鳜鱼病毒核酸随机引物扩增与克隆↑（*）

从感染鳜鱼病毒（ Ｓｉｎｉｐｅｒｃａ ｃｈｕａｔｓｉ Ｖｉｒｕｓ，简称ＳＣＶ） 的鳜鱼体中分离病毒，提纯核酸作模板。通过ＲＡＰＤ 法，用带酶切位点的引物进行病毒核酸随机引物扩增，获得扩增带谱。从带ＥｃｏＲⅠ酶切位点的引物扩增产物中，用琼脂糖凝胶电泳回收大小约为０ ．４ 和０ ．５ Ｋｂｐ 的片段，经ＥｃｏＲⅠ酶切处理制备成带粘性末端的片段，分别插入质粒ｐＵＣ１９ 的ＥｃｏＲⅠ位点。ＥｃｏＲⅠ酶对重组子进行了酶切鉴定，获得２ 种带插入ＳＣＶ 核酸ＰＣＲ扩增产物的重组子。     

两种不同地方种群尼罗罗非鱼遗传差异和分子遗传标记↑（*）

用ＡｐａⅠ，Ｂｃ１Ⅰ，Ｂｇ１Ⅱ，ＤｒａⅠ，ＥｃｏＲⅠ，ＥｃｏＲＶ，ＨｉｎｄⅢ，ＫｐｎⅠ，ＰｓｔⅠ，ＰｖｕⅡ，ＳａｃⅠ，ＸｂａⅠ，ＸｈｏⅠ等１３种限制性核酸内切酶对来自尼罗河上游和下游的两种不同地方种群尼罗罗非鱼的线粒体ＤＮＡ（ｍｔＤＮＡ）进行酶切分析，得到这两种罗非鱼的ｍｔＤＮＡ分子大小为：上游尼罗为１６９０９±９０ｂｐ，下游尼罗为１６９１２±１００ｂｐ。研究中还发现我国养殖的这两种不同地方种群尼罗罗非鱼，每一群体内个体间均存在ｍｔＤＮＡ酶切片段长度多态现象，而且群体间在ｍｔＤＮＡ水平上具有一定差异，依此构建了这两种不同地方种群尼罗罗非鱼ｍｔＤＮＡ限制性内切酶酶切图谱，建立了识别它们的分子遗传标记。     

条纹斑竹浙江省鲨线粒体DNA的研究

用６种限制性内切酶分析了４条条纹斑竹鲨的线粒体ＤＮＡ，ＰｓｔＩ，ＨｐａＩ，ＸｂａＩ，ＥｃｏＲＩ，ＥｃｏＲＶ，ＢｇｌⅡ在条纹斑竹鲨ｍｔＤＮＡ分子上分别具有０至２个切点，ｍｔＤＮＡ分子大小为１６．６ｋｂ，根据单酶和双酶完全酶解片段的大小，构建了条纹斑竹浙江省ｍｔＤＮＡ的限制性酶切图谱。     

对虾病毒HHNBV DNA构建质粒的研究与分析

用ＰＵＣ１８构建了对虾病毒ＨＨＮＢＶＤＮＡ重组质粒，提取后经斑点杂交及酶切分析，证实质粒中插入片段为病毒ＤＮＡ，其中０５＃质粒的插入片段大于２Ｋｂ，与质粒的分子量相近。对０５＃质粒酶切后的Ｓｏｕｔｈｅｒｎ杂交亦取得了满意的结果。同时，还对０５＃质粒的插入片段进行了内切酶图谱分析，共进行了ＥｃｏＲｌ、Ｈｉｎｄｌｌｌ、Ｓｍａｌｌ、Ｐｓｔｌ、ＰＵＶｌ、Ｈｉｎｄｌｌ６种限制性内切酶分析，结果表明，只有Ｐｓｔｌ在插入片段的一端有一个酶切位点，酶切位点的确切位置有待于进一步确定。     

镢鱼病毒核酸随机引物扩增与克隆

从感染鳜鱼病毒（Ｓｉｎｉｐｅｒｃａ ｃｈｕａｔｓｉ Ｖｉｒｕｓ，简称ＳＣＶ）的鳜鱼体中分离病毒，提纯核作模板。通过ＲＡＰＤ法，和带酶切位点的引物进行病毒核酸随机引物扩增，获得扩增带谱。从带ＥｃｏＰＩ酶切位点的引物扩增产物中，用琼脂糖凝胶电泳回收大小约为０．４和０．５Ｋｂｐ的片段，经ＥｃｏＲＩ酶切处理制备成带粘性未端的片段，分别插入质粒ｐＵＣ１９的ＥｃｏＰＩ位点。ＥｃｏＲＩ酶对重组子进行了酶切鉴定，     

