异育银鲫呼肠孤病毒的分离与鉴定  

Isolation and identification of a reovirus strain from Gibel carp

作　　者：巴翠玉 张培军[2] 赵福广[1] 杜晓燕[3] 焦雪[1] 李月红[1] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林省卫生监测检验中心,吉林长春130000 [3]吉林省水产科学研究院,吉林长春130033

出　　处：《华南农业大学学报》2017年第2期22-26,共5页Journal of South China Agricultural University

基　　金：国家自然科学基金(30972191);吉林省农业产业技术体系(201506);948计划(2014Z34)

摘　　要：【目的】发现异育银鲫Carassius auratus gibelio新病原,为其病害防控提供理论基础。【方法】从吉林省某养殖场采集异育银鲫出血病疑似病样,对其进行细菌分离及寄生虫观察、鲤疱疹病毒Ⅱ型的PCR检测和人工感染试验,然后用感染滤液接种鲤上皮瘤细胞(EPC),并对其进行电镜观察。对病毒基因组进行SDS-PAGE电泳分析、RT-PCR鉴定及序列测定。【结果】发病异育银鲫无细菌及寄生虫感染,鲤疱疹病毒Ⅱ型的PCR检测无特异性条带,将过滤后的患病鱼组织滤液感染健康异育银鲫,7 d内死亡率高达86.7%。盲传4代后出现明显的细胞病变。负染后电镜下可观察到病毒粒子,直径约70 nm,病毒颗粒近球形,无囊膜结构,初步判断为呼肠孤病毒(暂命名JL-4)。SDS-PAGE结果揭示,JL-4基因组由11条dsRNA组成,呈现水生呼肠孤病毒基因组典型特征。将RT-PCR扩增产物的序列进行聚类分析,结果显示,JL-4与呼肠孤病毒HZ08株S6序列相似性高达99%,证明该分离株为呼肠孤病毒。【结论】从患病异育银鲫中分离到1株呼肠孤病毒。【Objective】To discover a new pathogen of Gibel carp（ Carassius auratus gibelio）,and to provide a theoretical basis for the disease prevention and control. 【Method】Gibel carps suspected having hemorrhagic disease were collected from a farm in Jilin Province. Tissues were observed for bacterial infection and parasite contamination. PCR was used to detect Cyprinid herpes virus-2. Artificial infection was conducted,the infection filtrate was vaccinated to the carp epithelial tumor cells（ EPC）,and the EPC were observed by an electron microscope. The virus genome was analyzed by SDS-PAGE and identified by RT-PCR and sequencing. 【Result 】 Neither bacterial nor parasitic infection was detected.Cyprinid herpes virus-2 specific bands were not detected by PCR. After healthy Gibel carps were infected with tissue filtrate of sick fish,the mortality rate reached 86. 7% within seven days. Clear cytopathy was detected after four generations via blind passage. The virus particles were observed by an electron microscope after negative staining,the particle was spherical with around 70 nm in diameter and with no envelope. The virus was initially determined as a reovirus strain（ temporarily named JL-4）. The SDS-PAGE results showed that JL-4 possessed 11 segments of dsRNA,which was the typical characteristic of Aquareovirus genome. Cluster analysis for the sequences of the RT-PCR amplification product showed that S6 sequences from JL-4 and reovirus HZ08 had 99% similarity, confirming that JL-4 was reovirus.【Conclusion】One reovirus strain was isolated and identified from Gibel carp.

关 键 词：异育银鲫 呼肠孤病毒 鲤上皮瘤细胞 分离与鉴定 SDS-PAGE 

分 类 号：S941[农业科学—水产养殖]