Shrimp White Spot Syndrome Virus Research Articles

Protective effect of microbial immunostimulants and antiviral plants against WSSV in Litopenaeus vannamei cultured under laboratory conditions

Abstract The effect of a mixture of antiviral plants (Echinacea purpurea and Uncaria tomentosa) and microbial immunostimulants (Pediococcus parvulus and Candida parapsilosis) on growth performance, survival, white spot syndrome virus (WSSV) prevalence, and immune response of L. vannamei was evaluated under laboratory conditions. Powdered plants (PP) were included in the commercial feed pellet at 0.0, 1.0, 2.0, and 4.0 g kg feed− 1 and microbial immunostimulants (MI) were included at 0.0, 2.1, 4.2, and 8.4 mg kg feed− 1. A bioassay was conducted for 21 days. Feed supplemented with PP and MI did not influence survival and shrimp growth. However, the prevalence of WSSV in shrimp with low viral load decreased from 100% in the control group to 0% in animals fed with the PP and MI mixture. Additives increased significantly the phenoloxidase activity but not hemocyte and superoxide anion amount. The PP and MI mixture, added to feed, has the potential to be used prophylactically as an antiviral against WSSV in cultured shrimp.

The DNA Virus White Spot Syndrome Virus Uses an Internal Ribosome Entry Site for Translation of the Highly Expressed Nonstructural Protein ICP35

Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (∼300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5' untranslated region of icp35. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the Renilla luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Tunicamycin was used to show that the α subunit of eukaryotic initiation factor 2 is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the Spodoptera frugiperda 40S ribosomal protein RPS10 decreased icp35 IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation.

Isolation and characterization of halophilic Bacillus sp. BS3 able to produce pharmacologically important biosurfactants

ObjectiveTo characterize the pharmacological importance of biosurfactants isolated from halophilic Bacillus sp BS3. MethodsHalophilic Bacillus sp. BS3 was isolated from solar salt works, identified by 16S rRNA sequencing and was used for screening their biosurfactant production. Characters of the biosurfactant and their anticancer activity were analyzed and performed in mammary epithelial carcinoma cell at different concentrations. ResultsThe biosurfactant were characterized by TLC, FTIR and GC–MS analysis and identified as lipopeptide type. GC–MS analysis revealed that, the biosurfactant had various compounds including 13–Docosenamide, (Z); Mannosamine, 9– and N,N,N',N'–tetramethyl. Surprisingly the antiviral activity was found against shrimp white spot syndrome virus (WSSV) by suppressing the viral replication and significantly raised shrimp survival (P<0.01). Anticancer activity performed in the mammary epithelial carcinoma cell at different concentrations of biosurfactants, among the various concentrations of biosurfactants such as 0.000 25, 0.002 5, 0.025, 0.25 and 2.5μg, the 0.25 μg concentration suppressed the cells significantly (P<0.05) to 24.8%. ConclusionsBased on the findings, the present study concluded that, there is a possibility to develop eco–friendly antimicrobial and anticancer drugs from the extremophilic origin.

White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV-positive farms: post-larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7-100% nucleotide sequence identity with four strains in the GenBank database--China (AF332093), Taiwan (AF440570 and U50923) and Thailand (AF369029). This is the first broad study of WSSV infection in L. vannamei in Taiwan.

Improved primary cell culture and subculture of lymphoid organs of the greasyback shrimp Metapenaeus ensis

Despite numerous attempts, no continuous shrimp cell lines have yet been established. Here we report on an improved primary cell culture system as evidence of the routine and successful development of primary cell monolayers from the lymphoid organs of greasyback shrimp Metapenaeus ensis, using a newly formulated 1.5×L-15 based shrimp medium III. The shrimp medium III showed significantly better results than the other two shrimp media of I (0.5×L-15 based, hemolymph-imitated) and II (2×L-15 based) did. In medium III, the migration of lymphoid cells from the explants was initiated at 2–3h after seeding, and a 70%–80% confluent cell monolayer was formed within 16–24h and remained viable for over 20days. Moreover, the lymphoid cells cultured in medium III maintained their susceptibility to shrimp white spot syndrome virus (WSSV) and the WSSV could replicate in the infected lymphoid cells, an important feature for primary cell activity. In the attempts to passage the lymphoid cell culture, non-mammalian-derived enzyme complex of HyQTase, enzyme-free cell dissociation solution (ECDS), and the physical shocks with cold PBS (phosphate-buffered saline, 4°C) are important to markedly increase efficiency for both detachment (90%–100%) and reattachment (54.1±2.0% for HyQTase and 60.2±2.7% for ECDS) in comparison with other enzymes and methods tested. Using HyQTase and ECDS, we successfully passaged the lymphoid cell monolayers twice and then the cell density became too low to be further sub-cultured due to the absence of active proliferation of the in vitro cultured shrimp cells. The tested digestive enzymes of trypsin, collagenase type II, dispase, hyaluronidase, elastase and pronase were highly toxic to shrimp cells, and produced unfavorable subculture results in our procedures. The establishment of the above-mentioned primary cell culture and subculture systems will lay a solid foundation for further investigation of shrimp cell immortalization and development of stable shrimp cell lines.

