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.     

白斑综合征病毒感染克氏原螯虾后的PCR检测及组织病理学研究

采用投喂感染白斑综合征病毒(White Spot Syndrome Virus,WSSV)对虾肌肉的方式,对养殖克氏原螯虾(Procambarus clarkii)进行人工感染,以确定WSSV对养殖克氏原螯虾的易感性。结果发现,投喂病虾感染组螯虾的死亡率达到90%,而对照组未出现死亡。采用PCR对试验组螯虾的肌肉进行WSSV检测,发现投喂感染组的阳性检出率为100%,对照组的阳性检出率均为0。PCR检测结果发现,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏六种组织的PCR结果均为WSSV阳性,而对照组的各组织检测结果均为阴性。组织切片的光镜观察也证实,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏及血淋巴等组织均发生了不同程度的病变。     

养殖克氏原螯虾体内白斑综合征病毒的绝对定量分析

近年来克氏原螯虾的养殖受到WSSV的威胁,病毒在宿主组织中的绝对定量对于了解病毒的致病性具有重要意义,但克氏原螯虾组织中WSSV的绝对定量分布还有待研究。实验调查了湖北省5个主养区克氏原螯虾WSSV的感染率,结果表明80%以上克氏原螯虾都携带有WSSV。采用WSSV-VP28蛋白特异性抗体对克氏原螯虾提取蛋白进行Western Blot检测,在WSSV-PCR阳性样品中可检测到VP28特异性条带,在WSSV-PCR阴性样品中没有检测到相应条带。采用实验室建立的WSSV绝对定量PCR方法,对携带病毒的克氏原螯虾6个组织(鳃、胃、肠、血淋巴细胞、肝胰腺和心脏)进行检测。结果表明,在鳃、胃和肠可检测到较多病毒量(约108拷贝/mg),其次是血淋巴细胞(107拷贝/mg)、肝胰腺(106拷贝/mg),在心脏中病毒的含量最低(103拷贝/mg),表明病毒的复制存在组织特异性。结果显示WSSV主要存在于消化系统中,预示着克氏原螯虾可能主要在摄食过程中感染WSSV;不同地区克氏原螯虾组织病毒携带量表现出一定差异,预示着WSSV感染可能受到环境因素的影响。     

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.     

Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection

Three members of the tetraspanin/TM₄SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis . The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM₄SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene ( FcCD9 ) was specifically expressed in the hepatopancreas. While for the CD63 gene ( FcCD63 ), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63 , the tetraspanin-3 gene ( FcTetraspanin-3 ) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9 , FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM₄SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.     

Assessment of transmission risk in WSSV-infected shrimp Litopenaeus vannamei upon cooking

White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp-producing countries have launched regulations to prevent import of WSSV-infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV-infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV-infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0-min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0-min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported.     

Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus

White spot syndrome virus (WSSV) is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimp are cultured, as well as in the Gulf of Mexico and SE USA. The virulence of six geographic isolates of WSSV was compared using Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. For challenge studies, virus infected tissues were given per os to L. vannamei postlarvae and Fa. duorarum juveniles. Resultant WSSV infections were confirmed by histological examination. The cumulative mortality of L. vannamei postlarvae reached 100% after challenge with each of the six geographic isolates of WSSV. However, the Texas isolate caused mortalities more rapidly than did the other shrimp isolates; the crayfish WSSV isolate was the slowest. In marked contrast, cumulative mortalities of juvenile Fa. duorarum reached only 35–60%, and varied among the geographic isolates of WSSV. Interestingly, in Fa. duorarum, the Texas WSSV isolate was also the most virulent, while the crayfish WSSV was the least virulent. The findings suggest that slight differences in virulence exist among geographic isolates of WSSV, and that susceptibility may vary with species and lifestages of the host.     

刀额新对虾原代淋巴细胞培养及其感染白斑综合征病毒（WSSV）的病理特征

为深入了解对虾白斑综合征病毒(WSSV)的致病机理和体外培养的对虾细胞对WSSV的敏感性,实验以1.5×L-15培养基培养刀额新对虾原代淋巴细胞,待细胞形成单层后接种WSSV,通过倒置显微镜、荧光显微镜、透射电子显微镜观察接种病毒后细胞的病理变化。结果显示,使用1.5×L-15培养基培养对虾淋巴组织,3 h后可观察到有细胞迁出,并能迅速形成单层,36 h后细胞的迁出汇合率可达80%,且能存活20 d以上。接种WSSV 24 h后,出现病变的细胞变圆、漂浮,细胞之间的网状结构消失,最后细胞破碎、溶解;接种WSSV 48 h后Hoechst 33342染色结果显示,感染的细胞核深染,且变形、膨大;电镜下,细胞核内含大量成簇分布的杆状病毒,细胞器被挤向细胞边缘,细胞膜轮廓模糊。研究表明,纯化的病毒粒子接种体外培养的淋巴细胞,能够使其产生明显病理变化,证明了WSSV对体外培养的淋巴细胞具有感染力,并且可在淋巴细胞中增殖。     

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.     

