White Spot Syndrome Virus Copies Research Articles

Influence of Agathi grandiflora active principles inhibit viral multiplication and stimulate immune system in Indian white shrimp Fenneropenaeus indicus against white spot syndrome virus infection

Five herbs including Adathoda vasica, Agathi grandiflora, Leucas aspera, Psoralea corylifolia, and Quercus infectoria were selected to screen the antiviral and immunostimulant activity against white spot syndrome virus (WSSV) and Vibrio harveyi respectively using different organic polar and non-polar solvents. Based on the initial screening results, ethyl acetate and methanolic extracts of A. grandiflora had strong antiviral and immunostimulant activities. Those extracts incubated with WSSV injected Fenneropenaeus indicus got only 20% mortality and no PCR positive signals were seen in two step PCR amplification. The methanolic extracts of A. grandiflora were further purified through silica column chromatography and the fractions screened again for antiviral and immunostimulant activity. The secondary screening results revealed that, the fractions of F5 to F7 had effectively controlled the WSSV multiplication and V. harveyi growth. The pooled fractions (F5 to F7) was structurally characterized by gas chromatograph-mass spectrometry (GC-MS) analysis and few compounds were identified including 3,7.11,15-Tetramethyl-2-Hexane-1-ol, pytol and 1,2-Benzenedicarboxylic acid, diisooctyl ester. The pooled fractions were mixed with the basal feed ingredients at the concentration of 100 (D-1), 200 (D-2), 300 (D-3) and 400 (D-4) mg kg−1 and the diets fed to the F. indicus (9.0 ± 0.5 g) for 30 days. After the completion of feeding trail, they were challenged with virulent WSSV and studied the cumulative mortality, molecular diagnosis by quantitative real time PCR (qRT-PCR), biochemical, haematological and immunological parameters. The control diet fed F. indicus succumbed to death 100% within 3 days whereas the D-3 and D-4 helped to reduced the cumulative mortality of 60–80% respectively. The qRT-PCR revealed that, the WSSV copy number was gradually decreased when increasing concentration of A. grandiflora extract active fraction in the diets. The diets D-3 and D-4 helped to reduce the protein and carbohydrate levels significantly (P < 0.01) from the control diet fed groups. Moreover these diets help to decrease the coagulation time of maximum 61% from control groups and improve the total haemocyte count of maximum 51.82 × 105 cells ml−1 in D4 diet fed F. indicus. Finally immunological parameters including prophenol oxidase (proPO) activity, intracellular superoxide anion production and intra-agar lysozyme activity was significantly (P ≤ 0.001) improved in the D-3 and D-4 fed F. indicus after WSSV challenge.

Regulation of the immediate-early genes of white spot syndrome virus by Litopenaeus vannamei kruppel-like factor (LvKLF)

Kruppel-like factors (KLFs) belong to a subclass of Cys2/His2 zinc-finger DNA-binding proteins, and act as important regulators with diverse roles in cell growth, proliferation, differentiation, apoptosis and tumorigenesis. Our previous research showed that PmKLF from Penaeus monodon is crucial for white spot syndrome virus (WSSV) infection, yet the mechanisms by which PmKLF influences WSSV infection remain unclear. This study cloned KLF from Litopenaeus vannamei (LvKLF), which had 93% similarity with PmKLF. LvKLF formed a dimer via the C-terminal zinc-finger motif. Knockdown of LvKLF expression by dsRNA injection in WSSV-challenged shrimps was found to significantly inhibit the transcription of two important immediate-early (IE) genes, IE1 and WSSV304, and also reduced WSSV copy numbers. Moreover, reporter assays revealed that the promoter activities of these two WSSV IE genes were substantially enhanced by LvKLF. Mutations introduced in the promoter sequences of IE1 and WSSV304 were shown to abolish LvKLF activation of promoter activities; and an electrophoretic mobility shift assay demonstrated that LvKLF binds to putative KLF-response elements (KRE) in the promoters. Taken together, these results indicate that LvKLF transcriptional regulation of key IE genes is critical to WSSV replication.

