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。     

中国对虾对白斑综合征病毒的类免疫反应与验证

对中国对虾(Fenneropenaeus chinensis)能否产生类免疫保护作用进行了初步探讨。将健康的中国对虾接种经不同辐照强度γ射线灭活的白斑综合征病毒(white wpot wyndrome virus,WSSV),连续投喂接种20 d后,对其存活个体进行抗WSSV性能测试,检验其存活情况。结果显示,至216 h实验组(射线辐照剂量分别为5 kGy、10 kGy、15 kGy和20 kGy)存活率分别为45.5%、54.2%、68%和65.4%,存活对虾的病毒携带量分别为(14.0±11.9)、(14.1±4.3)、(24.5±15.0)和(15.1±7.8)copies/ng DNA,死亡对虾病毒携带量分别为(2.1±2.0)×105、(5.2±4.4)×105、(3.8±4.4)×105和(6.2±7.7)×105copies/ng DNA,阳性对照组存活率为20%,其存活和死亡对虾病毒携带量分别为(24.7±7.4)×105copies/ng和(5.8±6.9)×105copies/ng DNA。以上结果说明,中国对虾可能存在类免疫保护作用,而且通过接种经γ射线灭活的WSSV能够提高对虾后续抗感染能力。研究结果旨为采用免疫学手段预防对虾白斑综合征提供参考。     

低剂量对虾白斑综合征病毒粗提液对斑节对虾体内潜伏期病毒及血细胞的影响

利用含3×103、6×102、2×102copies/mL的对虾白斑综合征病毒(white spot syndromevirus,WSSV)粗提液和PBS液人工注射感染携带病毒量约1×105copies/g的斑节对虾,通过实时定量PCR法、显微镜观察法研究了感染后斑节对虾发病死亡率、肌肉内WSSV含量及总血淋巴细胞数和不同种类血细胞的比例组成变化。结果表明,3种浓度下斑节对虾累积死亡率分别为93.33%±2.89%、56.67%±5.77%和45.00%±5.00%,对照组没有发生死亡。注射3×103copies/mL的浓度组,在注射后30 min时,对虾肌肉内的病毒含量达到最低值(3.54×104copies/g),而后持续上升至48 h时达到最高值(3.12×108copies/g)后再次下降;另两个感染组,在注射后1 h时,肌肉内病毒含量达到最低值(分别为1.11×105、9.54×104copies/g),在感染后6 h时出现一个次高值(分别为1.58×105、1.11×105copies/g),而后又降低,在48 h时达到最高值(分别为1.48×107、5.46×106copies/g)后下降;对照组对虾肌肉内病毒含量没有出现显著变化。不同感染浓度组,斑节对虾总血淋巴细胞数波动幅度和出现峰值的时间不同,特别是注射3×103copies/mL组,对虾总血淋巴细胞数在12和48 h时出现极低值(分别为3.48×106、4.05×106/mL);3个感染组除个别时间点外,对虾总血淋巴细胞数的变化规律与肌肉中WSSV的扩增规律成负相关。4个处理组,半颗粒细胞比例在感染初期呈现明显上升的趋势,后期比例虽有起伏但均维持在较高水平;颗粒细胞和透明细胞比例在感染初期均呈现明显下降的趋势,中期均略有上升,但后期颗粒细胞比例显著低于初期,而透明细胞比例则与初期无显著性差异。     

凡纳滨对虾白斑综合征血液病理研究

对自然发病、投喂和注射感染的凡纳滨对虾白斑综合征（white spot syndrome，WSS）血液病理进行研究，结果发现：不同感染方式患病对虾的血液病理变化相似，表现为：1．患病对虾血细胞总数、透明细胞数量极显著减少，小颗粒细胞、大颗粒细胞极显著增加。2．显微病理变化主要表现为血涂片中血细胞明显减少且分布不均匀，破损或解体的细胞增多，呈典型的溶血状态。3．超微病理变化表现为，大部分血细胞坏死，少数血细胞呈不典型的凋亡。患病对虾的血细胞核中可见大量白斑综合征病毒（white spot syndrome virus，WSSV）粒子。病理变化表明血细胞是WSSV的主要靶细胞。     

