| 摘要: |
| 【目的】确定陕西省略阳县某大鲵养殖场高致死性疾病的病原，分析其主要衣壳蛋白(MCP)的基因特征。【方法】采集患病大鲵，观察其临床症状，取其脏器组织，处理后分别分离细菌和接种鲤鱼上皮瘤(EPC)细胞，观察细胞病变，分离病毒；对病原进行增殖，采用蔗糖密度梯度(20%，30%，40%，50%，60%)离心法进行纯化，电镜观察纯化样品。克隆分离病毒的MCP基因，测定其序列并进行系统进化树分析。【结果】患病大鲵背部有溃疡、头部胀大且有出血点，尾部有轻微的溃烂。取病鲵内脏组织样品分离病原，未分离到细菌；处理的内脏组织感染EPC细胞，出现细胞病变。电镜观察证实，在蔗糖密度梯度为50%～60%的样品中，病毒粒子的纯度最高，且结构较为完整；病毒粒子的直径约150 nm，与虹彩病毒相似。MCP PCR扩增得到了预期长度(1 392 bp)的特异性产物。NCBI BLAST结果显示，扩增的MCP序列与蛙病毒属的感染性造血器官坏死病毒(EHNV)、食用蛙病毒（RSV）、普通产婆蟾病毒（CMTV）以及梭鲈虹彩病毒（PPIV）的MCP全长的同源性最高，达到99%；与中华鳖虹彩病毒 (STIV)、蛙病毒3型(FV3)、虎纹蛙虹彩病毒(RTV)等MCP全长的同源性为98%。系统发育分析结果显示，导致该例大鲵发病的病毒为蛙病毒属的虹彩病毒。【结论】造成陕西省略阳县某大鲵养殖场大鲵大量死亡的病原为蛙病毒属的虹彩病毒，将该株病毒命名为大鲵虹彩病毒略阳分离株(CGSIV LY1112)。 |
| 关键词: 大鲵 虹彩病毒 纯化 MCP 系统进化树 |
| DOI： |
| 分类号: |
| 基金项目:陕西省科学技术计划项目(2012K01-18) |
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| Isolation,purification and major capsid protein gene sequence analysis of Iridovirus isolated from Chinese giant salamander,Andrias davidianus |
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ZHANG Xing-lang,ZHOU Xiao-yuan,ZHANG Hui
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| Abstract: |
| 【Objective】The study aimed to determine the causative pathogen of diseased Chinese giant salamander Andrias davidianus with highly mortality and examine its major capsid protein (MCP) genetic characteristics.【Method】After observation of clinical symptoms of the diseased Chinese giant salamander,the tissues infected were disposed to isolate bacterium and inoculate with EPC cells,separately.Then the doubtful virus was cultured with EPC,and sampled to purify by sucrose density gradient centrifugation (20%,30%,40%,50%,and 60%).Electron microscope was used to observe the purified viral particles.MCP clone plasmid of the purified virus was detected by PCR,and phylogenetic tree was constructed based on nucleotide sequences of MCP.【Result】The gross lesions with ulcer in the back and swelling of head with petechial hemorrhages were observed.No pathogenic bacterium was isolated from organs including liver,spleen and kidney.After treatment,the visceral tissue suspension was infected with EPC,and cytopathic effect was observed.Electron microscopy showed that the most abundant and intact virus particles occurred in the 50%-60% sucrose density fraction,the average diameter was 150 nm,and the shape was highly similar to that of iridovirus.With the primers designed according to the sequences of major capsid protein (MCP) from the GenBank,specific products with predicted size of 1 392 bp were obtained from the cloned plasmid.Comparative analysis of nucleotide sequences was performed with the GenBank using BLAST database network service.The results indicated that the putative gene products from sick giant salamander shared high identities with homologous of 99% with epizootic hematopoietic necrosis virus (EHNV),Rana esculenta virus (RSV),common midwife toad virus (CMTV),pike-perch Iridovirus (PPIV),and with homologous of 98% with soft shelled turtle Iridovirus (STIV) and FV3 and Rana tigrina virus (RTV).Phylogenetic tree indicated that the pathogen in present study was a species within the genus Ranavirus.【Conclusion】The pathogen was a virus belonging to the genus of Ranavirus and it was named tentatively Lueyang isolates of Chinese giant salamander Iridovirus (CGSIV LY201112). |
| Key words: Andrias davidianus Iridovirus purification MCP phylogenetic tree |
