In vitro digestibility of specific dsRNA by enzymes of digestive tract of shrimp Litopenaeus vannamei / Digestibilidad in vitro del dsRNA específico por enzimas del tracto digestivo del camarón Litopenaeus vannamei

ABSTRACT Objective. The digestibility of specific dsRNA by action of the enzymes of digestive tract of the whiteleg shrimp Litopenaeus vannamei was determined in vitro. Materials and methods. Digestive enzyme activity (amylase, lipase, protease, DNase and RNase) was measured in the stomach, digestive gland, and anterior, middle, and posterior intestine of juvenile shrimp, and the digestibility of DNA, RNA and the dsRNA-ORF89, specific to WSSV, was determined by in vitro assays, as well as electrophoretic and densitometric analyses. Results. The highest enzymatic activity was found in the digestive gland: amylase (81.41%), lipase (92.60%), protease (78.20%), DNase (90.85%), and RNase (93.14%). The highest digestive capacity against DNA, RNA, and dsRNA was found in the digestive gland (5.11 ng of DNA ​​per minute, 8.55 ng of RNA per minute, and 1.48 ng dsRNA per minute). Conclusions. The highest digestibility of dsRNA-ORF89, specific to WSSV, was found in the digestive gland, whereas the lowest digestibility was observed in the posterior intestine. This is the first report regarding the digestibility of dsRNA-ORF89 by whiteleg shrimp digestive tract enzymes, with potential therapeutic importance in shrimp culture to prevent WSSV disease through balanced feed.

RESUMEN Objetivo. La digestibilidad del dsRNA específico para el virus de la mancha blanca (WSSV) por acción de las enzimas del tracto digestivo del camarón Litopenaeus vannamei fue analizada in vitro. Materiales y métodos. Se midió la actividad de enzimas digestivas (proteasa, amilasa, lipasa, ADNasa y ARNasa) en el estómago, la glándula digestiva, el intestino anterior, medio y posterior en juveniles de camarón patiblanco y se evaluó la digestibilidad de ácidos nucleicos ADN, ARN y dsRNA-ORF89 especifico contra el virus WSSV, por análisis electroforéticos y densitometría. Resultados. La actividad enzimática más alta se encontró en la glándula digestiva del camarón: amilasa (81.41%), lipasa (92.60%), proteasa (78.20%), ADNasa (90.85%) y ARNasa (93.14%). Se evidenció la capacidad digestiva del camarón patiblanco contra el ADN, ARN y dsRNA-ORF89 encontrando en la glándula digestiva la mayor digestión (5.11 ng de ADN por minuto, 8.55 ng de ARN por minuto y 1.48 ng de dsRNA por minuto). Conclusiones. La mayor digestibilidad del dsRNA-ORF89, específico contra el virus WSSV, se encontró en la glándula digestiva y la menor en el intestino posterior. Este es el primer informe relacionado con la digestibilidad del dsRNA-ORF89 por las enzimas del camarón patiblanco con potencial importancia terapéutica en el cultivo de camarón para prevenir la enfermedad del WSSV a través del alimento balanceado.

Comparative transcriptome and potential antiviral signaling pathways analysis of the gills in the red swamp crayfish, Procambarus clarkii infected with White Spot Syndrome Virus (WSSV)

Abstract Red swamp crayfish is an important model organism for research of the invertebrate innate immunity mechanism. Its excellent disease resistance against bacteria, fungi, and viruses is well-known. However, the antiviral mechanisms of crayfish remain unclear. In this study, we obtained high-quality sequence reads from normal and white spot syndrome virus (WSSV)-challenged crayfish gills. For group normal (GN), 39,390,280 high-quality clean reads were randomly assembled to produce 172,591 contigs; whereas, 34,011,488 high-quality clean reads were randomly assembled to produce 182,176 contigs for group WSSV-challenged (GW). After GO annotations analysis, a total of 35,539 (90.01%), 14,931 (37.82%), 28,221 (71.48%), 25,290 (64.05%), 15,595 (39.50%), and 13,848 (35.07%) unigenes had significant matches with sequences in the Nr, Nt, Swiss-Prot, KEGG, COG and GO databases, respectively. Through the comparative analysis between GN and GW, 12,868 genes were identified as differentially up-regulated DEGs, and 9,194 genes were identified as differentially down-regulated DEGs. Ultimately, these DEGs were mapped into different signaling pathways, including three important signaling pathways related to innate immunity responses. These results could provide new insights into crayfish antiviral immunity mechanism.

