防治对虾白斑综合征病毒(WSSV)的主要措施

对虾白斑综合征病毒(wh ite spot syndrom e virus,W SSV)是迄今为止危害最为严重的一种对虾病毒,发病对虾3~10 d死亡率可达到100%[1]。该病毒自1992年爆发以来,已在世界范围内传播开来,每年给对虾养殖业造成巨大经济损失,至今仍未得到完全控制,成为目前对虾养殖业可持续发展的     

抗白斑综合征病毒(WSSV)感染途径研究进展

白斑综合征病毒(White Spot Syndrome Virus,WSSV),是引起养殖对虾大规模死亡的病原。在过去的10年中,各国学者不断地寻找预防和控制WSSV感染的方法及途径。本文主要介绍了中和抗体与噬菌体展示单链抗体、免疫增强剂、重组蛋白疫苗和灭活疫苗、遗传育种、RNAi等几种方法的研究现状,为进一步开发抗WSSV药物和饵料,更有效地防治WSSV及相关科研提供参考。     

中国对虾和日本对虾对白斑综合征病毒(WSSV)敏感性的比较

用10⁵拷贝、10⁴拷贝和10³拷贝3个剂量的白斑综合征病毒(WSSV)粗提液分别对中国对虾和日本对虾进行人工注射感染,比较两种对虾对WSSV敏感性的差异,以探讨日本对虾白斑综合征(WSS)低发病率的原因,为对虾的病害防治提供参考。结果显示:用10⁵拷贝、10⁴拷贝和10³拷贝的WSSV粗提液注射后,中国对虾的平均存活时间分别为54.21±0.60 h、71.26±4.26 h和75.04±5.73 h;日本对虾平均存活时间分别为77.61±4.45 h、105.84±6.36 h和168.82±13.15 h。中国对虾和日本对虾的存活时间均随着病毒剂量的降低而延长。同剂量病毒注射下,日本对虾组的存活时间均长于中国对虾组,差异显著(P<0.05)。试验结果表明,日本对虾对WSSV的抵抗力较中国对虾强。     

对虾白斑综合征病毒(WSSV)高分子量结构蛋白的分离

采用蔗糖密度梯度离心从患病对虾组织中提取WSSV,应用SDS-PAGE对WSSV结构蛋白进行了分析。采用12％分离胶,将WSSV样品煮沸5min,应用SDS-PAGE分离了WSSV的中低分子量结构蛋白,并将该结果与其他学者已发表的结果进行了比较。首次通过延长样品煮沸时间,采用8％分离胶,应用SDS-PAGE分离到了WSSV100kD以上的13条高分子量结构蛋白,计算出了每条蛋白带的分子量及其在总蛋白中的百分含量。其中WSSV高分子量结构蛋白的分离丰富了WSSV的研究范围,对今后深入的研究工作具有重要意义。     

7种消毒剂对对虾白斑综合症病毒(WSSV)DNA作用效果的比较

将中国对虾白斑综合症病毒 (WSSV)病毒液与万福金安含氯消毒剂、高锰酸钾、甲醛、过氧化氢、甲酚皂、酒精、氯仿作用。不同浓度消毒剂与病毒液作用不同时间后提取 DNA作病毒核酸斑点杂交检测。经分析比较表明 ,上述消毒剂中含氯消毒剂在有效氯含量高于 2 .975× 10 -3、高锰酸钾浓度高于 5 .0 0 0× 10 -3 ,5 min内 ;有效氯含量高于 1.190× 10 -3、高锰酸钾浓度在 5 .0 0 0× 10 -3 ,30 min即可破坏核酸     

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。     

PCR检测对虾白斑综合征病毒(WSSV)中使用UNG防遗留污染

设计了一对引物用于PCR检测对虾白斑综合征病毒，PCR扩增过程中用尿嘧啶-DNA糖基酶(UNG)防遗留污染。使用UNG时，该对引物的适宜dUTP和Mg^2 浓度分别为0．4mmol／L和2．0mmol／L；UNG存在时，PCR检测WSSV DNA的最低量为1pg，UNG的使用使PCR的检测灵敏度降低了1个数量级；进行正常PCR扩增前，0．5U UNG可消除至少10ng含dU的WSSV DNA的PCR扩增产物。     