氨氮胁迫下白斑综合征病毒对凡纳滨对虾的致病性

为了评价养殖水环境中氨氮(NH_4-N)对凡纳滨对虾(Litopenaeus vannamei)的危害性,开展了NH_4-N胁迫对凡纳滨对虾感染白斑综合征病毒(WSSV)后的死亡率、WSSV增殖速率和对虾主要免疫相关酶活性影响的实验。在NH_4-N胁迫质量浓度为15.6 mg·L-1,分别注射2×105和2×106个WSSV粒子,结果显示,NH_4-N胁迫下注射2×105个WSSV粒子的凡纳滨对虾第144小时死亡率达到53.3%,显著高于无胁迫组(40.0%)。对虾鳃组织WSSV荧光定量PCR检测结果显示,NH_4-N胁迫下凡纳滨对虾鳃组织内WSSV的增殖加快。此外,免疫相关酶活性结果显示,NH_4-N浓度突变会促使对虾血清中酚氧化酶(PO)、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活性短暂升高后持续降低。由此可见,NH_4-N胁迫会加快WSSV在患病凡纳滨对虾体内的增殖,导致更高死亡率,这可能是因为胁迫造成了对虾免疫相关酶活性降低和抗病原感染能力下降。     

聚β-羟基丁酸酯对中国明对虾抗WSSV能力的影响

为探索聚β-羟基丁酸酯(PHB)对感染白斑综合征病毒(WSSV)的水生动物存活率、病毒含量的影响,实验采用单因子浓度梯度法,对感染WSSV的中国明对虾投喂添加了不同浓度聚β-羟基丁酸酯(0.0%、0.5%、1.0%、2.5%、5.0%、10.0%)的饵料,统计相同时间点对虾死亡数量、存活率、相对免疫保护率(RPS),并利用real-time PCR测定死亡对虾体内病毒绝对含量。结果显示,PHB对中国明对虾存活率、平均存活时间及体内病毒拷贝数均有一定影响,主要表现在与对照组(0.0%)相比,随PHB浓度的升高,实验组对虾存活率和平均存活时间呈现先上升后下降趋势。各组平均存活时间为82.23、90.71、95.55、91.15、85.56及79.40 h,1.0%浓度组实验对虾平均存活时间与0.5%及2.5%浓度组无显著差异(P0.05),但是显著高于其余各组(P0.05);各组累计死亡率均为100%。另外还发现各组平均病毒拷贝数为1.08×107,1.15×107,4.75×107,1.27×107,1.14×107,3.29×106个/ng DNA;对照组与1%浓度组病毒含量具有显著差异(P0.05)。结果表明,PHB能提高中国明对虾抗WSSV的能力且1%PHB浓度为最适浓度。     

WSSV人工感染量和饵料对中国明对虾存活时间的影响

为揭示不同白斑综合征病毒(white spot syndrome virus,WSSV)病毒量对于中国明对虾存活时间和存活率的影响,实验设计了逐尾、定量人工感染实验,在确保每尾中国明对虾进食特定量WSSV毒饵后进行观察、分析.结果显示,分别喂食含5.2×108 copies、1.0×109copies、2.1×109 copies WSSV的毒饵,对虾平均存活时间分别是389.3、323.3和187.3 h,差异极显著(P＜0.01);对虾最终累计死亡率都为100％.研究表明,致死量范围内,WSSV的感染量越低,对虾的平均存活时间越长.为了揭示饵料对中国明对虾抗病性能的影响,对已感染WSSV的中国明对虾投喂不同饵料.结果显示,分别喂食活卤虫、鲜蛤肉和配合饲料,对虾平均存活时间分别是281.7、173.9和164.9 h;喂食活卤虫的实验组平均存活时间显著高于喂食配合饲料和鲜蛤肉的实验组(P＜0.01);喂食配合饲料和鲜蛤肉的对虾平均存活时间无显著差异(P＞0.05);3组累计死亡率都为100％,结果表明,与配合饲料和鲜蛤肉相比,喂食活卤虫更能增强对虾抗WSSV的能力.     

白斑综合征病毒感染与对虾的免疫防御反应

对虾白斑综合征病毒（WSSV）感染对虾后，最典型的免疫反应是对虾开放循环系统的血淋巴细胞数量急剧下降，血淋巴凝结功能下降，感染部位聚集了大量的血淋巴细胞，且以颗粒细胞为主。WSSV可感染对虾颗粒细胞和小颗粒细胞，其中小颗粒细胞感染率高、感染速率快，感染后大颗粒细胞占血细胞总数的比例可增加到50％；血淋巴中的总糖、总碳水化合物、总蛋白和游离氨基酸显著提高，超氧化物歧化酶、过氧化氢酶和诱导性一氧化氮合成酶的活性显著降低。自然状态下广泛存在WSSV潜伏感染，潜伏感染的存在会导致存在免疫反应情况下的感染复发，并且有助于病毒的传播；不同WSSV感染状态下过氧化物（POD）差异显著，其平均值由大到小依次为：潜伏感染虾样、中度感染虾样、严重感染虾样。而其抗菌活性（UA）、溶菌活性（UL）、酶氧化酶活性（PO）、碱性磷酸酶（ALP）和凝集效价（HAT）差异不显著；潜伏感染个体对再次接种WSSV有“类免疫应答”的抗性，这种抗性不是来源于发病期对虾的天然抗性，而是WSSV感染后的一种免疫系统增强。宿主细胞凋亡可能是感染对虾在高温时反而维持较高成活率的主要机制。免疫增强剂可对对虾防御WSSV感染产生影响，脂多糖、葡聚糖、肽聚糖、岩藻依聚糖和双链核糖核酸都已被证实可提高对虾抗病毒感染的免疫保护。WSSV主要囊膜蛋白VP28可诱导对虾对WSSV感染产生抗性降低累积死亡率，高效价的病毒抗血清具有良好的保护作用；对虾抗菌肽也可通过抑制病毒的复制而起保护作用。     