Pathogenicity of white-spot syndrome virus in Macrobrachium nipponensis via different infection routes / 生物工程学报

Macrobrachium nipponensis is delicious and has high economic value, but its susceptibility to white-spot syndrome virus (WSSV) is unknown. Susceptibility, morbidity, and multiplication of WSSV in M. nipponense were studied by epidemiological survey, infection experiment and qPCR. M. nipponense was the natural host of WSSV, and the natural carrying rate was about 8.33%. M. nipponense could be infected with WSSV via oral administration, muscle injection and immersion, and the cumulative infection rate of 10 d exposure was 100%, and the cumulative mortality rates were 100%, 75% and 0%, respectively. The infection of WSSV is fast by muscle injection. The virus content after 5 day's injection is 1 000 times higher than that of the first day of infection, and the mortality rate reached 100% after 8 days. The median lethal dose (LD₅₀) measured as the mortality of infected M. nipponense via injection indicated the LD₅₀ in the concentration of WSSV of 2.71×10⁵ virions/μL. In shrimp farming, M. nipponense can be infected by ingesting WSSV infected shrimp or dead shrimp, and also by soaking in WSSV-containing water and thus become a vector, consequently affecting the spread and pathogenicity of WSSV.

Immunostimulatory activity of brown seaweed-derived fucoidans at different molecular weights and purity levels towards white spot syndrome virus (WSSV) in shrimp Litopenaeus vannamei.

Fucoidan used as immunostimulant is commonly in the crude form. In this study, we investigated the effect of fucoidan in both crude and purified forms in their immunostimulatory activity. In addition, we studied the effect of low- and high-molecular weight fucoidan as hydrolysis products toward immunostimulatory activity. Four kinds of fucoidan were assayed for immunostimulant activity on the shrimp Litopenaeus vannamei. The parameters observed in the assay includes the mortality number, haemocyte, gene-related immunity (phenoloxidase, superoxide dismutase and transglutaminase) in the shrimps infected with viral WSSV. The assay results showed that pure fucoidan exhibited higher activity compared with that of crude fucoidan. Sulfate and carbohydrate content of HMW fucoidan are 7.8 % and 82.54 % with an estimated molecular weight of 8.28 x104 Dalton, and low molecular weight (LMW) fucoidan has 1.2% and 65.23% with an estimated molecular weight of 7.53 x104 Dalton. The transcriptional level of the immunity-related genes was found higher after feeding the infected shrimps with purified and HMW fucoidan. In particular, all of fucoidan forms increased the phenoloxidase gene transcription, suggesting that fucoidan have significant role in the production of phenoloxidase.

Hypoxia increases susceptibility of Pacific white shrimp to whitespot syndrome virus (WSSV) / Condição de hipóxia aumenta a susceptibilidade do camarão branco do Pacíficoao vírus da mancha branca (WSSV)

The present study aimed to evaluate the mortality, reactive oxygen species production (ROS) and total hemocyte counts (THC) of the marine shrimp Litopenaeus vannamei infected with the white spot syndrome virus (WSSV) at three levels of oxygen saturation. For this, 360 shrimp (20±2g) were distributed in 24 tanks (60L), divided in two groups (infected and non-infected), which were subjected to 30, 60 and 100% of dissolved oxygen saturation (in quadruplicate). During 96 hours after infection, daily hemolymph samples were collected for hemato-immunological parameter evaluation (THC and ROS) and dead animals were removed and computed to assess cumulative mortality rates. In the infected group, animals subjected to 100% saturation showed higher ROS production (P<0.05) after 48 hours, while THC was significantly reduced (P<0.05), regardless of oxygen saturation. The hypoxia resulted in high mortality when compared to 100% saturation condition. In the uninfected group, no significant differences were observed in all evaluated parameters. Thus, the hypoxia condition increased the susceptibility of shrimp to the infection of WSSV, which may be partly related to the low ROS production showed by the animals subjected to 30% oxygen saturation.

