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.     

Larvae and early post-larvae of Penaeus monodon (Fabricius) experimentally infected with white spot syndrome virus (WSSV) show no significant mortality

The pathogenicity of white spot syndrome virus (WSSV) was tested with different developmental stages of Penaeus monodon, i.e. nauplius, protozoeae, mysis, early post-larvae (PL1-10), late post-larvae (PL11-20) and juveniles. WSSV challenge was done by immersion and oral routes. No disease occurred in the larvae and early post-larvae but they were positive for WSSV by nested polymerase chain reaction (PCR) assay. Significant mortality was observed in late post-larvae and juveniles and both single and nested PCR assays gave positive results with these samples. The results demonstrated that WSSV virulence in P. monodon increases with advancing stages of development and that WSSV infection does not result in disease for larvae and post-larvae younger than PL10.     

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

A flow‐through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL^?1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1‐step polymerase chain reaction (PCR) and in between that of the 1‐ and 2‐step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post‐larvae, WSSV was first detected 14, 16 and 18 h post‐infection (hpi) by FTA, immunodot and one‐step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one‐step PCR, respectively. The FTA was more sensitive (25/27) than one‐step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions.     

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.     

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.     

Analysis of viral load between different tissues and rate of progression of white spot syndrome virus (WSSV) in Penaeus monodon

At present the most common and most devastating disease of shrimp is caused by the white spot syndrome virus (WSSV), which has spread throughout the world mainly by different species of crustaceans carrying the virus. After experimental injection of Penaeus monodon with a known copy number of WSSV in the abdominal muscle, the rate of viral progression in different tissues at 12, 24, 36 and 48 hpi (hours post infection) was assessed using quantitative real‐time PCR. At 12 hpi the viral load was highest in haemocytes followed by pleopod, muscle and gills whereas at 48 hpi, the gills, the main target of WSSV, showed the highest viral load followed by pleopod, muscle and haemocytes. Viral copy number in the haemocytes was the lowest beyond 12 hpi indicating a remarkable reduction in the rate of viral replication in haemocytes compared with other tissues. The viral load in haemocytes, though increased again beyond 36 hpi, never surpassed the load in the other tissues. The real‐time PCR assay with its high sensitivity and wide dynamic range make it ideal for detecting low‐level WSSV infections that can occur in apparently healthy P. monodon.     

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.     

White spot syndrome virus (WSSV) prevalence associated with disease resistance among wild populations of black tiger shrimp,Penaeus monodon (Fabricius)

White spot disease caused by white spot syndrome virus (WSSV) is the major issue of huge economic destruction globally in the shrimp aquaculture industry. In the present investigation, WSSV prevalence associated with disease resistance was studied among wild black tiger shrimp, Penaeus monodon (Fabricius) from four distant geographic locations along the East coast of India during 2009–2010. Results suggested that the WSSV prevalence in wild P. monodon was the highest (56.2%) in Chennai, Tamil Nadu followed by Digha, West Bengal (10.9%), Visakhapatnam, Andhra Pradesh (0.6%) and Chilika, Orissa (0%). Quantitative data suggested that the mean copy number of WSSV among these four places was 1.4 × 10⁶, 4.6 × 10⁴, 1.6 × 10² and 2.3 × 10² copies μg^?1 shrimp genomic DNA respectively. The disease resistant prevalence using the 71 bp microsatellite DNA marker was the highest among Chilika, Orissa (63.6%) and Visakhapatnam, Andhra Pradesh (63.5%). Higher WSSV prevalence in Chennai, Tamil Nadu and Digha, West Bengal corresponded to lower disease resistant prevalence (24% and 40.2%). Conclusively, probably collection of broodstock of P. monodon from places like Chilika and Visakhapatnam would be a much safer approach for the development of specific pathogen‐resistant shrimp aquaculture.     

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.     

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     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

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.     

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.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.