Studies of the pathogenicity of WSSV to the cultured shrimp Litopenaeus vannamei under chlorpyrifos stress

In order to evaluate the hazard of chlorpyrifos to the cultured shrimp Litopenaeus vannamei,the lethal effect of the shrimp infected with white spot syndrome virus(WSSV)under the chlorpyrifos stress was tested,while the quantity of WSSV in the gill tissue and acetylcholinesterase acticity in the muscle tissue were analyzed.The 96 h median lethal concentration(LC50)of chlorpyrifos to the shrimp was determined.The results showed that the 96 h LC50 is 0.758 μg/L(0.521-0.987 μg/L),and as time prolonged,the values of LC50 significantly declined,which showed a positive concentration-response relationship.On this basis,the stress concentration of chlorpyrifos of 0.2 μg/L was determined.After the shrimp were exposed to 0.2 μg/L chlorpyrifos for 4 days,they were infected with WSSV by injection.It showed that the cumulate mortality of chlorpyrifos-WSSV treatment(83.33%±4.7%)was significantly higher than that of ethanol-WSSV group(40.00%±0.9%).Meanwhile,the amount of WSSV was also determined by Real-time Relative PCR at 48,72 and 96 h post-injection.WSSV quantity of chlorpyrifos-WSSV treatment was about 4 times that of ethanol-WSSV control at 72 h.WSSV quantity of chlorpyrifos-WSSV treatment was significantly increased at 96 h,which was 4.9 times that of chlorpyrifos-WSSV treatment at 72 h,and 5.9 times that of ethanol-WSSV treatment at 96 h.The acetylcholinesterase(AchE)activity in shrimp muscle tissue under chlorpyrifos stress was 20% lower than that in control group.This showed that the proliferation rate of WSSV in shrimp increased rapidly under chlorpyrifos stress,which raised the mortality rate of the shrimp.

Effects on survival time of Fenneropenaeus chinensis challenged by different doses of WSSV and diets

White spot syndrome virus(WSSV)is one kind of destructive virus affecting shrimp industry in the world.Output of Fenneropenaeus chinensis which once was a staple species has been greatly affected by outbreak of WSSV since 1993 in China.To produce WSSV-resistance shrimp strains is an important approach to revive the shrimp industry.To select survived F.chinensis as broodstock via WSSV challenge test can increase the efficiency of breeding.However,mortality of shrimps could reach nearly 100% once they were infected with WSSV,which increases the difficulty for shrimp breeding.In this study for the purpose to reveal effect of different WSSV amount on F.chinensis,juvenile F.chinensis were infected with a certain amount of WSSV individually and the survival time and survival rate were observed.The results showed that survival time of shrimps infected with 5.2×108 copies,1.0×109 copies and 2.1×109 copies of WSSV respectively was 389.3,323.3 and 187.3 h and significant different(P0.01).The cumulative mortalities of all the shrimps finally reached 100%.The survival time increased while shrimps were fed with less WSSV bait.For the purpose to reveal the effect of diets on the ability for disease resistance,shrimps infected with WSSV were fed with different diets of live brine shrimps,commercial bait and fresh clam.It was showed that the survival time of infected shrimps fed with live brine shrimps,fresh clam and commercial bait respectively was 281.7,173.9 and 164.9 h.The survival time of infected shrimp fed with live brine shrimps was significantly higher than that fed by commercial bait and fresh clam respectively(P0.01).There was no significant difference of survival time between infected shrimps fed with commercial bait and fresh clam(P0.05).Cumulative mortalities of all the groups of infected shrimps that fed with different diets were 100%.Live brine shrimp is better than commercial bait and fresh clam for the purpose of enhancing shrimp WSSV resistance ability.