O presente estudo teve por finalidade avaliar a mortalidade e a contagem total de hemócitos (CTH) e espécies reativas de oxigênio (EROs) de camarão Litopenaeus vannamei infectados com o vírus da mancha branca (WSSV) e submetidos a três níveis de saturação de oxigênio. Para tanto, 360 camarões (20±2g) foram distribuídos em 24 tanques (60L), divididos em dois grupos, infectados e não infectados e submetidos a 30, 60 e 100% de saturação de oxigênio (em quadruplicata). Após a infecção, diariamente foram coletadas amostras de hemolinfa dos animais para avaliação dos parâmetros hematoimunológicos (CTH e EROs) e foi estimada a mortalidade, por 96 horas. No grupo com infecção, os animais submetidos à saturação de 100% apresentaram um aumento na produção de EROs (P<0,05) após 48 horas, ao mesmo tempo em que a CTH demonstrou uma redução (P<0,05) independentemente da saturação do oxigênio, e a condição de hipóxia acarretou maiores mortalidades quando comparada à do grupo com 100% de saturação. No grupo sem infecção, não foram observadas diferenças significativas nos parâmetros avaliados nem mortalidade. Dessa forma, pode-se concluir que a hipóxia aumentou a susceptibilidade do camarão à infecção com o vírus da mancha branca, que pode estar, em parte, relacionada com a baixa contagem de hemócitos e produção de EROs observadas nos animais submetidos a essa condição.

Multiple proteins of White spot syndrome virus involved in recognition of β-integrin.

The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that β-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of β-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with β-integrin. The β-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of β-integrin. Identification of proteins in WSSV that are associated with β-integrin will assist development of new agents for effective control of the white spot syndrome.

Multiple proteins of White spot syndrome virus involved in recognition of β-integrin.

The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that β-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of β-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with β-integrin. The β-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of β-integrin. Identification of proteins in WSSV that are associated with β-integrin will assist development of new agents for effective control of the white spot syndrome.

Screening, isolation and optimization of anti-white spot syndrome virus drug derived from marine plants

<p><b>OBJECTIVE</b>To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host-pathogen interaction model.</p><p><b>METHODS</b>Thirty species of marine plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.</p><p><b>RESULTS</b>Nine plant isolates exhibited significant survivability in host. The drug MP07X thus formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1 000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.</p><p><b>CONCLUSIONS</b>The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug.</p>

Screening, isolation and optimization of anti-white spot syndrome virus drug derived from terrestrial plants

<p><b>OBJECTIVE</b>To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host-pathogen interaction model.</p><p><b>METHODS</b>Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. The best anti-WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.</p><p><b>RESULTS</b>Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.</p><p><b>CONCLUSIONS</b>The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug.</p>

Screening, isolation and optimization of anti-white spot syndrome virus drug derived from marine plants

Objective: To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various marine floral ecosystems and to evaluate the efficacy of the same in host–pathogen interaction model.Methods:ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti-WSSV property in Litopenaeus vannamei. By means of chemical processes, the purified anti-WSSV plant isolate, MP07X was derived. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.Results:Thirty species of marine plants were subjected to Soxhlet extraction using water, formulated showing 85% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of MP07X required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 1000 mg/kg body weight/day survived at the rate of 85%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.Conclusions:Nine plant isolates exhibited significant survivability in host. The drug MP07X thus The drug MP07X derived from Rhizophora mucronata is a potent anti-WSSV drug.

Screening, isolation and optimization of anti-white spot syndrome virus drug derived from terrestrial plants

Objective: To screen, isolate and optimize anti-white spot syndrome virus (WSSV) drug derived from various terrestrial plants and to evaluate the efficacy of the same in host–pathogen interaction model.Methods:Thirty plants were subjected to Soxhlet extraction using water, ethanol, methanol and hexane as solvents. The 120 plant isolates thus obtained were screened for their in vivo anti–WSSV property in Litopenaeus vannamei. The best anti–WSSV plant isolate, TP22C was isolated and further analyzed. The drug was optimized at various concentrations. Viral and immune genes were analysed using reverse transcriptase PCR to confirm the potency of the drug.Results: Seven plant isolates exhibited significant survivability in host. The drug TP22C thus formulated showed 86% survivability in host. The surviving shrimps were nested PCR negative at the end of the 15 d experimentation. The lowest concentration of TP22C required intramuscularly for virucidal property was 10 mg/mL. The oral dosage of 750 mg/kg body weight/day survived at the rate of 86%. Neither VP28 nor ie 1 was expressed in the test samples at 42nd hour and 84th hour post viral infection.Conclusions:The drug TP22C derived from Momordica charantia is a potent anti-white spot syndrome virus drug.

In vivo biochemical changes occurring at different time intervals in white spot syndrome virus infected shrimp, treated with anti-WSSV drug derived from marine plants.