Assessment of WSSV prevalence and distribution of disease-resistant shrimp among the wild population of Penaeus monodon along the west coast of India

Shrimp aquaculture is threatened by many diseases, among which white spot disease (WSD) caused by white spot syndrome virus (WSSV) is the leading one. Information related to the geographical distribution and seasonal prevalence of WSD is necessary to obtain a clear understanding of the disease biology in shrimp. Identification of WSD-resistant individual shrimp with DNA markers is also an important technique to develop better WSD-free shrimp health management. The present study aim is to estimate the occurrence of WSSV in Penaeus monodon qualitatively and quantitatively during three different seasons during the years 2011 to 2013 along the west coast of India. Additionally, the disease resistance prevalence using previously developed 71bp microsatellite and 457bp RAPD-SCAR DNA markers is also investigated. Samples were collected throughout the year from four locations along the west coast of India: Kochi, Kerala; Mangalore, Karnataka; Vasco-da-Gama, Goa; and Veraval, Gujarat. The results depicted that the average WSSV prevalence, as determined by the nested PCR method and taken cumulatively over the four locations, was the lowest (0%) during the post-monsoon season and the highest (31.6%) during the monsoon season. The WSD prevalence was observed to increase when the latitude was decreased along the west coast of India (from Veraval to Kochi). Out of the three different seasons, the average WSSV copy number was the highest (approximately 103copiesμg−1 shrimp genomic DNA) during the monsoon season. The disease-resistant prevalence, as determined using the developed DNA markers, was found to be the highest in Vasco-da-Gama (59.5%) and the lowest in Kochi (40.9%). The present study suggests better options for the efficient collection of disease-free and disease-resistant brood stocks, which would be a more cost-effective and safer approach toward disease prevention over conventional trends of seed generation from unselected wild brood stock.

Development of an immuno‐based colorimetric assay for white spot syndrome virus

White spot syndrome virus (WSSV) is a major cause of infectious disease in cultured shrimp. A fast and reliable method for detecting and monitoring the amount of WSSV during farming would be extremely useful. This work describes a sandwich immunoassay that uses anti-GST-VP26, a WSSV-binding protein (WBP), and modified streptavidin magnesphere paramagnetic particles (SMPPs) to develop the technique. The WBP was immobilized on SMPPs and later bound to different copies of WSSV. The binding was detected using anti-GST-VP26 conjugated to alkaline phosphatase. This enzymatic reaction successfully changed the test solution to a concentration-dependent yellow color that was measured at 405nm. The sensitivity of this method was between 1.6×10(4) and 1.6×10(7) copies µL(-1) of WSSV. In this study, the color for detection and semiquantitative analysis is easily observed and measured and can lead to the development of a test kit for screening WSSV during shrimp farming.

White spot syndrome virus (WSSV) and prevalence of disease resistance in a commercially cultured population of Penaeus monodon Fabricius, 1798 (Decapoda, Dendrobranchiata)

White spot disease (WSD) caused by the white spot syndrome virus (WSSV) is the leading cause for huge economic destruction in the shrimp aquaculture industry. The present study investigates the prevalence of WSSV and disease-resistant shrimps among a commercially cultured population of Penaeus monodon Fabricius, 1798. Results suggest that WSSV prevalence among 32 culture ponds affected by various diseases was 58.2% cumulatively in 2009 and 2010. Real-time PCR yielded 1.46 × 108, 1.94 × 106 and 3.6 × 104 copies of WSSV per μg of genomic DNA of shrimp in severe, moderate and low infected shrimps, successively. Among the shrimps collected, the prevalence of disease resistance was 31.4% and 37.1% consecutively in 2009 and 2010. It was found that 34% of the shrimps were disease resistant while considering both years. This study shows the importance of using disease-resistant seeds for better shrimp health management.

Involvement of Viral MicroRNA in the Regulation of Antiviral Apoptosis in Shrimp

Viruses, in particular DNA viruses, generate microRNAs (miRNAs) to control the expression of host and viral genes. Due to their essential roles in virus-host interactions, viral miRNAs have attracted extensive investigations in recent years. To date, however, most studies on viral miRNAs have been conducted in cell lines. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blotting, the expression of viral miRNAs was tissue specific in vivo. The results indicated that the viral miRNA WSSV-miR-N24 could target the shrimp caspase 8 gene, and this miRNA further repressed the apoptosis of shrimp hemocytes in vivo. As a result, the number of WSSV copies in shrimp in vivo was significantly increased compared with the control level (WSSV only). Therefore, our study presents the first report on the in vivo molecular events of viral miRNA in antiviral apoptosis.