Label-free Fluorescent Detection of Loop-mediated Isothermal Amplification of Nucleic Acid Using Pyrophosphate-selective Xanthene-based Zn(II)-coordination Chemosensor

Abstract A label-free fluorescent detection method for the loop-mediated isothermal amplification (LAMP) of nucleic acids was first developed by using the pyrophosphate-selective xanthene-based Zn(II)-coordination chemosensor 1–2Zn(II). In this method, the production of inorganic pyrophosphate (PPi), a by-product of the LAMP reaction, was detected by 1–2Zn(II) with a large fluorescence enhancement. On combination with the fluorescent plate reader technique, this method allows us to detect the shrimp white spot syndrome virus in a high-throughput manner with high sensitivity and selectivity.

Characterization of the Interaction Between Arginine Kinase and siRNA

RNAi, a crucial pathway in animals to defend against virus infection, is mediated directly by RNA-induced silencing complex (RISC) in an ATP-dependent manner. The RISC comprises one strand of short interfering RNA (siRNA) and multiprotein including Argonaute protein, which can cleave target RNAs. However, the proteins interacted with siRNA are not extensively explored. In this study, an antiviral siRNA (vp28-siRNA) targeting the vp28 gene of shrimp white spot syndrome virus was characterized. Based on the biotin/streptavidin affinity screening, it was found that the shrimp arginine kinase was specifically bound with the vp28-siRNA. The co-immunoprecipitation assays revealed that the siRNA was directly interacted with arginine kinase, suggesting that arginine kinase was an essential component of RNA-induced silencing complex. Therefore, our study presented a novel finding on the RISC components, which would be helpful to reveal the molecular events in the RNAi pathway.

Comprehensive characterization of viral miRNAs involved in white spot syndrome virus (WSSV) infection

Guided by miRNAs, RNAi plays an important role in virus-host interactions by fine-tuning gene expression. Many viral and cellular miRNAs are involved in virus infection, though no comprehensive general model for miRNAs derived from invertebrate DNA viruses exists for their function in eukaryotic systems, despite extensive research on miRNAs. To address this issue, the miRNAs from shrimp white spot syndrome virus (WSSV), a DNA virus with a 305 kb double-stranded circular DNA genome, were characterized. Based on WSSV miRNA microarray and northern blot analyses, WSSV was shown to possess the capacity to encode 40 distinct viral miRNAs, a miRNA content roughly 360 times greater than that of humans. These findings suggested that the high content of viral miRNAs might greatly contribute to viral variability in response selective pressures in the host environment. Transcription analysis revealed that 80% of WSSV miRNAs were expressed during early stages of viral infection, indicating their importance in initial infective processes. Additionally, biogenesis of viral miRNAs was demonstrated to be dependent on host Drosha and Dicer 1, mediated by Ago 1, and viral miRNAs, including WSSV-miR211 and WSSV-miR212, were required for successful WSSV infection. During WSSV infection, numerous viral genes were likely targeted by WSSV miRNAs. The current study presented the first comprehensive view of viral miRNAs encoded by an invertebrate DNA virus, providing insight into the molecular events of virus-host interactions.

Involvement of interaction between viral VP466 and host tropomyosin proteins in virus infection in shrimp

The accumulating evidence indicates that the viral structural proteins play critical roles in virus infection. However, the interaction between the viral structural protein and host cytoskeleton protein in virus infection remains to be addressed. In this study, the viral VP466 protein, one of the major structural proteins of shrimp white spot syndrome virus (WSSV), was characterized. The results showed that the suppression of VP466 gene expression led to the inhibition of WSSV infection in shrimp, indicating that the VP466 protein was required in virus invasion. It was found that the VP466 protein was interacted with the host cytoskeleton protein tropomyosin. As documented, the VP466–tropomyosin interaction facilitated the WSSV infection. Therefore our findings revealed a novel molecular mechanism in the virus invasion to its host, which would be helpful to better understand the molecular events in virus infection in invertebrate.

