Spot Syndrome Virus In Shrimp Research Articles

Dietary probiotic Bacillus OJ and isomaltooligosaccharides influence the intestine microbial populations, immune responses and resistance to white spot syndrome virus in shrimp ( Litopenaeus vannamei)

In order to reveal the potential of Bacillus OJ (PB) as probiotic and the synergistic effects of PB and isomaltooligosaccharides on intestine microbial populations, immune responses and disease resistance of shrimp ( Litopenaeus vannamei), a 28-day feeding experiment was conducted in shrimp by feeding PB at doses of 0, 10 8, and 10 10 CFU/g feed with or without 0.2% isomaltooligosaccharides in shrimp diets. After a 14-day feeding experimental period, six shrimp per tank were sampled for bacterial quantification and immunity determination. Then all the shrimp left were challenged by white spot syndrome virus. Results showed that, with the increasing doses of PB in diets, shrimp survivals and immune parameters generally increased whereas the counts of total viable bacteria and Vibrio decreased. Shrimp fed with PB at dose of 10 10 CFU/g of feed produced significantly higher survivals and immune parameters than the control groups. Although the effect of 0.2% isomaltooligosaccharides alone was negligible, 0.2% isomaltooligosaccharides in combination with 10 8 CFU/g feed PB produced significantly positive synergistic effects on shrimp immune responses and disease resistance. While the doses of PB increased to 10 10 CFU/g feed, the synergistic effects significantly decreased. Then, our results confirm the potential of PB as dietary probiotic and the synergistic effects of PB and isomaltooligosaccharides in shrimp.

Silencing VP28 Gene of White Spot Syndrome Virus of Shrimp by Bacterially Expressed dsRNA

An in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria to provide a practical control of white spot syndrome virus (WSSV) in shrimp was developed. The bacterially synthesized dsRNA specific to VP28 gene of WSSV promoted gene-specific interference with the WSSV infection in shrimp. Virus infectivity was significantly reduced in WSSV-challenged shrimp injected with VP28-dsRNA and 100% survival was recorded. The inhibition of the expression of WSSV VP28 gene in experimentally challenged animals by VP28-dsRNA was confirmed by RT-PCR and Western blot analyses. Furthermore, we have demonstrated the efficacy of bacterially expressed VP28-dsRNA to silence VP28 gene expression in SISK cell line transfected with eukaryotic expression vector (pcDNA3.1) inserted with VP28 gene of WSSV. The expression level of VP28 gene in SISK cells was determined by fluorescent microscopy and ELISA. The results showed that the expression was significantly reduced in cells transfected with VP28dsRNA, whereas the cells transected with pcDNA-VP28 alone showed higher expression. The in vivo production of dsRNA using prokaryotic expression system could be an alternative to in vitro method for large-scale production of dsRNA corresponding to VP28 gene of WSSV for practical application to control the WSSV in shrimp farming.

Screening the antiviral activity of Indian medicinal plants against white spot syndrome virus in shrimp

Twenty species of Indian traditional medicinal plants were selected for this study. They were Aegle marmelos, Allium sativum, Aristolochia indica, Azadirachta indica, Cassia fistula, Catharanthus roseus, Curcuma longa, Cynodon dactylon, Lantana camara, Melia azedarach, Mimosa pudica, Momordica charantia, Morus alba, Ocimum americanum, Phyllanthus amarus, Phyllanthus emblica, Psidium guajava, Solanum nigrum, Tridax procumban and Tylophora indica. These plants were used to extract antiviral substances with petroleum ether, benzene, diethyl ether, chloroform, ethyl acetate, methanol, ethanol and distilled water separately using a Soxhlet apparatus. Each extract was tested for its activity against WSSV in marine shrimp and fresh water crabs. Only five plants among these twenty ( A. marmelos, C. dactylon, L. camara, M. charantia and P. amarus) showed antiviral activity against WSSV. The aqueous extract of C. dactylon showed strong antiviral activity at the concentration of 100 mg/kg of animal body. The methanol extract of M. charantia showed significant antiviral activity at the concentration of 150 mg/kg of animal body weight. The aqueous extract of L. camara and P. amarus and methanol extract of A. marmelos showed partial antiviral activity at the concentration of 150 mg/kg of animal body weight.

