Taura Syndrome Research Articles

Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.

Evaluation of the preservation of shrimp samples with Davidson’s AFA fixative for infectious myonecrosis virus (IMNV) in situ hybridization

The potential negative effect of prolonged storage of shrimp tissues in Davidson’s AFA fixative on in situ hybridization (ISH) signal was demonstrated previously for Taura syndrome virus (TSV), which has a single-stranded RNA genome. In this study we evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA (Alcohol, Formaldehyde, Acetic acid) fixative will degrade its double-stranded RNA genome resulting in false negative ISH reactions. Twenty-one shrimp (3 g) specific-pathogen-free Litopenaeus vannamei were used in this study. Three shrimp were used as negative control and 18 shrimp were inoculated with a tissue homogenate prepared from frozen IMNV-infected L. vannamei obtained from Brazil in 2003 (positive control). Shrimp were collected at Day 12 post-injection and fixed in Davidson’s AFA for five different preservation times (1, 2, 4, 7 and 10 days). After the different fixation times, the Davidson’s AFA was replaced with several changes of 70% ethanol until the pH was stable. IMNV lesions were confirmed in all positive control shrimp by routine H & E histology and ISH. Myonecrosis lesions were strongly positive by ISH at all five preservation times evaluated. Hence, in the present report it was found that the length of time (up to 10 days) in Davidson’s AFA did not have a deleterious effect on the ISH reaction for IMNV.

Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV)

The gene sequence encoding VP3 capsid protein of Taura syndrome virus (TSV) was cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant GST-VP3 (rVP3) fusion protein was obtained and further purified by electro-elution before use in immunizing Swiss mice for production of monoclonal antibodies (MAb). One MAb specific to glutathione-S-transferase (GST) and 6 MAb specific to VP3 were selected using dot blotting and Western blotting. MAb specific to VP3 could be used to detect natural TSV infections in farmed whiteleg shrimp Penaeus vannamei by dot blotting and Western blotting, without cross reaction to shrimp tissues or other shrimp viruses, such as white spot syndrome virus (WSSV), yellow head virus (YHV), monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). These MAb were also used together with those specific for WSSV to successfully detect TSV and WSSV in dual infections in farmed P. vannamei.

Multiplex RT-PCR assay for simultaneous detection of six viruses of penaeid shrimp

In the present study, multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneously detection of six major shrimp viruses including yellow-head virus (YHV), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), infectious hypodermal and hematopoietic necrosis virus (IHHNV) and monodon baculovirus (MBV). The six primer sets could amplify viral nucleic acids resulting in PCR products with different sizes. They were highly specific and did not cross-hybridize with other viral or shrimp nucleic acids. The sensitivity of the multiplex RT-PCR was 0.15pg for IHHNV, 0.15pg for TSV, 1.00pg for HPV, 1.5pg for MBV, 5.00pg for WSSV and 10.00pg for YHV. In the field application, 42 samples including whole tissue of post-larvae and hepatopancreas of Penaeus monodon collected from ponds in the central and southern parts of Thailand during 2002-2005 were examined by multiplex RT-PCR. The results revealed that a single infection was dominant and WSSV was the highest prevalence at that time. Dual infection was found in one sample. This developed multiplex RT-PCR will be useful for simultaneous detection of six major viruses of penaeid shrimp and benefit to shrimp cultured industry.

SHRIMPMAP: A LOW-DENSITY, MICROSATELLITE-BASED LINKAGE MAP OF THE PACIFIC WHITELEG SHRIMP, LITOPENAEUS VANNAMEI: IDENTIFICATION OF SEX-LINKED MARKERS IN LINKAGE GROUP 4

A preliminary low-density linkage map (ShrimpMap) based solely on simple sequence repeats (SSRs) or microsatellite genetic markers was constructed for specific pathogen-free (SPF) shrimp, Litopenaeus vannamei, from the United States Marine Shrimp Farming Program (USMSFP). Marker loci originated mostly from genomic libraries cloned using DNA from ovaries of adult shrimp or cDNA libraries cloned using RNA of juveniles challenged with either Taura syndrome virus (TSV) or white spot syndrome virus (WSSV). Out of approximately 150 polymorphic markers initially tested with a small test panel consisting of 16 individuals, 100 (83 genomic SSRs and 17 EST-SSRs) were useful for genotyping with the entire mapping panel, a three-generation pedigree comprising two grandparents, two parents and 89 progeny. High frequency of null alleles (approx. 25% of markers) was observed in shrimp microsatellites. Chi-square goodness-of-fit tests revealed that 30 of the 100 markers showed segregation distortion, 17 of which were highly significant distorted (P < 0.001). Linkage analysis using CRIMAP with a LOD score of 3.0 provided a map with coverage for 14 linkage groups consisting of 67 linked markers spaced every ∼22 cM with an observed genome length of 689.1 cM and an estimated genome length for L. vannamei of 2675.7 cM. Variable results were obtained using a LOD score of 5.0. Sex-linked microsatellites were identified in linkage group 4 (LG4) of ShrimpMap, a linkage group that also includes 28 s rRNA, nonLTR retrotransposon reverse transcriptase and other markers with no homology to any sequence in the GenBank database. The map includes the first EST-SSR markers mapped for L. vannamei. Human ESTs also amplified in shrimp DNA and two of them were useful for linkage analysis. Work is underway to place additional EST-SSR markers on to ShrimpMap to serve as a framework map for penaeid species to facilitate integration of linkage and physical maps, study conservation and evolutionary genomics in penaeids, and search for candidate genes associated with fitness traits in wild and cultured shrimp. Moreover, the mapped EST-SSRs provide valuable comparative genomic links not only between L. vannamei and other penaeid species but also between shrimp and other invertebrate and vertebrate genomes.