Efficiency of two fragments of VP28 against White Spot Syndrome Virus in Litopenaeus vannamei

White Spot Syndrome Virus (WSSV) is a major pathogen in shrimp culture, which is widespread in China, Southeast Asia and India continent, causing a catastrophic economical loss in shrimp aquaculture. In the present study, VP28 caused effective approaches in the systemic infection of WSSV in shrimp. To define the key regions of VP28 responsible for shrimp immune, N-terminal and C-terminal fragments of VP28 were constructed and expressed in Escherichia coli. The protective efficacy of injection and oral feeding of two fragments in Litopenaeus vannamei was investigated. Quantitative real-time PCR was used to determine the mRNA expression level of STAT, Dicer, Argonaute and Lysozyme in different tissues, and relevant enzyme activity was determined from the hemolymph after immunization. Vaccination experiments indicated that C-terminal VP28 shows significant effect against WSSV compared with that of N-terminal VP28. Dicer, STAT, Argonaute and Lysozyme in C-terminal VP28 treated groups have higher mRNA expression compared with that of N-terminal VP28 treated groups. Results suggested that C-terminal VP28 might play a crucial role in the pathway of viral attack.

Water temperature influences viral load and detection of White Spot Syndrome Virus (WSSV) in Litopenaeus vannamei and wild crustaceans

The Pacific white shrimp Litopenaeus vannamei is particularly affected by White Spot Syndrome Virus (WSSV) as this virus can cause high mortality in infected populations. Presently, there are no known treatments for shrimp affected by WSSV and management tools for preventing this disease are limited to the exclusion of the virus from cultured shrimp populations. Previous studies have shown that warm-water culture conditions inhibit the replication rate of WSSV, as well as two other important shrimp viruses in L. vannamei. The purpose of this study was to evaluate the effect of thermal stress on the replication rate of WSSV in shrimp held in warm water (29 ± 0.5 °C), compared to the replication rate of WSSV in shrimp held in cool water (18 ± 0.5 °C), looking for improve virus detection in epidemiological programs. Furthermore, post larvae and captured wild crustaceans were screened for the WSSV after being held in warm water for 2 days (48 h). The results indicate that water temperature had a profound effect on the replication rate of WSSV in L. vannamei and a protocol for WSSV screening after thermal stress is proposed. Our results support the findings of previous studies and further point out to the potential application of environmental temperature as a management strategy to selecting WSSV-free spawning shrimp within the shrimp farming industry in Mexico and possibly in other producing countries.

Gene transfection and expression in the primary culture of crayfish hemocytes

Lack of efficient gene delivery and expression methods is the major obstacle for crustacean research in the cellular level. Here, we reported the construction of an expression vector with a strong promoter from shrimp white spot syndrome virus. This vector could efficiently express foreign genes in the primary culture of crayfish hemocytes with the transfection efficiency between 5 and 10% by electroporation. Our findings provide a method for in vitro gene functional study in primary crayfish cells.

The antiviral vp28-siRNA expressed in bacteria protects shrimp against white spot syndrome virus (WSSV)

White spot syndrome virus (WSSV) has been a major viral pathogen responsible for large economic losses to shrimp aquaculture all over the world. The RNAi mediated by siRNA contributes a novel strategy to control this viral disease. However, the efficient approach to deliver the siRNA into shrimp in vivo remains to be addressed. In our study, the vp28-siRNA, having a specific activity against WSSV, was expressed in an RNase III-deficient strain of Escherichia coli HT115 (DE3). Then the inactivated bacteria expressing the vp28-siRNA were delivered into Marsupenaeus japonicus shrimp by injection. The results showed that the vp28-siRNA could be detected in shrimp hemocytes. As shown by quantitative PCR, the vp28-siRNA inhibited the replication of WSSV in shrimp, which presented a better antiviral activity of siRNA. Further evidence indicated that the mortality of WSSV-infected shrimp was significantly reduced ( P < 0.01) by the vp28-siRNA. Therefore this study presented a novel efficient therapeutic approach to control WSSV by using siRNA expressed in bacteria.

Transcriptional Analysis for Oral Vaccination of Recombinant Viral Proteins against White Spot Syndrome Virus (WSSV) in Litopenaeus vannamei