Studies on the inactivation of white spot syndrome virus of shrimp by physical and chemical treatments, and seaweed extracts tested in marine and freshwater animal models

Studies on the inactivation of white spot syndrome virus of shrimp by physical and chemical treatments, and seaweed extracts tested in marine and freshwater animal models

Development of an immunochromatographic test to detect White Spot Syndrome Virus of shrimp

A rapid, single step, sensitive and specific immunochromatographic test (ICT) is described for detecting white spot syndrome virus (WSSV) using anti-WSSV monoclonal antibodies (MAbs), 2E6 and 2A3, labeled with colloidal gold as a detection reagent and anti-WSSV MAb 1D5 as a capture antibody immobilized on nitrocellulose membrane (NCM). The test process was completed within 10 min without incubation or any other equipment. A reddish color at the site of immune reaction on NCM indicated a positive result and 1 μg/ml of WSSV could be detected. The assay sensitivity could be increased to 0.01 μg/ml by silver enhancement. The sensitivity of ICT was similar to that of dot-blot nitrocellulose enzyme immunoassay (DB-NC-EIA). However, ICT is a method that is more convenient and can be quickly performed pond side without false positive results.

Development of dot-immunogold filtration assay to detect white spot syndrome virus of shrimp

The dot-immunogold filtration assay (DIGFA) detects white spot syndrome virus (WSSV) by using a nitrocellulose membrane (NCM) as a support of WSSV and anti-WSSV monoclonal antibodies (MAbs) 1D5 and 6A4 labelled with red color colloidal gold. The principle of filtration for the rapid reaction of antigen and antibody is adopted in order for the test process to be completed within 3 min without incubation or any equipment. A reddish dot indicative of a positive result is present visibly. In comparison with dot-blot nitrocellulose enzyme immunoassay (DB-NC-EIA), the sensitivity of DIGFA is similar. However, DIGFA is a method that can be performed conveniently pond side providing a faster result without the production of false positive results.

Neutralization of white spot syndrome virus of shrimp by antiserum raised against recombinant VP28

An attempt was made to neutralize white spot syndrome virus of shrimp using the antiserum against recombinant VP28 protein of WSSV. The dilutions of virus stock ranged from 1 × 10 1 to 1 × 10 8 were used to determine the dilution resulting in 90–100% mortality in Penaeus monodon by intramuscular injection of WSSV. A constant amount of WSSV (at 1 × 010 4) was incubated with various antiserum concentrations and injected into shrimp. No shrimp died in the negative control injected only with NTE buffer. The shrimp in the positive control, which were injected with WSSV only showed 100% mortality at 10-day post infection (d p.i.). The WSSV preincubated with preimmune serum caused 100% mortality of shrimp at 10 d p.i. None of the shrimp injected with the preimmune serum only died. When the WSSV was preincubated with two or one time diluted anti r-VP28, shrimp mortality was 100% and 75%, respectively, at 50 d p.i. apparently the anti r-VP28 at these dilutions is not able to neutralize the WSSV. When the WSSV was preincubated with undiluted anti r-VP28, none of the shrimp died, indicating that WSSV can be neutralized by the anti r-VP28 in a dose-dependent manner.

Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp

White spot syndrome virus (WSSV) is type species of the genus Whispovirus of the new family Nimaviridae. Despite the elucidation of its genomic sequence, very little is known about the virus as only 6% of its ORFs show homology to known genes. One of the structural virion proteins, VP15, is part of the nucleocapsid of the virus and shows homology to some putative baculovirus DNA binding proteins. These DNA-binding or histone-like proteins are thought to be involved in the condensation and packaging of the genome in the nucleocapsid. Using bacterially expressed VP15 fusion proteins in ELISA and Far-Western experiments showed that VP15 interacts with itself, forming homomultimers, but not with the other major structural proteins of the WSSV virion. Antibodies against phosphorylated proteins revealed that VP15 originating from different sources was not phosphorylated. WSSV VP15 binds non-specifically to double-stranded DNA, but has a clear preference to supercoiled DNA suggesting that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. This research shed further light on the composition of WSSV virions and the function of one of its nucleocapsid proteins.

Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) method is a novel, sensitive and rapid technique which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for white spot syndrome virus (WSSV) detection was designed. A set of four primers, two outer and two inner primers, were designed from WSSV genome DNA. Reaction time and temperatures were optimized for 60 min at 65 °C, respectively. The detection limit (DL) using the LAMP method was up to 1 fg, when compared to 10 fg by nested polymerase chain reaction (PCR). Thus, standardized LAMP procedure was used to detect the presence of WSSV in the heart, stomach and lymphoid organ from infected shrimp. The study has developed a diagnostic procedure which is a rapid and highly sensitive for WSSV detection in shrimp.