Viral MicroRNAs Targeting Virus Genes Promote Virus Infection in Shrimp In Vivo

Viral microRNAs (miRNAs), most of which are characterized in cell lines, have been found to play important roles in the virus life cycle to avoid attack by the host immune system or to keep virus in the latency state. Viral miRNAs targeting virus genes can inhibit virus infection. In this study, in vivo findings in Marsupenaeus japonicus shrimp revealed that the viral miRNAs could target virus genes and further promote the virus infection. The results showed that white spot syndrome virus (WSSV)-encoded miRNAs WSSV-miR-66 and WSSV-miR-68 were transcribed at the early stage of WSSV infection. When the expression of WSSV-miR-66 and WSSV-miR-68 was silenced with sequence-specific anti-miRNA oligonucleotides (AMOs), the number of copies of WSSV and the WSSV-infected shrimp mortality were significantly decreased, indicating that the two viral miRNAs had a great effect on virus infection. It was revealed that the WSSV wsv094 and wsv177 genes were the targets of WSSV-miR-66 and that the wsv248 and wsv309 genes were the targets of WSSV-miR-68. The data demonstrate that the four target genes play negative roles in the WSSV infection. The targeting of the four virus genes by WSSV-miR-66 and WSSV-miR-68 led to the promotion of virus infection. Therefore, our in vivo findings show a novel aspect of viral miRNAs in virus-host interactions.

A novel peroxinectin involved in antiviral and antibacterial immunity of mud crab, Scylla paramamosain

Peroxinectin (PX) with cell adhesion and peroxidase activities is important in invertebrate immune responses. We identified a novel PX homolog from Scylla paramamosain (designated as Sp-PX) through transcriptome sequencing. The full-length of cDNA sequence was 3,165bp. And there was a peroxidase domain in the deduced protein sequence. A cell-adhesive sequence (KGD motif) was also found in the N-terminus. The predicted molecular mass of the mature protein is 83.9kDa, with an estimated pI of 6.21. At the amino acid level, Sp-PX shared much higher similarities with other crustaceans PX proteins. And Sp-PX also exhibited some similarities with other peroxidase family members. According to real-time polymerase chain reaction, Sp-PX was mainly distributed in the hemocytes. The gene expression levels in the hemocytes of the normal and white spot syndrome virus (WSSV)-challenged crabs were compared via high-throughput RNA sequencing technology, and the results showed that Sp-PX was upregulated at 48h post-WSSV challenge. Subsequently, how Sp-PX responds to WSSV stimulus was explored through time-course experiments. The Sp-PX transcripts dramatically increased and reached the highest level at 12h post-injection, whereas Sp-PX transcripts were recovered at 96h post-challenge. Meanwhile, it was found that the WSSV copies proliferated significantly after a period of latent viral infection for 48h. In addition,Sp-PX transcripts were also upregulated after Vibrio harveyi or Staphylococcus aureus challenge. Overall, Sp-PX not only participates in antibacterial immunity but also plays a crucial role in the antiviral immune responses of mud crab at the early stage of WSSV infection.

Protection of Shrimp Penaeus monodon from WSSV Infection Using Antisense Constructs

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3-VP24, pPn-VP24), VP28 (pH3-VP28, pPn-VP28), TS (pH3-TS, pPn-TS), and RR2 (pH3-RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90% survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.

Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 μg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 μg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.

Host defense against DNA virus infection in shrimp is mediated by the siRNA pathway

The RNA interference (RNAi) system of eukaryotes using siRNAs has been documented as an immune response against invasion by RNA viruses. However, whether the siRNA pathway can be triggered by the infection with DNA viruses in animals remains to be investigated. In the present study, we show that Marsupenaeus japonicus shrimp can generate an antiviral siRNA (vp28-siRNA) in response to infection by a double-stranded DNA virus, white spot syndrome virus (WSSV). After challenging with WSSV, vp28-siRNA is detected in all the WSSV-infected organs and tissues of shrimp as early as 24 h postinfection (p.i.). The results indicate that the host Dicer2 and Ago2 proteins are required for the biogenesis and function of vp28-siRNA, respectively. We show further that vp28-siRNA predominates in the cytoplasm of shrimp hemocytes at 48 h p.i. Knockdown of Dicer2 by special siRNA or inhibition of vp28-siRNA with locked nucleic acid antisense oligonucleotides both lead to a significant increase in WSSV copy number at 24-48 h p.i. Our study highlights a novel aspect of the siRNA pathway in the immune response of animals against infection by DNA viruses.