White spot syndrome virus (WSSV), is the most contagious pathogen of cultured shrimp that causes mass mortality, leading to huge economic loss to the shrimp industry. The lack of effective therapeutic or prophylactic measures has aggravated the situation, necessitating the development of antiviral drugs. With this objective, the antiviral activity of the drug, (MP07X -derived from the marine plant) in the host, Litopenaeus vannamei was evaluated. The biochemical changes aggravated by WSSV in the host, and the in vivo efficacy of the drug in the host – pathogen interaction were analyzed. The survival percentage of the treated (with MP07X) WSSV infected host was 85 %. Significant results were obtained from the cytotoxicity assays of the drug in both the brine shrimp and host. A total of 9 biochemical parameters such as, total protein, total carbohydrate, total glucose, total free amino acid, total fatty acid, fructose 1, 6 diphosphatase, aldolase, glucose 6 phosphatase and glucose 6 phosphate dehydrogenase were examined for healthy (NEG), WSSV infected (POS) and test sample (TS) shrimps. Significant differences (p < 0.01) were observed between the POS, NEG and TS in the biochemical variables at different time intervals post infection with WSSV. In the case of POS, significantly (p < 0.01) reduced variables were observed when compared to the NEG. In contrast, significant (p < 0.01) elevations were observed in the TS after a certain time interval due to the anti-WSSV activity of MP07X. Neither the VP 28 gene nor the immediate early genes (ie 1) were expressed in the host at the 42nd and 84th hrs. Thus, in accordance with the above results it can be concluded that acute WSSV infection triggers alterations in biochemical parameters in L. vannamei and at the same time the drug is efficient enough to combat the deadly virus and can increase the survivability of the host.

Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.

Presença do vírus da síndrome da mancha branca em crustáceos decápodes silvestres em lagoas costeiras no Sul do Brasil / Presence of the white spot syndrome virus (WSSV) in wild decapods crustaceans in coastal lagoons in southern Brazil

A presença do vírus da síndrome da mancha branca (em inglês WSSV) nas principais espécies de camarões, siris e caranguejos de cinco lagoas que recebem o efluente de fazendas afetadas pela enfermidade foi detectada por nested PCR, e inclusões virais nos camarões por histologia. Pela nested PCR encontrou-se a presença de WSSV em 13 de 16 (81,2 por cento) amostras de camarões da espécie Farfantepenaeus paulensis, em 13 de 14 (92,8 por cento) de Litopenaeus schmitti, em uma de duas de Farfantepenaeus brasiliensis (50 por cento), em 13 de 15 (86,6 por cento) de siri da espécie Callinectes danae e em 11 de 12 (91,6 por cento) de Callinectes sapidus, e não foi detectada no caranguejo Chasmagnathus granulata em 10 amostras. Inclusões características de WSSV foram observadas em três amostras histológicas de 50 (6,0 por cento) no epitélio gástrico e cuticular e nas brânquias de dois exemplares de F. paulensis e um de L. schmitti. É o primeiro relato da presença de WSSV em camarões L. schmitti e no siri C. danae silvestres. As principais espécies de camarões e siris dos ambientes de entorno das fazendas foram contaminadas pelo WSSV, constituindo-se em vetores potenciais do vírus.

The presence of white spot syndrome virus (WSSV) in the main species of shrimps, blue crabs, and burrowing crabs of five lagoons where shrimp farm effluents are discharged, was analyzed by nested PCR and the presence of virus inclusions in the shrimps was analyzed through histopathology. The nested PCR analysis indicated the presence of WSSV in 13 of 16 (81.2 percent) samples of the shrimp species of Farfantepenaeus paulensis, in 13 of 14 (92.8 percent) of Litopenaeus schmitti, in one of two of Farfantepenaeus brasiliensis (50 percent), in 13 of 15 (86.6 percent) of blue crab species of Callinectes danae and in 11 of 12 (91.6 percent) of Callinectes sapidus and none was detected in the 10 samples of the burrowing crab Chasmagnathus granulata. The inclusion characteristics of WSSV were observed in three samples of 50 (6.0 percent) in the gastric and cuticular epithelium and in the gills of two specimens of F. paulensis and one of L. schmitti. The presence of WSSV in L. schmitti wild shrimp and in the C. danae blue crab is reported for the first time in the present work. The results indicate that the main species of shrimps and blue crabs of the environment surrounding the farms were infected by WSSV, and they may be considered potential vectors of the virus.