氨氮和亚硝基氮共同胁迫对凡纳滨对虾感染WSSV的影响

为了评价养殖水环境中氨氮和亚硝基氮对凡纳滨对虾(Litopenaeus vannamei)的危害性,开展了氨氮和亚硝基氮共同胁迫对凡纳滨对虾感染WSSV后的死亡率、WSSV在患病对虾体内增殖速率和对虾主要免疫相关酶活性影响的研究。实验设置氨氮(NH+4)和亚硝基氮(NO-2)的共同胁迫浓度均为20 mg·L-1,分别注射10-4和10-5稀释度的WSSV提取液。结果显示,胁迫下感染10-4WSSV的凡纳滨对虾144 h死亡率达到100%,显著高于无胁迫组(76.67%),相同实验条件下高浓度病毒感染组死亡率高于低浓度组。对虾鳃组织WSSV荧光定量PCR检测结果显示,氨氮和亚硝基氮共同胁迫下凡纳滨对虾体内WSSV的增殖加快,感染48 h后胁迫组病毒量是无胁迫组的1.6倍,72 h时病毒量达到无胁迫组的2.0~3.7倍。此外,免疫相关酶活性结果显示,氨氮和亚硝基氮浓度突变会促使对虾血清中酚氧化酶(PO)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)活性先短暂升高然后降低。由此可见,氨氮和亚硝基氮共同胁迫会加快WSSV在患病凡纳滨对虾体内的增殖,导致更高死亡率,这可能是因为胁迫造成了对虾免疫相关酶活性的降低和抗病原感染能力下降所致。     

中国明对虾抗菌肽基因应答WSSV侵染的表达及其SNP分析

通过白斑综合征病毒(WSSV)感染实验,利用实时定量PCR技术研究了中国明对虾(Fenneropenaeus chinensis)应答病毒侵染后,已知的3种抗菌肽(对虾肽)在肝胰腺、肌肉、肠和鳃4种组织中的差异表达情况.结果显示,虽然3种抗菌肽表现出明显的组织表达特异性,即在不同组织中的表达趋势和表达丰富度存在明显的差异,但是就同一个组织而言,3种抗菌肽在1～120 h WSSV侵染区间内的表达趋势基本一致,在0 h(未侵染病毒)时,3种抗菌肽的表达量极低(为0);在6～24 h期间,检测到明显的表达量;48～120 h期间,3种抗菌肽的表达量总体呈现下降的趋势.这暗示3种抗菌肽在对虾机体内可能具有相似的生物学功能.在此基础上,本研究对各类型中国明对虾抗菌肽的SNP位点进行了筛选,进一步对不同SNP类型与抗WSSV或易感WSSV的关联程度进行了分析,结果显示3种抗菌肽基因的SNP位点很少,且在抗性和易感对虾群体内不存在明显的偏向分布.     

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.     

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

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.     

A Profound Effect of Hyperthermia on Survival of Litopenaeus vannamei Juveniles Infected with White Spot Syndrome Virus

This study was conducted to examine the effect of increasing seawater temperature on White Spot Syndrome Virus (WSSV) infection in juvenile Pacific White shrimp ( Litopenaeus vannamei ). Infection by WSSV was achieved using two methods, intramuscular injection and per os (oral) administration. Forty injected and 20 per os infected animals were kept in heated tanks at 32.3 ± 0.8 C, and the same number of WSSV infected animals were maintained in tanks at ambient temperature (25.8 ± 0.7 C). Despite the route of exposure, there were no survivors among the animals kept at ambient temperature; whereas, in heated tanks the survival of the WSSV infected juvenile shrimp was always above 80%, suggesting the existence of a beneficial effect from hyperthermia that mitigated the progression of WSSV disease. Moreover, this beneficial effect was not attributable to viral inactivation. Infected animals kept at 32 C had histologically detectable lymphoid organ spheroids suggestive of a chronic viral infection but were PCR negative (hemolymph) for WSSV. These findings might be related to low viral replication in WSSV-infected shrimp held at the higher environmental temperature. When the WSSV-infected shrimp were transferred from 32 C to ambient temperature, the mortality from WSSV ensued and was always 100%. Although the mechanism related to the beneficial effect of heating was not determined, our results indicate that increasing the water temperature modifies dramatically the natural history of the WSSV disease and the survival curves of WSSV-infected juvenile Pacific White shrimp.     

