Quantitative analysis of an experimental white spot syndrome virus (WSSV) infection in Penaeus monodon Fabricius using competitive polymerase chain reaction

White spot syndrome virus (WSSV) has been a major pathogen of cultured Penaeus monodon Fabricius in Malaysia since 1994. As quantitative study on the replication of WSSV is in its infancy, competitive polymerase chain reaction (PCR) was used for quantitative study of an experimental WSSV infection per os in growout P. monodon . Gills, abdominal integument and abdominal muscle were selected for viral quantification. Infection was detectable as early as 14 h postinfection (h p.i.) in both gills and integument, but the infection in muscle was only detected at 24 h p.i. Gill tissue had the highest viral load, followed by integument and muscle. Typical viral growth curves were obtained for all organs with distinct phases of eclipse (0–24 h p.i.), logarithmic (24–48 h p.i.) and the plateau (48–120 h p.i.). Cumulative mortality rapidly increased from 48 h p.i. and reached 100% at the end of the plateau phase at 120 h p.i. Gross signs of white spots and reddish discoloration were also obvious in moribund individuals from the plateau phase. Based on the three phases of viral growth, WSSV infection was classified into light, moderate and heavy infection stages.     

Evidence for the presence of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild Penaeus monodon (Fabricius) broodstock,in the southeast coast of India

A survey on the presence of the viruses of two economically significant diseases, white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild‐collected Penaeus monodon broodstock, was conducted during different seasons of the year in two major coastal areas of southeast India. The broodstock were collected along the coast of Tamil Nadu and Andhra Pradesh during summer, premonsoon, monsoon and post‐monsoon seasons for three consecutive years. A total of 7905 samples were collected and subjected to MBV screening, and 6709 samples that were screened as MBV negative were diagnosed for WSSV. MBV was detected using rapid malachite green staining and WSSV by nested polymerase chain reaction. Prevalence data of the viruses were analysed using the EpiCalc 2000 program at 95% confidence interval. Samples collected from the Andhra Pradesh coast displayed a slightly higher prevalence of WSSV and MBV infection than those collected from Tamil Nadu, although this difference was not statistically significant (P > 005). In addition, it was found that the prevalence of both WSSV and MBV infections fluctuated according to season. Data on prevalence of these viruses in broodstock would be useful to develop strategies for shrimp health management along the southeast coast of India.     

Limited evidence for genetic variation for resistance to the white spot syndrome virus in Indian populations of Penaeus monodon

There has been a highly detrimental impact of the white spot syndrome virus (WSSV) on black tiger shrimp (Penaeus monodon) aquaculture in India. Currently, no cost‐effective measures are available for controlling the disease. One alternative is to improve WSSV resistance through a selective breeding programme for disease‐resistant shrimp, provided that genetic variation exists for this trait. The aim of this study was to evaluate the evidence for genetic variation in resistance to WSSV in P. monodon sourced from Indian populations. Post‐larval shrimp (n=1950) from 54 full‐sibling families were challenged with WSSV using WSSV‐infected mince meat. The heritability was estimated using four different statistical models fitted to the resulting time to death data, including two linear models and two Weibull proportional hazard frailty models. None of the estimated heritabilities were significantly different from zero. We suggest three possible explanations for these results: there actually is very little variation between P. monodon in WSSV resistance and all individuals are highly susceptible to the disease; there is genetic variation in resistance to WSSV in P. monodon but we did not find it in our experiment because the level of challenge in the experiment was too high to allow genetic differences to be expressed; the variation is due to mutations conferring resistance, which are at a low frequency in the population, and we did not sample a broad enough genetic base to capture these mutations.     

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 10⁵ viral DNA copies.     

TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan‐based real‐time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L^?1) for 10 days and challenged with WSSV by feeding WSSV‐positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post‐infection. The TaqMan real‐time PCR assay showed a highly dynamic detection limit that spanned over 6 log₁₀ concentrations of DNA and high reproducibility (standard deviation 0.33–1.42) and small correlation of variability (CV) (1.89–3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L^?1 of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L^?1 of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L^?1 (TAN) had increased susceptibility to WSSV.     

白斑综合症病毒（WSSV）对中国对虾卵及各期幼体人工感染的试验研究

用感染白斑综合症毒病的中国对虾头胸甲，对健康的仔虾进行了人工投喂感染实验，同时从病虾中分离出病毒悬液作为毒种，无节幼体，蚤体 幼体和糠虾体进行不同温度条件下的人工浸浴实验，结果表明，卵和无节幼体，蚤状幼体，糠虾幼对病毒悬液不敏感,闰理切片观察未见病毒粒子，光镜病理切片可观空到鳃上皮，前肠上皮有包涵体样病变，同时在肝胰组织中发现了肝胰腺细小病毒（HPV）包涵体。     

斑节对虾白斑综合症病毒部分基因组文库及核酸探针检测法

通过分离纯化白斑综合症病毒（ＷＳＳＶ）粒子,抽提病毒ＤＮＡ。用限制性内切酶ＥｃｏＲⅠ或ＳａｌⅠ酶切后,克隆入质粒ｐＢｌｕｅｓｃｒｉｐｔⅡＫＳ中,从而建立了ＷＳＳＶ部分基因组文库。估计ＷＳＳＶ基因组ＤＮＡ在１６５ｋｂ以上。将ＷＳＳＶ ＥｃｏＲⅠ克隆片段标记制备为探针。进行Ｓｏｕｔｈｅｒｎ杂交、打点杂交和原位杂交,其结果证明了克隆片段对ＷＳＳＶ特异,并为检测ＷＳＳＶ提供了方法。通过对部分基因组文库序列     

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.     

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.     

Indian isolates of white spot syndrome virus exhibit variations in their pathogenicity and genomic tandem repeats

To detect genomic variation of white spot syndrome virus (WSSV) isolates from different geographical regions of India, the variable number of the tandem repeat (VNTR) region of the ORF 94 (Thailand WSSV isolate – GeneBank Accession No. AF369029 ) was analysed using five specific sets of primers. Analysis of 70 WSSV‐positive samples showed the presence of 14 different genotypes of WSSV with VNTRs ranging from 2 to 16 tandem repeats with the majority (85.47%) having 6–12 tandem repeats. Occurrence of different genotypes of WSSV was found to be neither correlated to any specific geographical region nor to the different growth stage of the tiger shrimp, Penaeus monodon. Pathogenicity studies conducted with 25 isolates of WSSV revealed the presence of virulent and avirulent strains of WSSV in Indian shrimp farms. However, an unambiguous link could not be established between the different genotypes and their virulence.     

Detection of monodon baculovirus and white spot syndrome virus in apparently healthy Penaeus monodon postlarvae from India by polymerase chain reaction

The simultaneous presence of monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in apparently healthy postlarvae of Penaeus monodon from different hatcheries in India was studied by nested polymerase chain reaction (PCR). MBV could be detected in 54% of the samples. However, only 15% of samples were positive by non-nested reaction. WSSV could be detected in 75% of samples, 19% being positive by non-nested reaction. The results show simultaneous presence of WSSV and MBV in many samples at various degrees of infection. Only 14% of the samples analysed were negative for both viruses.     

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.     

Characterization and application of monoclonal antibodies against white spot syndrome virus

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG₁ class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.     