Molecular epidemiological investigation of infectious hypodermal and hematopoietic necrosis virus and Taura syndrome virus in Penaeus Vannamei cultured in China

The Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV) are two important shrimp viruses in cultured shrimp in America. These two viruses were transmitted to China at the beginning of the 21st century. In this study, 214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection. The results showed that there were a high prevalence of IHHNV (65.42%) and low prevalence of TSV (3.27%) in the tested samples. Several samples were found to be co-infected with these two viruses. A 3 kb fragment of 7 positive IHHNV samples and a structure protein region (ORF2) of three TSV positive samples were amplified and sequenced. The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii. Phylogenetic analysis showed that TSV isolates were clustered into two groups, Asia and America group, which was genetically correlated to geographic distribution.

Proteomic analysis of differentially expressed proteins in Penaeus vannamei hemocytes upon Taura syndrome virus infection

To understand molecular responses of crustacean hemocytes to virus infection, we applied 2-DE proteomics approach to investigate altered proteins in hemocytes of Penaeus vannamei during Taura syndrome virus (TSV) infection. At 24 h postinfection, quantitative intensity analysis and nano-LC-ESI-MS/MS revealed 11 forms of 8 proteins that were significantly up-regulated, whereas 9 forms of 5 proteins were significantly down-regulated in the infected shrimps. These altered proteins play important roles in host defense (hemocyanin, catalase, carboxylesterase, transglutaminase, and glutathione transferase), signal transduction (14-3-3 zeta), carbohydrate metabolism (acetylglucosamine pyrophosphorylase), cellular structure and integrity (beta-tubulin, beta-actin, tropomyosin, and myosin), and ER-stress response (protein disulfide isomerase). Semiquantitative RT-PCR and Western blot analysis confirmed the upregulation of 14-3-3 at both mRNA and protein levels. Interestingly, several altered protein spots were identified as fragments of hemocyanin. Mass spectrometric analysis showed that the hemocyanin spots at acidic and basic regions represented the C- and N-terminal hemocyanin fragments, respectively. As three-quarters of C-terminal fragments were up-regulated, whereas two-thirds of N-terminal hemocyanin fragments were down-regulated, we therefore hypothesize that C- and N-terminal hemocyanin fragments may have differential roles in hemocytes. Further investigation of these data may lead to better understanding of the molecular responses of crustacean hemocytes to TSV infection.

Specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for Taura syndrome virus by colorimetric dot–blot hybridization

The goal of this study was to develop a field diagnosis system based on isothermal reverse transcription-loop-mediated amplification (RT-LAMP) for shrimp Taura syndrome virus (TSV), placing emphasis on specific and simple detection of the LAMP amplicons. After a single-tube RT-LAMP reaction for TSV was established, colorimetric dot–blot hybridization (DBH) was adopted to detect signals only from the target-derived amplicons. The results showed that the modified DBH offered unambiguous and sensitive detection of the TSV RT-LAMP amplicons without the UV cross-linking and denaturation steps. Together, TSV RT-LAMP-DBH assay reached the same dilution point as reverse transcription-nested polymerase chain reaction-agarose gel electrophoresis (RT-nPCR-AGE) for TSV detection. Specificity of the assay was demonstrated by the absence of DBH signal from yeast tRNA and various shrimp viruses. TSV RT-LAMP-DBH was applied to 125 Penaeus vannamei and demonstrated a very good concordance (kappa value, 0.823) with RT-nPCR-AGE assay in detection efficiency. Furthermore, a one-step guanidinium thiocyanate (GuSCN) homogenization method was established to provide RNA extraction efficiency comparable to that of the TRIzol Reagent for RT-LAMP. Requiring simply a heating apparatus, the GuSCN RNA extraction-isothermal RT-LAMP-DBH protocol has the potential for further development for diagnosis of diseases in the field.