中国对虾皮下及造血组织坏死杆状病毒（HHNBV）DNA探针的制备及应用

ＨＨＮＢＶ是１９９３年中国对虾暴发性流行病的主要病原之一。从纯化的病毒中提取ＤＮＡ，用ＥｃｏＲｌ限制性内切酶酶切成ＤＮＡ片段，与ＰＵＣ１８体外重组，建立ＨＨＮＢＶＤＮＡ文库。从中筛选出三个重组质粒（５＃、８＃、９＃），它们所插入的片段大小分别为：２．８Ｋｂ、０．８Ｋｂ、１．６Ｋｂ，它们分别用光敏生物素标记成探针，与感染ＨＨＮＢＶ的病虾ＤＮＡ呈阳性反应，以此利用制备的ＨＨＮＢＶＤＮＡ探针的高敏感性和特异性来检测虾病。     

鲇鱼线粒体DNA的酶切图谱

采用差速离心法及ＤＮａｓｅⅠ、ＲＮａｓｅ消化法制备并纯化了鲇（Ｓｉｌｕｒｕｓａｓｏｔｕｓ）肝脏线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ，ｍｔＤＮＡ）。用８种限制性内切酶对ｍｔＤＮＡ进行了分析。ＢｇⅡ、ＥｃｏｒⅠ、ＰｓｔⅠ、ＢｇｌⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＨｉｎｄⅢ、ＸｈｏⅠ在鲇ｍｔＤＮＡ分子上分别具１、１、１、２、２、７、７和０个切点。ｍｔＤＮＡ分子量约１０．８４×１０￣６道尔顿，大小为１７．５４ｋｉｌｏｂａｓｅｐａｉｒｓ。根据单酶和双酶解片段的数目和分子量，建立了鲇ｍｔＤＮＡ的限制性酶切图谱。     

草鱼线粒体DNA限制性内切酶分析

本文报道了用ＢａｍＨⅠ，ＢｇｌⅠ，ＢｇｌⅡ，ＣｌａＩ，ＥｃｏＲＩ，ＨｉｎｄⅢ，ＰｖｕⅡ，ＳａｃⅠ，ＸｂａⅠ，ＸｈｏⅠ等十种限制性内切酶酶切草鱼线粒体ＤＮＡ，计算出各酶切片段长度及草鱼ｍｔＤＮＡ大小。其中六种酶有多个切点，在所检测的样品中未发现碱基替换。     

鲢鳙线粒体DNA的九种限制性内切酶酶切图谱的比较

以十一种限制性内切酶对鲢鱼ｍｔＤＮＡ进行单酶完全酶解的切点数：ＳａＩ和ＢｇｌⅡ为０；Ｐｓｔ Ｉ．Ｋｐｎ Ｉ和Ｓａｃ ｉ为１；Ｈｐａ和Ｘｂａ Ｉ为２；ＢａｍＨ Ｉ为３；Ｂｇｌ Ｉ和ＥｃｏＲ Ｉ为４。以九种限制性内切酶对鳙鱼ｍｔＤＮＡ进行单酶完全酶解，其切点数：Ｐｓｔ Ｉ．Ｘｈｏ Ｉ．Ｓａｌ Ｉ．Ｋｐｎ Ｉ．Ｂｇｌ Ⅱ均为１；Ｈｐａ Ｉ和Ｘｂａ Ｉ为２；Ｓａｃ Ｉ为３；Ｂｇｌ Ｉ为４。采用琼脂糖凝胶电     

鳙小卫星DNA的克隆

鳙基因组ＤＮＡ 经内切酶Ｓａｕ３ＡⅠ完全酶切后，与Ｂａｍ ＨⅠ－ ｐＵＣ１８ 质粒连接，转化ＤＨ５α后，获得９３３ 个重组体。用人源小卫星探针３３ ．１５ 对重组体进行原位杂交筛选，获得１４ 个阳性克隆，取其中的１ 个阳性克隆（ 即小卫星ｐＢＣ１７４） 作探针，与５ 尾鳙和４ 尾鲢基因组ＤＮＡ 杂交，能产生具有高度变异性的、个体特异性的ＤＮＡ 指纹图，表明克隆的鳙小卫星ｐＢＣ１７４ 可用于鱼类ＤＮＡ 指纹分析。     

Experimental infection of European crustaceans with white spot syndrome virus (WSSV)