The incidence of suspected white spot syndrome virus in semi‐intensive and extensive shrimp farms in Bangladesh: implications for management

The study was conducted to assess key factors influencing suspected white spot syndrome virus (WSSV) disease and associated shrimp production and economic performance in three contrasting black tiger shrimp (Penaeus monodon) culture technologies promoted by the United States Agency for International Development funded Shrimp Quality Support Project (SQSP) in Bangladesh. A total of 350 traditional, 315 Modified Traditional Technology1 (MTT1), 36 MTT2 and 88 Closed System Technology (CST) farmers from 10 sub‐districts in three districts of Khulna division were surveyed following random sampling at the end of the project. Binomial probit regression analysis revealed that smaller newly constructed ponds (known locally as gher) were less susceptible to WSSV, provided aquatic weeds were controlled using chemicals. Removal of sludge from ghers also had a positive effect, irrespective of technology and location. It was also shown that stocking of screened shrimp postlarvae (PL) does not guarantee protection against WSSV (t = 1.39, P > 0.05). Higher shrimp production was obtained by farmers practicing CST, followed by those operating MTTs and traditional technology respectively. Farmers who adopted CST also gained higher profitability followed by those operating MTT1, MTT2 and traditional technology.     

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L^?1 were subjected to salinities of 50 g L^?1, 35 g L^?1, 20 g L^?1, 10 g L^?1 and 7 g L^?1 or 5 g L^?1 and simultaneously exposed to 10^5.5 SID₅₀ mL^?1 of WSSV for 5 h, after which the salinity was brought back to 35 g L^?1. Shrimp that were transferred from 35 g L^?1 to 50 g L^?1, 35 g L^?1 and 20 g L^?1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L^?1 to 10 g L^?1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L^?1, 7 g L^?1 and 5 g L^?1, was 6.7%, 46.7% and 53.3%, respectively (P < 0.05)_. For testing the effect of moulting, shrimp in early premoult, moulting and post‐moult were immersed in sea water containing 10^5.5 SID₅₀ mL^?1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post‐moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L^?1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.     

白斑综合征病毒囊膜蛋白vp28基因在毕赤酵母中的表达

白斑综合征病毒(whitespotsyndromevirus,WSSV)是危害东南亚及北美洲沿岸养殖对虾的重要病原。近十年来,国内外对该病毒的形态结构、基因组全序列、宿主范围、传播途径、致病性和组织细胞特异性等作了大量的研究[1-7],并已确定该病毒的侵染与其囊膜蛋白vp28有关[8]。实验根据已发表的白斑综合征病毒囊膜蛋白vp28基因序列[9]设计一对引物,通过PCR扩增出该囊膜蛋白的基因片段。将该片段连接到毕赤酵母表达载体pPICZ上,在大肠杆菌中筛选到含目的基因的重组质粒pPICZVP28,用电穿孔法将重组质粒转化到毕赤酵母菌株X33中,获得了转化子X33 …     

Experimental susceptibility of different life-stages of the giant freshwater prawn, Macrobrachium rosenbergii (de Man), to white spot syndrome virus (WSSV)

Studies were conducted by injecting/feeding white spot syndrome virus (WSSV) derived from infected shrimp, Penaeus monodon (Fabricius), to different life-stages, namely post-larvae, juveniles, sub-adults and adults of Macrobrachium rosenbergii (de Man). The disease was also induced in brood stock, and the eggs and larvae derived from these animals were subsequently tested for WSSV infection. All the stages except egg used for the experiment were found WSSV positive in histopathology, cross infection bioassay and polymerase chain reaction (PCR) analysis. Experimentally infected post-larvae and juveniles showed a high percentage of mortality and an increased rate of cannibalism. The cumulative mortality in post-larvae was up to 28%; with 28–40% cannibalism resulting in a maximum loss of up to 68%. In juveniles, observed mortality and cannibalism were 10–20% and 6.7–30.0%, respectively, and the maximum loss recorded was 50%. In sub-adults, mortality ranged from 2.8 to 6.7%, cannibalism was up to 20% and the total loss was up to 26.7%. Sub-adults and adults were found to be more tolerant to the infection as evidenced by the mortality pattern. A nested (two-step) PCR resulted in a 570-bp product specific to WSSV in all stages, except the eggs.