Antimicrobial effect of Calotropis procera active principles against aquatic microbial pathogens isolated from shrimp and fishes

Objective: To study the influence of Calotropis procera (C. procera) active principles against aquatic microbial pathogens isolated from shrimp and fishes. Methods: C. procera leaf powder was serially extracted with hexane, ethyl acetate and methanol and screened by antibacterial, antifungal and antiviral activity against aquatic pathogens which isolated from shrimp/fish. After initial screening, the active extract was purified through column chromatography and again screened. Finally the active fractions were characterized by phytochemical analysis and GC-MS analysis. Results: In vitro antibacterial, antifungal and antiviral screening revealed that, the ethyl acetate extracts were effectively suppressed the bacterial pathogens Pseudomonas aeruginosa (P. aeruginosa), Vibrio harveyi (V. harveyi) and Aeromons hydrophila (A. hydrophila) of more than 20 mm zone of inhibition; the fungi Fusarium sp and the killer virus WSSV. The ethyl acetate extracts of C. procera incubated WSSV was failed to multiply its progeny in the in vivo system of shrimp P. monodon. The shrimp had 80% survival after WSSV challenge from the control group significantly (P<0.001) and also PCR detection confirmed that no WSSV transcription found in shrimp haemolymph. After purified the ethyl acetate extracts again antimicrobial screening performed and it concluded that the fraction namely F-II was effectively suppressed the bacterial growth and WSSV due to its enriched active principles such as cardiac glycosides, Phenols, alkaloids, Tannin and quinines. Surprisingly this fraction, F-II was effectively controlled the WSSV at 90% level at a highest significant level (P<0.001). Finally the structural characterization by GC-MS analysis revealed that, the F-II fraction contained Phenols including several other compounds such as 2,4-bis(1,1-dimethylethyl)-, Methyl tetradecanoate, Bicyclo[3.1.1] heptane, 2,6,6-trimethyl-, (1α,2β,5α)-and Hexadecanoic acid etc. Conclusions: The present study revealed that there is a possibility for developing new eco-friendly antibacterial and antiviral drugs from C. procera against aquatic important pathogens.

First report of White spot syndrome virus in farmed and wild penaeid shrimp from Lagoa dos Patos estuary, southern Brazil

In this study, we detected White spot syndrome virus (WSSV) in wild Farfantepenaeus paulensis collected in the Lagoa dos Patos estuary and cultivated Litopenaeus vannamei. This is the first report of WSSV in F. paulensis from Lagoa dos Patos and farmed L. vannamei shrimps in Rio Grande do Sul.

Molecular cloning and recombinant expression of the VP28 carboxyl-terminal hydrophilic region from a brazilian white spot syndrome virus isolate

In the present study, a fragment of the VP28 coding sequence from a Brazilian WSSV isolate (BrVP28) was cloned, sequenced and expressed in E. coli BL21(DE3) pLysS strain in order to produce the VP28 carboxyl-terminal hydrophilic region. The expression resulted in a protein of about 21 kDa, which was purified under denaturing conditions, resulting in a final highly purified BrVP28 preparation. The recombinant protein obtained can be used in several biotechnology applications, such as the production of monoclonal antibodies which could be used in the development of diagnostic tools as well as in the studies on the characterization of white spot syndrome virus (WSSV) isolated in Brazil.

Prevalence of Three Shrimp Viruses in Zhejiang Province in 2008 / 中国病毒学·英文版

White spot syndrome virus(WSSV), Taura syndrome virus(TSV)and Infectious hypodermal and haematopoietic necrosis virus(IHHNV)are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang(93.3%), liuao(66.7%), Jianshan(46.7%)and Xianxiang(46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.

Genotyping of White Spot Syndrome Virus in Chinese Cultured Shrimp during 1998-1999 / 中国病毒学·英文版

Recent studies showed that white spot syndrome virus(WSSV)isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15,and variable number of tandem repeats(VNTR)within ORF94.In this study,genotyping was performed according to these three variable regions among WSSV isolates collected during 1998/1999 from Southern China.These WSSV isolates contain a deletion of 1168,5657,5898,9316 and 11093 bp,respectively in the variable region ORF23/24compared with WSSV-TW,and a deletion of 4749 or 5622 bp in the variable region ORF14/15 relative to TH-96-II.Four types of repeat units(RUs)(6,8,9 and 13 RUs)in ORF94 were detected in these isolates,with the shortest 6 RUs as the most prevalent type.Our results provide important information for a better understanding of the spatio-temporal transmission mode and the WSSV genetic evolution lineage.

Characterization and Diagnostic Use of a Monoclonal Antibody for VP28 Envelope Protein of White Spot Syndrome Virus / 中国病毒学·英文版

The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV)was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.