Identification and characterization of Alix/AIP1 interacting proteins from the black tiger shrimp,Penaeus monodon

Apoptosis is proposed to be a major cause of death in shrimp viral infections. From our previous study, an apoptosis-related gene, Pm-Alix, was identified from the black tiger shrimp. Its expression was high in defence-related tissues including haemocytes and the lymphoid organ. To clarify its possible role in shrimp, we used Pm-Alix as bait in a yeast two-hybrid analysis to search for Alix interacting proteins in shrimp. Two cDNA sequences discovered had homology to a predicted ubiquitin C of the purple sea urchin, Strongylocentrotus purpuratus, and to a guanylyl cyclase of the red swamp crayfish, Procambarus clarkii. In vitro pull-down assays confirmed positive interaction between Pm-Alix and both proteins. Tissue distribution analysis revealed that Pm-Alix and the two binding partners were widely expressed in various tissues but more highly expressed in haemocytes. However, no significant positive or negative correlation was found in the expression of these genes as shrimp approached morbidity and death after challenge with white spot syndrome virus. Thus, the results suggested that Alix and its interacting partners did not play a direct role related to shrimp death.     

中国对虾雄性生殖系统感染WSSV在其垂直传播中的作用

通过人工感染实验,对中国对虾(Fenneropenaeus chinensis)雄性亲虾进行投喂感染。在确定其携带WSSV粒子后,将被WSSV感染的精荚人工移植到健康的雌虾纳精囊内。在无其他病源的情况下,促其产卵繁殖,统计各组子代的受精率、孵化率及无节幼体至溞状幼体的变态率贸?式PCR技术对亲虾及子代进行WSSV检测。结果表明,受WSSV感染的精荚能够把病毒传播给健康雌虾,雌虾能产出携带WSSV的卵子,培育出带毒幼体。各组子代的受精率、孵化率及变态率的统计结果表明,感染组和对照组在受精率上没有明显区别,受WSSV感染的精卵细胞可以正常结合。对照组受精卵的孵化率明显高于感染组,差异显著(P=0.045<0.05)。对照组无节幼体的变态率也高于感染组。说明WSSV的入侵对受精卵及幼体的发育有影响,WSSV感染导致部分受精卵及幼体不能正常发育或死亡。     

卤虫在WSSV病毒病传播中的媒介作用

2003年8月对卤虫进行投喂(浸泡)感染，并进行中国明对虾投喂实验。各组对虾阳性感染率的单因素方差分析比较结果表明，卤虫可以携带有活性的WSSV病毒粒子，并可通过摄食导致对虾间接地携带病毒，但病毒感染能力有限。卤虫病理切片观察结果显示，攻毒后卤虫细胞核没有显著深染及核肿大现象，但无论是横切还是纵切均显示有上皮脱落、部分组织结构松散、细胞结构不完整现象。本试验确定卤虫成体可以携带WSSV并通过投喂感染对虾，使其潜在携带病毒，仔虾阶段投喂成体卤虫应经过严格检疫，制作含有经过检疫卤虫的微囊饵料作为卤虫及其他鲜活饵料的替代品或许是有效的防病措施。     

Oral vaccination trials with crayfish, Procambarus clarkii, to induce resistance to the white spot syndrome virus

The potential of oral vaccination against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. The protective effect of binary ethylenimine (BEI)-inactivated WSSV was tested by oral vaccination, followed by an oral challenge with WSSV. The crayfish fed with feed pellets coated with BEI-inactivated WSSV showed a resistance to WSSV on the seventh day post vaccination (dpv). The relative percentage survival values were 60%, 70% and 75% for the vaccinated once, twice and thrice with inactivated WSSV. Following an intramuscular injection experiment, no mortality was recorded in the inactivated WSSV group and the negative control at 17 days post challenge. The cumulative mortalities in the heated WSSV group and WSSV group were 100%. Shrimp that survived the WSSV challenge on the seventh day after cessation of oral vaccination were positive for the presence of WSSV by a polymerase chain reaction assay specific for WSSV. This result indicated that inactivated WSSV could protect crayfish against WSSV by oral delivery.     