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。     

白斑综合征病毒(WSSV)3种PCR检测方法的灵敏度比较

为了探讨不同PCR检测方法的灵敏度,分别利用TaqMan实时定量PCR、世界动物卫生组织(OIE)公布的巢式PCR引物(简称OIE)、黄海水产研究所种质资源与工程育种研究室(GB)设计的引物(简称GB)及2种巢式PCR对应的一步法PCR,对具有不同白斑综合征病毒(White Spot Syndrome Virus,WSSV)含量的中国明对虾(Fenneropenaeus chinensis)样品进行检测.结果显示,当使用已知病毒含量的标准品进行检测时,TaqMan实时定量PCR方法可以检测到l0个WSSV拷贝;OIE巢式PCR与GB巢式PCR方法分别可检测到104和103个WSSV拷贝;单独使用OIE巢式PCR的外引物和内引物扩增时,分别可检测到5×104和2.5×104个WSSV拷贝;单独使用GB巢式PCR的外引物和内引物进行一步法PCR扩增时,分别可检测到104和5×103个WSSV拷贝.使用上述PCR方法分别对44份未知WSSV含量的样品进行验证,定量PCR方法检测阳性率为84.09%,OIE巢式PCR与GB巢式PCR方法检测的阳性率分别为18.18%和27.27%;单独使用OIE巢式PCR的外引物和内引物扩增检测的阳性率均为15.91%;单独使用GB巢式PCR的外引物和内引物扩增检测的阳性率分别为18.18%和20.45%.根据以上结果,PCR方法检测WSSV的灵敏度由高到低依次为:定量PCR、巢式PCR、一步法PCR.     

不同温度下凡纳滨对虾和中国明对虾对白斑综合征病毒(WSSV)耐受性比较

本研究分别对凡纳滨对虾"壬海1号"(Litopenaeus vannamei"Renhai No.1")和中国明对虾"黄海2号"(Fenneropenaeus chinensis"Huanghai No.2")在3种温度条件下(24℃、28℃和32℃),采用单尾定量口饲感染白斑综合征病毒(White spot syndrome virus,WSSV)的方法进行感染实验,比较2种对虾在不同温度情况下对WSSV的耐受性差异(L代表凡纳滨对虾,F代表中国明对虾)。结果显示,L-24℃和F-24℃组的平均存活时间分为(184.05±69.56)h和(101.68±38.45)h;L-28℃和F-28℃组的平均存活时间分别为(100.25±26.79)h和(73.38±22.22)h,相同温度组内均存在显著性差异(P0.05);截至第15天,L-32℃和F-32℃组的存活率分别为45.74%和23.47%。3个温度组对虾在50%的死亡率时的存活时间分别为178 h和98 h、98 h和74 h、292 h和78 h;死亡高峰时间分别为第5天和第4天、第5天和第4天、第10天和第4天。另外,分别在感染后的12 h、1 d、2 d、3 d、4 d、5 d、6 d、7 d、15 d共9个时间点对每组对虾进行活体取样,利用实时荧光定量RT-PCR技术对其进行病毒载量检测,从对虾体内肌肉组织病毒载量的角度探寻不同对虾抗病性能的差异。6 d时,L-24℃和F-24℃组对虾肌肉内病毒载量分别达到(2.97×10~6±7.44×10~6)和(8.08×10~6±3.22×10~6)copies/ng DNA,差异极显著(P0.01),L-28℃和F-28℃组分别达到(6.73×10~6±1.49×10~6)和(1.20×10~7±6.15×10~5)copies/ng DNA,差异极显著(P0.01);15 d,L-32℃和F-32℃组分别达到(5.18×10~4±4.32×10~4)和(3.78×10~4±8.97×10~4)copies/ng DNA,差异显著(P0.05)。研究表明,2种对虾在3种温度环境下感染WSSV后,凡纳滨对虾耐受WSSV能力要高于中国明对虾;不同温度下同种对虾肌肉体内WSSV的增殖能力从强到弱依次为28℃组、24℃组和32℃组。     