鲤病毒病原的感染性测定

为查明养殖鲤（Cyprinus carpio）突然大批发病死亡的原因，对鲤病样品进行了细胞攻毒、空斑测定、电镜观察，以及鱼体感染等实验。先以患病鲤的组织匀浆液，经过滤后，分别接种到草鱼鳍条细胞（GCF）、鲤上皮瘤细胞（EPC）等14种培养细胞中。利用倒置显微镜观察显示，在1-2d内，该病鱼组织匀浆液可使其中9种细胞出现典型的细胞病变。收集出现病变的细胞液（即病毒悬液），进一步进行病毒滴度检测、空斑测定和鱼体感染实验。结果显示，在GCF细胞上的病毒滴度为10^7.3TCID50／mL；在FHM，TSB和GCO等细胞中可产生直径1～4mm的圆形空斑，空斑的大小与宿主细胞的种类和接种的病毒浓度有关。通过对感染细胞制备的超薄切片和病毒负染样品进行电镜观察，显示这是一类呈典型子弹头样的弹状病毒颗粒。感染了病毒悬液的鲫和鲤先后在第2天和第3天开始出现病症，间隔1～2d后发病的鱼开始死亡，至第14天，两种感染鱼的死亡率均达到83．3％。收集人工感染后濒死的鲫和鲤，分别制备组织匀浆液，回接感染鱼类培养细胞，24h内能使其出现与原发病鲤组织匀浆液所引起的类似的细胞病变。因此证实患病鲤是由病毒病原感染所致。[中国水产科学，2006，13（4）：617—623]     

聚合酶链反应（PCR）检测养殖对虾的白斑症病毒（WSSV）感染

对虾WSSV病是亚洲对虾养殖业中的一个棘手问题。本研究采用Kimura引物 ,用PCR技术对不同生长期的中国对虾 (Penaeuschinensis)进行了WSSV的检测 ,同时也检测了对虾发病时养殖池中多见的野生厚蟹 (Helicesp .)和矛尾刺虎鱼 (Acanthogobiushasta)。检测结果表明 :分别在检测的 5尾亲虾中的 1尾 ,6尾仔虾中的 1尾 ,5尾稚虾中的 3尾及所检测的 5尾病虾和 2只厚蟹中获得到 982bp的PCR扩增产物 ,说明为WSSV感染阳性。在检测的 2尾矛尾刺虎鱼中均未获得PCR扩增产物 ,说明为WSSV感染阴性。在亲虾、虾苗以及虾池内的野生厚蟹中检测到WSSV感染的阳性结果表明 :WSSV感染的亲虾有可能是病毒的储主 ,WSSV感染的野生厚蟹有可能是病毒中间宿主或病毒的携带者 ,它们在对虾WSSV病的感染、传播中起了重要的作用     

Virulence status,viral accommodation and structural protein profiles of white spot syndrome virus isolates in farmed Penaeus monodon from the southeast coast of India

The objective of this study was to investigate the reason for variation in the virulence of white spot syndrome virus (WSSV) from different shrimp farms in the Southeast coast of India. Six isolates of WSSV from farms experiencing outbreaks (virulent WSSV; vWSSV) and three isolates of WSSV from farms that had infected shrimps but no outbreaks (non‐virulent WSSV; nvWSSV) were collected from different farms in the Southeast coast of India. The sampled animals were all positive for WSSV by first‐step PCR. The viral isolates were compared using histopathology, electron microscopy, SDS‐PAGE analysis of viral structural proteins, an in vivo infectivity experiment and sequence comparison of major structural protein VP28; there were no differences between isolates in these analyses. A significant observation was that the haemolymph protein profile of nvWSSV‐infected shrimps showed three extra polypeptide bands at 41, 33 and 24 kDa that were not found in the haemolymph protein profile of vWSSV‐infected shrimps. The data obtained in this study suggest that the observed difference in the virulence of WSSV may not be due to any change in the virus, rather it could be due to the shrimp defence system producing certain factors that help it to accommodate the virus without causing any mortality.     

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.