自然养殖水体投喂CpG ODN和表面展示VP28的解脂耶罗维亚酵母对凡纳滨对虾(Litopenaeus vannamei)抗WSSV的免疫保护作用

用添加CpG寡聚核苷酸(CpG ODN)和表面展示VP28的解脂耶罗维亚酵母(VP28-yl)的饵料投喂凡纳滨对虾,进行田间中试实验。投喂30 d后进行WSSV感染实验,评估其对凡纳滨对虾的免疫保护作用。投喂实验结束后,CpG ODN投喂组对虾的相对增重率达到(65.8±7.8)% (P<0.05),这暗示CpG ODN可能具有促生长作用。WSSV攻毒后,CpG ODN和VP28-yl投喂组对虾中WSSV拷贝数与对照组相比均显著降低(P<0.05),相对免疫保护率分别可达到26.7%和36.7%。在投喂结束和WSSV刺激后,CpG ODN组对虾中的呼吸爆发水平均显著升高(P<0.05)。而在VP28-yl投喂组,WSSV引起的细胞凋亡则显著受到抑制(P<0.05)。此外,WSSV刺激后,STAT基因在CpG ODN组和VP28-yl组对虾中的表达水平均显著上调(P<0.05),分别在第5天和第3天达到最大值,而对照组中则显著下调。研究结果表明,CpG ODN和VP28-yl增强了凡纳滨对虾抗病毒免疫力,对养殖对虾病毒性疫病的防控具有显著作用,可以作为免疫增强剂添加在饵料中,具有在养殖生产中推广使用的前景。     

Studies on pathogenicity and prevalence of white spot syndrome virus in mud crab, Scylla serrata (Forskal), in Zhejiang Province, China

Mud crab, Scylla serrata (Forskal), is the most commercially important marine crab species in China. In recent years, serious diseases have occurred in major mud crab culture regions in SE China. PCR detection of white spot syndrome virus (WSSV) in diseased mud crabs collected from Zhejiang Province during 2006–2008 showed a prevalence of 34.82%. To study the pathogenicity of WSSV to mud crab, healthy mud crabs were injected intramuscularly with serial 10‐fold dilutions of a WSSV inoculum. The cumulative mortalities in groups challenged with 10^?1, 10^?2, 10^?3 and 10^?4 dilutions were 100%, 100%, 66.7% and 38.9% at 10 days post‐injection, respectively. All moribund and dead mud crabs except the control group were positive for WSSV by PCR. Based on the viral load of the WSSV inoculum by quantitative real‐time PCR, the median lethal dose (LD50) of WSSV in S. serrata was calculated as 1.10 × 10⁶ virus copies/crab, or 7.34 × 10³ virus copies g^?1 crab weight. The phenoloxidase, peroxidase and superoxide dismutase activities in haemolymph of WSSV‐infected moribund crabs, were significantly lower than the control group, whereas alkaline phosphatase, glutamate‐pyruvate transaminase and glutamic‐oxaloacetic transaminase were higher than in the control group. WSSV was mainly distributed in gills, subcuticular epithelia, heart, intestine and stomach as shown by immunohistochemical analysis with Mabs against WSSV. The epithelial cells of infected gill showed hypertrophied nuclei with basophilic inclusions. Numerous bacilliform virus particles were observed in nuclei of infected gill cells by transmission electron microscopy. It is concluded that WSSV is a major pathogen of mud crab with high pathogenicity.     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.     

Detailed monitoring of white spot syndrome virus (WSSV) in shrimp commercial ponds in Sinaloa, Mexico by nested PCR

WSSV has caused great losses to the global shrimp industry in recent years. This virus can infect shrimps asymptomatically. However, once the clinical signs are developed, mortalities can reach 100% in 3-10 days. PCR has been extensively used to detect WSSV in a specific and sensitive manner. Nested PCR is even more sensitive than single-step PCR and had been used for the detection of WSSV in asymptomatic populations. In this work, a detailed monitoring of WSSV by nested PCR in shrimp commercial ponds in Guasave County, State of Sinaloa, Mexico, is presented. Five ponds from two different farms were monitored for growth and presence of WSSV. At the beginning of the culture, ponds from both farms showed no or very slight WSSV presence. A 3-day period of rain occurred at both farms 10 and 14 weeks of culture for farms 1 and 2, respectively. At this time, WSSV was widely distributed in the shrimp populations of farm 1 according to nested-PCR data, although no visual symptoms were observed. In ponds of farm 2, WSSV was present at low level. However, the number of PCR-positive groups was drastically increased in both farms by nested and single-step PCR. Abrupt fluctuations in temperature and salinity were documented in farm 2 after the rain, which may have contributed to the increasing of viral load in the pond's shrimp populations. Twelve days after the rain period, estimated mortalities of 80% occurred in farm 1. Nevertheless, the study ponds at farm 2 culture continued normally for three more weeks and were harvested successfully (52% and 67% of survival for ponds 1 and 2, respectively). The removal of 40% and 50% of shrimp population 2-4 days after the raining period may have contributed to the thriving of the cultures. Analyses of the presence of WSSV in individuals of both sexes indicated that there is no preference for this virus to infect male or female shrimp. Also, no differences in weight were found between WSSV infected and non-infected individual shrimps, as well as nested-PCR positive against single-step PCR positive organisms. Nested PCR is more useful to monitor shrimp cultures than single-step PCR since it allows knowing how widely distributed the virus is in asymptomatically populations.