Eight European marine and freshwater crustaceans were experimentally infected with diluted shrimp haemolymph infected with white spot syndrome virus (WSSV). Clinical signs of infection and mortalities of the animals were routinely recorded. Diagnosis was by direct transmission electron microscopy (TEM), DNA hybridization (dot-blot and in situ hybridization) using WSSV probes and by PCR using WSSV specific primers. High mortality rates were noted between 7 to 21 days post-infection for Liocarcinus depurator , Liocarcinus puber , Cancer pagurus , Astacus leptodactylus , Orconectes limosus , Palaemon adspersus and Scyllarus arctus . Mortality reached 100%, 1 week post-infection in P. adspersus . When infection was successful, direct TEM observation of haemolymph revealed characteristic viral particles of WSSV, some observed as complete virions (enveloped), others as nucleocapsids associated with envelope debris. WSSV probes showed strong positive reactions in dot-blots and by in situ hybridization in sections and specific virus DNA fragments were amplified successfully with WSSV primers. White spot syndrome virus was pathogenic for the majority of the crustaceans tested. This underlines the epizootic potential of this virus in European crustaceans.     

应用PCR和RT-PCR技术对4种对虾病毒的检测

探讨了应用PCR和RT－PCR技术对4种主要的对虾病毒进行检测的方法。同时使用该方法检测了对虾天然饵料－－卤虫中的4种对虾病毒。结果显示，含病毒核酸的阳性对照样品分别扩增出了大小为824bp,705bp,260bp和216bp的预期产物，但未能从卤虫样品中检测到此4种病毒的存在。本文报道的病毒检测方法具有快速、灵敏、准确的特点，可以用于进出口贸易中对活体或冰冻对虾，虾苗，对虾饵料等进行相关对虾病毒的检疫，也为制定我国对虾病毒检疫检验规范提供了技术参考。     

3种市售养殖对虾白斑综合征病毒(WSSV)的核酸探针检测

对虾白斑综合征病毒是对虾养殖业危害最为严重的病毒, 每年给对虾养殖造成很大经济损失, 已成为对虾养殖业可持续发展的严重障碍。在过去的十几年中, 人们采取各种方法防止白斑综合征病毒的传播。中国明对虾(Fenneropenaeus chinensis )、凡纳滨对虾( Litopenae vannamei)、日本囊对虾(Marsup enaeus jap onicus)是我国主要的对虾养殖品种, 也是白斑综合征病毒的敏感宿主。按照煮沸- 乙醇沉淀法快速、简便提取市售30 只中国明对虾、28只凡纳滨对虾、29只日本囊对虾DNA, 然后用地高辛标记的核酸探针进行斑点杂交检测白斑综合征病毒,从而了解市场中养殖对虾携带白斑综合征病毒的情况。检测结果显示, 所有样品均未感染白斑综合征病毒, 说明目前白斑综合征病毒在一定程度上得到了有效控制。     

A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp

White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.     

中国对虾几个产卵场群体携带白斑综合征病毒状况调查

白斑综合征可以导致养殖对虾短时间内大面积死亡，是迄今为止对虾养殖业面临的最大挑战。本试验采用巢式PCR法对2001年采自黄渤海的中国对虾几个产卵场群体进行白斑综合征病毒检测，旨在较全面地了解黄渤海野生中国对虾携带病毒状况。各群体的阳性检出率分别为：朝鲜半岛南海岸群体55％；渤海湾群体35％；辽东湾群体94．7％；海州湾群体47．4％。结果显示，中国对虾几个产卵场群体均不同程度地携带白斑综合征病毒。辽东湾产卵场群体阳性检出率明显高于其他群体，推测人工孵化苗种放流、海湾的地理和水质条件与中国对虾的WSSV感染率相关。而中国对虾野生群体携带病毒对于对虾养殖业的影响是不容忽视的，笔者认为，只有从无特异病原(SPF)及抗特异病原(SPR)对虾养殖群体的建立着手才能从根本上避免由于对虾携带病毒而可能导致的病毒性疾病的暴发。同时，应该重视海区污染的治理，减少病毒病暴发的诱因。本试验建立了快速检测WSSV的PCR方法，1pg病毒核酸仍可检测到，为白斑综合征病毒病的防治及早期诊断提供了有效的手段。     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.     

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.     

乳山对虾养殖场白斑综合征病毒(WSSV)调查研究

2002年采用PCR-核酸探针斑点杂交法检测了乳山对虾养殖场1000余份样品携带白斑综合征病毒(WSSV)的情况。结果显示,639例对虾样品中阳性检出率为26·6%;77例蟹类样品中阳性检出率为18·2%;266例浮游动物样品中阳性检出率为38·3%,3~9月份浮游动物阳性率呈下降趋势,消毒后水体中浮游动物的阳性率仍很高;30例贝类样品检测均为阴性;204例底泥样品中,阳性检出率为17·6%,22例抽滤海水样品检测均为阴性。结果表明,虾、蟹类在传播WSSV中起着重要作用,贝类、海水传播WSSV的可能性很小,浮游动物、底泥在传播WSSV中的作用和机制应引起高度重视。     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).