This study was carried out for the molecular level identification of recombinant protein vaccine efficacy, by oral feeding against white spot syndrome virus infection, with the comparison of viral mRNA transcriptional levels in shrimp cells. For the determination of WSSV dilution ratio for the vaccination experiment by oral feeding, in vivo virus titration was carried out using different virus dilutions of virus stock (1×10(2), 2×10(2), and 1×10(3)). Among the dilution ratios, 2×10(2) diluted WSSV stock was chosen as the optimal condition because this dilution showed 90% mortality at 10 days after virus injection. Recombinant viral proteins, rVP19 and rVP28, produced as protein vaccines were delivered in shrimps by oral feeding. The cumulative mortalities of the shrimps vaccinated with rVP19 and rVP28 at 21 days after the challenge with WSSV were 66.7% and 41.7%, respectively. This indicates that rVP28 showed a better protective effect against WSSV in shrimp than rVP19. Through the comparison of mRNA transcriptional levels of viral genes from collected shrimp organ samples, it was confirmed that viral gene transcriptions of vaccinated shrimps were delayed for 4~10 days compared with those of unvaccinated shrimps. Protection from WSSV infection in shrimp by the vaccination with recombinant viral proteins could be accomplished by the prevention of entry of WSSV due to the shrimp immune system activated by recombinant protein vaccines.

Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice

Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections.

Probiotic microorganisms and antiviral plants reduce mortality and prevalence of WSSV in shrimp (Litopenaeus vannamei) cultured under laboratory conditions

The protective effect of a probiotic mixture (PM) and antiviral plants, against the white spot syndrome virus (WSSV) in Litopenaeus vannamei, was evaluated in three experiments. The PM was composed of four lactic acid bacteria (LAB) and one yeast strain. The plant mixture was composed of Ocimum sanctum and commercial antiviral plants (VPH®, HSV®). Shrimp in each experiment (weighing 2.7±0.7, 11.5±1.3, 11.70±2.5 g) were cultured in 120-L plastic tanks and fed twice a day with commercial feed plus additives (plants or bacteria and yeast). Animals were monitored for the occurrence of WSSV by single-step and nested PCR. The PM and powdered antiviral plants added to the commercial feed showed an increase in survival and a decrease in the prevalence of WSSV in shrimp. The results showed that both the PM and the powdered antiviral plants can provide protection for shrimp against WSSV.

Viral ubiquitin ligase WSSV222 is required for efficient white spot syndrome virus replication in shrimp

The E3 ligase WSSV222 of white spot syndrome virus (WSSV) is involved in anti-apoptosis regulation by ubiquitin-mediated degradation of tumour suppressor-like protein (TSL), a shrimp tumour suppressor. In the present study, WSSV222 gene expression was silenced by using specific small interfering RNA (siRNA) in Sf9 and BHK cells. Based on the results of the in vitro silencing, WSSV-challenged shrimp were treated with anti-WSSV222 siRNA to knock down WSSV222 protein expression. The survival rate of shrimp and the efficiency of WSSV replication were assessed to evaluate the efficacy of anti-WSSV222 siRNA in regulating WSSV infection in shrimp. The anti-WSSV222 siRNA reduced the cumulative mortality in shrimp challenged with 10(3) copies of WSSV and delayed the mean time to death in shrimp challenged with the higher dose of 10(6) copies. The results of real-time quantitative PCR showed that virus replication was delayed and reduced in WSSV-challenged shrimp treated with anti-WSSV222 siRNA in comparison with challenged shrimp treated with random-control siRNA. Co-immunoprecipitation assays revealed that WSSV222 silencing inhibited the degradation of TSL in WSSV-challenged shrimp, indicating the requirement for WSSV222 for efficient replication of WSSV in shrimp.

Membrane Binding Activity with Virus in White Spot Syndrome Virus-Infected Fenneropenaeus chinensis

Problem statement: White Spot Syndrome Virus (WSSV) is one of the most disastrous pathogens in shrimp culture, having caused high mortality in many cultured shrimp species. It is well known that the most important step of viral infection is cell attachment. Approach:Therefore determining the binding activity of shrimp cells to WSSV is an important to evaluate anti-infection ability and understanding the procedure of a viral infection. So this study aims to analyze binding activity of cell membrane with WSSV in shrimp of Fenneropenaeus chinensis (F. chienesis). WSSV was used to stimulate F. chinensis and binding activity in gills, muscle and hepatopancreas with virus within the procession of WSSV was measured. Results:The results showed that binding activity in gills with WSSV within infection stage (0-96 h) had no significant change. The highest binding activity was attained after infection 24-48 h in muscle. The binding activity in hepatopancreas with WSSV was lower during infection stage (0-96 h). Conclusion/Recommendation: The higher binding activity of WSSV to gills and muscle indicated that gills and muscle are the importance target tissue for WSSV infection. This suggested that receptor of WSSV existed in gills and muscle.