摘要
收集福建某渔场的患病石斑鱼苗制备组织切片,同时将患病石斑鱼苗组织匀浆后接种到条纹月醴细胞系（striped snakehead,SSN-1）,对发生细胞病变（cytopathic effects,CPE）的SSN-1细胞进行逆转录聚合酶链式反应（RT-PCR）、纯化负染,并制备细胞超薄切片,借助SSN-1细胞系从患病石斑鱼苗中分离病毒性神经坏死病毒（viral nervous necrosis virus,VNNV）。试验结果表明：在病鱼脑和视网膜中可见大量的空泡,接种SSN-1后2～3 d出现CPE,PCR检测可扩增出VNNV特异性带,提示这是VNNV病毒。负染及SSN-1细胞超薄切片的电镜观察显示,该病毒为直径25～30 nm的二十面体病毒粒子,在胞质中呈不完全晶体状排列,同时细胞质中有大量空泡,进一步说明分离株为VNNV。对该分离株进行细胞敏感性试验、脂溶剂敏感试验（氯仿处理）、热敏感试验（55℃30 min）、酸碱敏感试验（pH值为3和pH值为11各1 h）,试验结果表明：石斑鱼吻端细胞、鲤上皮瘤细胞、肥头鲤细胞在28℃、25℃、20℃时对该病毒都不敏感;该病毒对氯仿不敏感,对热和酸碱都比较敏感。研究结果表明,用SSN-1细胞系可以从患病鱼中分离VNNV。
Sick grouper fry in Fujian Province were collected. Vacuolation was obviously observed in brain and retina of diseased fry in histopathological section. The fry organs were homogenized and supernatant was inoculated to SSN-1 cell. CPE was developed on the 2 to 3 days after incubation. The result of RT-PCR showed that it may be VNNV. It was an icosahedral virus with a mean diameter of 25-30 nm by electron microscopy. The virus replicated in the cytoplasm and formed paracrystalline array in the infected SSN-1 cells. This confirmed that the isalated virus was VNNV. The cell sensitivity test indicated that grouper proboscis cell line（GP）, epithelima popuasum cuprini cell line （EPC）, pimephales promelas cell line（FHM） were not sensitive to the isolate. The chloroform susceptibility test, pH susceptibility test and heat stability（55 ℃ ,30 min） test were carried out in this paper. The results showed the isolate was resistant to chloroform, sensitive to pH, and heat which indicated that VNNV can be isolated from sick fish by SSN-1 cell culture.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2008年第3期409-413,共5页
Journal of Huazhong Agricultural University
基金
中华人民共和国质量监督检验检疫总局项目(2006IK013)资助
关键词
病毒性神经坏死病毒
分离
病毒特性
viral nervous necrosis virus
isolation
characterization of virus