Rotifer cellular membranes bind to white spot syndrome virus (WSSV)

Cell membranes from the rotifer, Brachionus urceus, were obtained by centrifugation and found to specifically bind white spot syndrome virus (WSSV) in vitro. This finding suggests that there is likely a WSSV receptor on the rotifer cell membrane and provides evidence that rotifers may be a host for WSSV.     

Effect of low water temperature on viral replication of white spot syndrome virus in Procambarus clarkii

This study evaluated the effect of low water temperature (10 ± 1 °C) on viral infection and replication of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, under standardized conditions. Crayfish were (i) maintained at 24 ± 1 °C before challenge and 10 ± 1 °C afterwards, or (ii) maintained at 10 ± 1 °C before challenge and 24 ± 1 °C afterwards. No mortality was observed when crayfish were held at 10 ± 1 °C after challenge, but mortality reached 100% when they were transferred to 24 ± 1 °C. Competitive PCR showed that viral levels at 10 ± 1 °C rose from 10⁶ to 10⁸ copies/mg of gill tissues, while at 24 ± 1 °C levels increased from 10⁶ to 10¹⁰ copies/mg of gill tissues during the same time interval. These results showed that a low water temperature of 10 ± 1 °C could reduce viral replication when compared to 24 ± 1 °C but could not prevent it.     

注射白斑综合征病毒对克氏原螯虾酚氧化酶活力的影响

将白斑综合征病毒(white spot syndrome virus,WSSV)、嗜水气单胞菌(Aeromonas hydrophila,Ah)、大肠杆菌(Escherichia coli)(DH5α)用注射法接种克氏原螯虾(Procambarus clarkii),在0~72 h之间定时检测克氏原螯虾血细胞和肝胰脏中酚氧化酶(Phenoloxidase,PO)活力变化。结果显示:(1)0.1 mg/m L和1 mg/m L胰蛋白酶处理样品后,样品间差异不显著。(2)加胰蛋白酶处理与未加胰蛋白酶相比,供试克氏原螯虾PO活力均升高。(3)未加胰蛋白酶与加胰蛋白酶表现出相似的特征,WSSV和Ah注射组与对照组相比均表现为,12~48 h PO活力显著高于对照组,并且在48 h达到最大值,72 h时基本恢复正常;注射DH5α组与对照组相比没有显著性变化。可见感染WSSV后,克氏原螯虾体内酚氧化酶活力发生了变化,由此推测,PO参与了螯虾体内抵御病毒的免疫反应。     

白斑综合征病毒(WSSV)提取物中43 kDa蛋白来源的MALDI-TOF分析

将纯化的WSSV粒子蛋白经SDS-PAGE分离后,对图谱中出现的43kDa蛋白进行质谱分析,发现该蛋白不是WSSV基因组编码,是来自其宿主的蛋白成分,与肌动蛋白有很高的同源性。提示该蛋白的有无及含量多少与纯化病毒粒子的状态有很大关系,可以作为纯化WSSV完整性的参考指标。     

