR扩增结果表明,该草鱼呼肠孤病毒新分离株属基因型Ⅱ型GCRV;通过绝对定量的方法,对病毒悬液的浓度进行了测定,为2.97×103 copy/μL。通过灌胃法,对3个组别的实验鱼分别感染不同浓度的病毒液,同时设置灌胃PBS的组别作为对照组。结果显示,3个实验组死亡率均在80%左右,而对照组仅出现一例死亡个体。实验组死亡个体体表发黑,腹部、鳍条基部以及鳃盖处均有明显的出血症状,为草鱼出血病的典型症状。随机选取死亡的个体进行RT-PCR检测,均能检测出Ⅱ型GCRV的条带。以上结果说明,灌胃法可以作为一种新的方法,用于草鱼的GCRV人工感染实验。Grass carp（Ctenopharyngodon idella） is an important freshwater economic fish in China and its production was 5.5×109 kg in 2014, accounting for 13% of global freshwater fishery production, which was the highest output and consumption of freshwater fish worldwide. Hemorrhagic disease caused by grass carp reovirus（GCRV） seriously affects the grass carp production. This study isolated and purified a new strain of GCRV from diseased grass carp from muscle of dead fish with typical symptoms of hemorrhagic diseases, whihc was further determined.by electron microscope and RT-PCR. A large number of virus particles with 70—80 nm in diameter were observed by electron microscope, which was diagnosed as genotype（GCRV-） by RT-PCR. The titer of virus was determined by absolute quantitative PCR（2.97×103 copy/μL）. The GCRV artificial infection experiments with 3 three different concentrations and one PBS control group were conducted with new method of lavage. The mortality rate of three experimental groups were all about 80%, while only one fish died in PBS control group. The typical symptoms of hemorrhagic disease were found in the abdomen, fin base and gill cover of dead fish. GCRV-was detected from four randomly selected dead fish using RT-PCR. These results indicate that lavage could be a new effective way for GCRV artificial infection experiment.

关 键 词：草鱼呼肠孤病毒 人工感染 水产养殖 RT-PCR 分型 

分 类 号：Q31[生物学—遗传学]