对虾白斑病毒感染的电子显微镜观察及DNA杂交证据  

INFECTION OF WHITE SPOT BACULOLIKE VIRUS(WSBV) IN PENAEUS CHINENSIS:EVIDENCE FROM ELECTRON MICROSCOPY AND DNA HYBRIDIZATION

作　　者：徐洪涛[1] 朴春爱[2] 王雷[1] 王文兴[3] 相建海[1] 王运涛[1] 

机构地区：[1]中国科学院海洋研究所,青岛266071 [2]中国预防医学科学院病毒学研究所,北京100052 [3]国家海洋局第一海洋研究所,青岛266003

出　　处：《微生物学报》2000年第3期252-256,共5页Acta Microbiologica Sinica

基　　金：海洋 8 63 81 9 0 2 0 5课题;中国科学院海洋研究所EMBL开放课题;中国科学院海洋研究所所长基金

摘　　要：中国大陆养殖的中国对虾从 1 993年开始至今连年爆发病毒性流行病 ,俗称“白斑病”(WSBV) ,死亡率近 1 0 0 % ,造成巨大经济损失。为进一步明确虾病暴发原因 ,利用电子显微镜技术 ,对 1 993年至 1 998年收集的发病中国对虾组织进行观察 ,发现病原体为直径 ( 1 2 5±7 6)nm、长约 ( 345± 1 6)nm大小的带包膜的非包涵体型杆状病毒。经氯化铯密度梯度超速离心技术获得了纯化的病毒核衣壳 ,大小为 ( 80± 1 3)nm× ( 380± 2 4 )nm。形态学特征与台湾地区发现的白斑杆状病毒 (WSBV)相似。利用地高辛标记的WSBVDNA探针对病虾标本及纯化病毒DNA进行斑点杂交检测 ,均呈阳性反应 ,而与正常对虾组织无杂交反应。从形态学及分子生物学角度证明WSBV感染是造成中国大陆养殖对虾连年暴发流行病的重要原因。Severe epizootic causing high mortalities in cultivated Penaeus chinensis has occurred every year in China's Mainland since 1993 with great economic loss. Naturally diseased P.chinensis sampled in Qingdao shrimp farming regions between 1993 and 1998 were examined by electron microscopy. A similar non occluded nuclear bacilliform virus like the white spot baculo like virus (WSBV) reported in Taiwan was mostly found in the gill, cuticular epidermis under the exoskeleton, stomach, lymphoid organ and other tissues of ectodermal and mesodermal origin from these diseased samples. The viron was enveloped with a mean size of (125±7 6)nm×(345±16)nm. Sometimes, a nipple like appendage could be found protruding from one extremity of the viron. Envelopes were all lost after purification from cesium chloride gradient ultracentrifugation. The cylindrical nucleocapsid in negatively staining preparations was (80±13)nm×(380±24)nm with 13～16 stripes perpendicular to the longitudinal axis of the nucleocapsid. Based on its pathogenic and morphological characteristics, this virus should be related to WSBV. A pair of primers created from information of WSBV genome DNA Sal I fragment produced a 355bp band using the WSBV isolate from P.chinensis in China's Mainland, as the DNA template. The specific PCR product was cloned, sequenced and labeled with digoxigenin(DIG)DNA labeling kit(Boehringer Mannherm). The sequence of the probe is identical to that of WSBV. All of the sampled diseased shrimps between 1993 and 1998 reacted with the DIG labeled probe by dot blot hybridization and no hrbridization was observed using DNA from health shrimp as templates. These results based on electron microscopy and DNA hybridization indicated that the shrimp epizootic occurred in China's Mainland was related to the WSBV infection previously occurred in Taiwan early 1992.

关 键 词：中国对虾 白斑杆状病毒 电子显微镜 DNA探针 

分 类 号：Q934.2[生物学—微生物学]