对虾白斑综合征病毒在螯虾动物模型的感染特性

应用克氏原螯虾作为动物模型研究对虾白斑综合征病毒（ＷＳＳＶ）的感染增殖特性，涉及感染温度，感染途径，继发感染，半数致死量（ＬＤ５０），免疫保护及保存期等，结果显示，２２－２５℃时，接种ＷＳＳＶ的螯虾一般于２－７d内死亡，温度升高对病毒增殖影响不显著，３０－３２℃时，平均死亡时间２－６d，温度降低对病毒增殖影响较显著，１５－１９℃时，接种螯虾平均２－１０d内死亡，８－１０℃时，平均死亡时间３－６d，感染途径分别用腹节肌肉，腹节皮下洲射及口服，均能使螯虾感染发病，而浸泡方式不能使螯虾发病，细菌分离和细菌定量结果表明，寄考于螯虾心脏，肝胰腺内的阴沟肠杆菌在感染后期大量增殖，菌量分别是正常螯虾的２５和３０倍，形成继发感染，用螯虾心脏，肝胰腺内的阴肠杆菌在感染后期大量增殖，菌量分别是政党螯虾的２５和３０倍，形成继发梁。用螯虾测定ＷＳＳＶ的ＬＤ５０为１０^-6.5.mL^-1种毒液，将病毒５６℃，３０min灭活后免疫螯虾，不能使螯虾形成免疫保护，ＷＳＳＶ匀浆液－３０℃冻存１年后失活，而－３０℃冻存于螯虾体内ＷＳＳＶ保存１年后仍有活力。     

Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60 000  g ) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80 000  g ) through a discontinuous iodixanol gradient (phosphate‐buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1 000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.     

Recent insights into host–pathogen interaction in white spot syndrome virus infected penaeid shrimp

Viral disease outbreaks are a major concern impeding the development of the shrimp aquaculture industry. The viral disease due to white spot syndrome virus (WSSV) observed in early 1990s still continues unabated affecting the shrimp farms and cause huge economic loss to the shrimp aquaculture industry. In the absence of effective therapeutics to control WSSV, it is important to understand viral pathogenesis and shrimp response to WSSV at the molecular level. Identification and molecular characterization of WSSV proteins and receptors may facilitate in designing and development of novel therapeutics and antiviral drugs that may inhibit viral replication. Investigations into host–pathogen interactions might give new insights to viral infectivity, tissue tropism and defence mechanism elicited in response to WSSV infection. However, due to the limited information on WSSV gene function and host immune response, the signalling pathways which are associated in shrimp pathogen interaction have also not been elucidated completely. In the present review, the focus is on those shrimp proteins and receptors that are potentially involved in virus infection or in the defence mechanism against WSSV. In addition, the major signalling pathways involved in the innate immune response and the role of apoptosis in host–pathogen interaction is discussed.     

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) was developed and field testing was conducted. A 604‐bp target sequence was selected from the vp28 gene of WSSV. A primer set was developed to amplify a 338‐bp DNA fragment at the junction of the NS2 and NS1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139‐bp PCR fragment from the β‐actin gene by alignment of this gene from Litopenaeus vannamei, Fenneropenaeus chinensis and Penaeus monodon. The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV, WSSV and β‐actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170 L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV, and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus‐free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV.     

Antiviral activity of esculin against white spot syndrome virus: A new starting point for prevention and control of white spot disease outbreaks in shrimp seedling culture

White spot syndrome virus (WSSV) is a pathogenic and threatening virus in shrimp culture for which there is no effective control strategy. Finding antiviral lead compounds for the development of anti-WSSV drugs is urgent and necessary; in this study, esculin from 12 monomeric compounds exhibited an excellent anti-WSSV activity. The results showed that esculin increased the survival rate of WSSV-infected shrimps by 59% and reduced the virus copy number in vivo over 90% at 100 μM. In the pre-treatment and post-treatment experiments, esculin could prevent and treat WSSV infection. Compared with the control group, the virus copy number decreased by 30% after 6 h of esculin pre-incubation with WSSV particles and inhibited horizontal transmission of WSSV to a certain extent. Considering that the antiviral activity of esculin was stable in the aquacultural water for 2 days, we evaluated the dosing pattern of continuous medication changes. Obviously, the survival rate of WSSV-infected shrimps was 0% at 108 h when no esculin exchange was made, while at 120 h the survival rate was over 40% at continuous medicine changes. In addition, esculin significantly increased the expression of antimicrobial peptides and thus improved the ability of shrimp to resist WSSV. Overall, our findings suggest that esculin has the potential to be developed into an anti-WSSV medicine.