Transcript Analysis of White spot syndrome virus Latency and Phagocytosis Activating Protein Genes in Infected Shrimp (Penaeus monodon)

Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20μgg(-1) body weight) of recombinant PAP. Relative percent survival of 10% was observed in shrimp immunized with the dose of 15μgg(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.

A new method for quantifying white spot syndrome virus: Experimental challenge dose using TaqMan real-time PCR assay

White spot syndrome virus (WSSV) is an important pathogen in shrimp aquaculture. The susceptibility of crayfish (Procambarus clarkii) was assessed by means of serial dilutions of a solution containing WSSV. A TaqMan real-time PCR was used to quantify the WSSV challenge dose in P. clarkii. The results showed that WSSV copies could be detected at concentrations from 1.365×10(4) to 1.129×10(9) copies/μl. The viral infectivity (LD(50)), measured as the mortality of infected crayfish, indicated 60% mortality in the 10(5) dilution group (1.524×10(5) copies/μl). TaqMan real-time PCR represents a novel standard method, based on the by quantitation of WSSV copies, for determining the appropriate concentration of WSSV for use in infection experiments.

Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus (wssv) and its use as a biosensor

White spot syndrome virus (WSSV) is a major cause of high mortality in cultured shrimp all over the world. VP26 is one of the structural proteins of WSSV that is assumed to assist in recognizing its host and assists the viral nucleocapsid to move toward the nucleus of the host cell. The objective of this work was to produce a polyclonal antibody against VP26 and use it as a biosensor. The recombinant VP26 protein (rVP26) was produced in E. coli (BL21), purified and used for immunizing rabbits to obtain a polyclonal antibody. Western blot analysis confirmed that the antiserum had a specific immunoreactivity to the VP26 of WSSV. This VP26 antiserum was immobilized onto a gold electrode for use as the sensing surface to detect WSSV under a flow injection system. The impedance change in the presence of VP26 was monitored in real time. The sensitivity of the biosensor was in the linear range of 160–160000 copies of WSSV, indicating that it is good and sensitive for analysis of WSSV. The specificity of the biosensor was supported by the observation that no impedance change was detected even at high concentrations when using Yellow Head Virus (YHV). This biosensor may be applied to monitor the amount of WSSV in water during shrimp cultivation.

Oral Vaccination of Baculovirus-Expressed VP28 Displays Enhanced Protection against White Spot Syndrome Virus in Penaeus monodon

White Spot Syndrome Virus (WSSV) is an infectious pathogen of shrimp and other crustaceans, and neither effective vaccines nor adequate treatments are currently available. WSSV is an enveloped dsDNA virus, and one of its major envelope proteins, VP28, plays a pivotal role in WSSV infection. In an attempt to develop a vaccine against WSSV, we inserted the VP28 gene into a baculovirus vector tailored to express VP28 on the baculovirus surface under the WSSV ie1 promoter (Bac-VP28). The Bac-VP28 incorporated abundant quantity (65.3 µg/ml) of VP28. Shrimp were treated by oral and immersion vaccination with either Bac-VP28 or wild-type baculovirus (Bac-wt). The treatment was followed by challenge with WSSV after 3 and 15 days. Bac-VP28 vaccinated shrimp showed significantly higher survival rates (oral: 81.7% and 76.7%; immersion: 75% and 68.4%) than Bac-wt or non-treated shrimp (100% mortality). To verify the protective effects of Bac-VP28, we examined in vivo expression of VP28 by immunohistochemistry and quantified the WSSV copy number by qPCR. In addition to that, we quantified the expression levels shrimp genes LGBP and STAT by real-time RT-PCR from the samples obtained from Bac-VP28 vaccinated shrimp at different duration of vaccine regime. Our findings indicate that oral vaccination of shrimp with Bac-VP28 is an attractive preventative measure against WSSV infection that can be used in the field.

Contribution of the Caspase Gene Sequence Diversification to the Specifically Antiviral Defense in Invertebrate

Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221–229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response.

Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein ( PAP) gene

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi ( V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO ( Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.