A rapid non-enzymatic method of DNA extraction for PCR detection of white spot syndrome virus in shrimp

The present study describes a simple method of extraction of white spot syndrome viral DNA (WSSV) from infected shrimp for the polymerase chain reaction (PCR) detection of WSSV. The DNA preparation using this method was found to be free from the host DNA, RNA and protein, and is suitable for different PCR protocols such as single-step PCR, nested PCR and single-tube semi-nested PCR. This method of extraction has worked successfully for extracting the WSSV-DNA from different organs (haemolymph, eyestalk, carapace, head muscle, heart, gills, appendages, heptopancreas, stomach, intestine, abdominal muscle and tail muscle) of WSSV-infected adult shrimp, and WSSV-infected larvae and postlarvae.

A phage-displayed peptide can inhibit infection by white spot syndrome virus of shrimp.

White spot disease, caused by white spot syndrome virus (WSSV), results in devastating losses to the shrimp farming industry around the world, and no effective treatments have been found. Control focuses on exclusion of the virus from culture ponds but, once introduced, spread is often rapid and uncontrollable. The purpose of this study was to select a phage-displayed peptide that might be able to prevent WSSV infection. A 10-mer phage display peptide library (titre 7.2 x 10(7)) was constructed and screened against immobilized WSSV. Selected peptides were assessed for specificity and efficiency of inhibition of virus infection. Of four peptides that specifically bound to WSSV one, designated 2E6, had a high specificity and blocked virus infection, with the possible critical motif for virus inhibition being VAVNNSY. The results suggest that peptide 2E6 has potential for exploitation as an antiviral peptide drug.

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested (Penaeus stylirostris, P. monodon, P. vannamei) ranged from 2.0 × 104 to 9.0 × 1010 WSSV copies μg–1 of total DNA (n=26). In whole moribund post‐larvae, 4.3 × 109 WSSV copies μg–1 of DNA were detected which is equivalent to 5.7 × 1010 WSSV copies g–1 of post‐larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT‐50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).

Detection of new hosts for white spot syndrome virus of shrimp using nested polymerase chain reaction

The presence of white spot syndrome virus (WSSV) of shrimp in various marine crustaceans was studied by using polymerase chain reaction (PCR). The incidence of the virus in non-cultured crustaceans from shrimp farms was also studied. The results indicate that wild-caught asymptomatic marine shrimp such as Metapenaeus dobsoni, Parapenaeopsis stylifera, Solenocera indica and Squilla mantis carry WSSV. This virus could be detected in apparently healthy marine crabs Charybdis annulata, C. cruciata, Macrophthalmus sulcatus, Gelasimus marionis nitidus and Metopograpsus messor. The virus could also be detected in asymptomatic Macrobrachium rosenbergii cultured inland far away from coast. Detection of carrier animals required two-step nested PCR.

Identification and phylogeny of a non-specific endonuclease gene of white spot syndrome virus of shrimp.

White spot syndrome virus (WSSV) is a taxonomically unclassified virus which causes a disease in shrimps worldwide. A 936 bp long open reading frame (ORF) was found on a 7.2 kb HindIII fragment of the DNA genome of WSSV located adjacent to the ribonucleotide reductase small subunit gene. This putative ORF showed homology to prokaryotic and eukaryotic endonucleases, which contain a non-specific endonuclease motif. Alignment with viral and eukaryotic endonuclease ORFs revealed that most catalytically and structurally important amino acid residues were present in the putative WSSV non-specific endonuclease gene. An unrooted parsimonous phylogenetic tree of non-specific endonucleases indicated that the WSSV ORF was located in a well bootstrap supported clade containing only arthopods, including one of WSSV's natural hosts, Penaeus japonicus. A similar conjunction was found for the only other viral homologue, present in Fowlpox virus, which was also found in a well bootstrap-supported clade with its natural host, Gallus gallus. This clustering of virus and host suggests that both WSSV and Fowlpox virus may have acquired their nuclease genes from their respective natural hosts. Because the motif for non-specific nucleases is found in only two viruses, this gene cannot be used to clarify the taxonomic position of WSSV. However, the presence of this type of nuclease rarely found in viruses adds a novel feature to WSSV.

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions.

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Identification of Two Major Virion Protein Genes of White Spot Syndrome Virus of Shrimp

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 × 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.