Comparison of white spot syndrome virus quantification of fleshy shrimp Fenneropenaeus chinensis in outdoor ponds between different growing seasons by TaqMan real-time polymerase chain reaction

In the present study, we used TaqMan real-time polymerase chain reaction to quantify and compare infection of white spot syndrome virus (WSSV) with shrimp production of Fenneropenaeus chinensis cultured in outdoor ponds along the west coast of the South Korea. In 2007, a total of 60 specimens in summer and 116 specimens in autumn were collected from 12 growing-out ponds and 12 harvest ponds respectively. Pond harvest data were obtained from farmers. Of the summer samples, all specimens were WSSV positive, with a wide range of 12.4–7.0 × 107 (mean 7.5 × 106) copies ng−1 DNA; shrimp production was 1.7 metric tonnes per hectare (mt ha−1). Of the 116 autumn-sample specimens, 81 (69.8%) were WSSV positive; WSSV infection had been decreased dramatically, to 0–7.2 (mean 3.5) copies ng−1 DNA. Shrimp production of autumn ponds was 2.1 mt ha−1. Statistical analysis indicated that the difference in WSSV infections detected in summer and autumn was highly significant (P<0.01). In summer, seven ponds (58.3%) with low-WSSV infection loads (0–1000 WSSV copies ng−1 DNA) had shrimp production of 2.7 mt ha−1; the others had shrimp production of only 0.2 mt ha−1. The mean shrimp production between the two infection levels showed a highly statistically significant difference (P<0.01).

Characterization of WSSV resistance in selected families of Litopenaeus vannamei

Disease outbreaks caused by the white spot syndrome virus (WSSV) have resulted in high economic losses in the shrimp-culture industry worldwide. After three generations of selective breeding for WSSV resistance, 29 families of Litopenaeus vannamei (G3) were selected and divided into three groups: relatively high-resistance, moderate-resistance, and susceptible families, which had average survival rates of 22.7±12.30, 9.08±1.46, and 0.78±1.70%, respectively, after WSSV infection. Appearance of the mortality peak in the relatively high-resistance families was postponed by 2–3days compared to the same in susceptible families and unselected shrimps. Using real-time quantitative polymerase chain reaction, the copy number of WSSV DNA in the muscles of surviving shrimps from the relatively high-resistance family 14 was significantly lower than that of the moderate-resistance family 17, the susceptible family 4, and the unselected family of shrimps at 36hourspostinfection (hpi) and thereafter; for example, at 48hpi, the DNA copy number was (23.87±2.56)×106copiesg−1 in the high-resistance families against (40.03±2.54)×106, (65.70±3.44)×106, and (47.87±3.95)×106copiesg−1 body weight, respectively, in the other groups. The WSSV copy number in the muscles of dead shrimps from the relatively high-resistance family 14 was significantly higher than that of the moderate-resistance family 17, the susceptible family 4, and the unselected family of shrimps at 48hpi and thereafter; for example, at 72hpi, the DNA copy number was (54.1±3.9)×106copiesg−1 compared to values of (31.5±2.6)×106, (11.6±2.9)×106, and (25.4±2.7)×106copiesg−1 body weight in the other groups, respectively. The results indicated that shrimps from the relatively high-resistance families could inhibit the replication of WSSV and therefore had apparent WSSV resistance. These results provide a direction for the future selective breeding of shrimps for WSSV resistance.

Protective immunity induced by CpG ODNs against white spot syndrome virus (WSSV) via intermediation of virus replication indirectly in Litopenaeus vannamei

The worldwide shrimp culture is beset with diseases mainly caused by white spot syndrome virus (WSSV) and suffered huge economic losses, which bring out an urgent need to develop the novel strategies to better protect shrimps against WSSV. In the present study, CpG-rich plasmid pUC57-CpG, plasmid pUC57 and PBS were employed to pretreat shrimps comparatively to evaluate the protective effects of CpG ODNs on shrimps against WSSV. The survival rates, WSSV copy numbers, and antiviral associated factors (Dicer, Argonaute, STAT and ROS) were detected in Litopenaeus vannamei. There were higher survival proportion, lower WSSV copy numbers, and higher mRNA expression of Dicer and STAT in pUC57-CpG-pretreatment shrimps than those in pUC57- and PBS-pretreatment shrimps after WSSV infection. The Argonaute mRNA expression in pUC57-CpG-, pUC57- and PBS-pretreatment shrimps after WSSV infection was significantly higher than that of shrimps post PBS stimulation on the first day. The ROS levels in pUC57-CpG-pretreatment shrimps post secondary stimulation of PBS were significantly higher than those post WSSV infection on the first day. These results together demonstrated that pUC57-CpG induced partial protective immunity in shrimps against WSSV via intermediation of virus replication indirectly and could be used as a potential candidate in the development of therapeutic agents for disease control of WSSV in L. vannamei.