Rapid and sensitive detection of Taura syndrome virus by reverse transcription loop-mediated isothermal amplification

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In this study, using the RT-LAMP method, a protocol for detecting Taura syndrome virus which is a causative agent of Penaeus vannamei was developed. Time and temperature conditions for detection of TSV were optimized for 60 min at 63 °C. The nucleic acids of other shrimp pathogens (yellow head virus; YHV and white spot syndrome; WSSV) were not amplified by this RT-LAMP system. The detection of TSV using RT-LAMP was 10 times more sensitive than the RT-PCR but less sensitive than nested RT-PCR. However this system was more convenient, rapid, and does not require sophisticated PCR machine.

A multiplex RT-PCR for simultaneous differentiation of three viral pathogens of penaeid shrimp

A multiplex reverse transcription polymerase chain reaction (mRT-PCR) was developed and optimized to simultaneously detect 3 viral pathogens of shrimp. Three sets of specific oligonucleotide primers for Taura syndrome virus (TSV), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) were used in the assay. The mRT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 231 bp for TSV, 593 bp for WSSV and 356 bp for IHHNV. No specific bands of the same size were amplified from other penaeid shrimp pathogenic viruses or bacteria. As little as 10 pg of TSV RNA and 100 pg of WSSV DNA and IHHNV DNA could be detected using gel electrophoresis. Studies are in progress to further test the specificity and sensitivity of this mRT-PCR method on viral isolates, as well as on clinical samples.

Characterization and diagnostic use of a recombinant single-chain antibody specific for the gp116 envelop glycoprotein of Yellow head virus

Yellow head virus (YHV) is an invertebrate nidovirus that can cause mass mortality of the cultured Penaeus monodon shrimp . A single-chain variable fragment (scFv) antibody directed against the gp116 envelop glycoprotein of YHV was constructed from hybridomas. Variable heavy (V H) and light (V L) chain genes were amplified from cDNA using antibody-specific primers, linked to generate a full-length gene via a standard peptide linker, ligated into the pET28a expression vector and transformed into E. coli. The expressed insoluble scFv antibody was solubilized, purified using immobilized metal affinity chromatography and rapid refolded; final yield 1–1.5 mg/l. Solid-phase non-competitive enzyme-linked immunosorbent assay (non-competitive ELISA) determined the affinity constant ( K A) to be 3.34 ± 0.38 × 10 8 l/mol. The sensitivity and specificity of scFv antibody was demonstrated by ELISA, dot blot and Western blot analysis. The detection limit determined by dot blot and indirect ELISA was 9 ng and 45 ng of purified YHV, respectively. Dot-blot assays revealed that the scFv antibody could detect YHV-infected shrimp at 24 h post-infection and did not cross-react with White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) proteins. The scFv antibody therefore might find application in rapid, simple and sensitive diagnostic tests to detect YHV in farmed shrimp.

Advances in Research of Necrotizing Hepatopancreatitis Bacterium (NHPB) Affecting Penaeid Shrimp Aquaculture

Necrotizing hepatopancreatitis (NHP) is a severe bacterial disease affecting penaeid shrimp aquaculture. NHP is caused by the gram-negative, pleomorphic, obligately intracellular NHP-bacterium (NHPB) that targets the hepatopancreas tissue of shrimp. NHPB is classified as an α -proteobacterium and is related to members of the Rickettsia. First reported in 1985 in a Texas shrimp farm, NHP has since affected shrimp farming in several North and South American countries, causing mortalities up to 95% and devastating economic losses to aquaculture crops. Because NHPB remains unculturable through traditional in vitro methods, the development of an in vivo cultivation system of NHPB in susceptible, specific pathogen-free Litopenaeus vannamei has allowed advancement of experimental research, including NHPB population biology and transmission dynamics. Current research on NHPB is discussed relative to management strategies of NHP disease in a shrimp pond. Compared to other important shrimp pathogens, NHPB is geographically limited, like that of yellowhead virus (YHV), but in contrast to the global distribution of Taura syndrome virus (TSV) and white spot syndrome virus (WSSV). Additionally, NHPB is less virulent than TSV and WSSV, and the transmission dynamics of TSV and YHV are much more complicated than that of NHPB and WSSV in penaeid shrimp.

Binding Mode of the First Aminoacyl-tRNA in Translation Initiation Mediated by Plautia stali Intestine Virus Internal Ribosome Entry Site

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.

Taura syndrome virus in specific pathogen-free Penaeus vannamei originating from Hawaii and in P. vannamei stocks farmed in France?

It is the opinion of the authors of the Comment on Do et al. (2006), that those authors incorrectly interpreted their test results, which are more likely the result of mislabeling of samples or within-laboratory contamination, and that the TSV isolates found in Penaeus vannamei in Korea in 2004 and 2005 did not originate from Hawaii as claimed by the authors, but from a country (or countries) in southeast Asia. Finally, we believe that the authors did not follow proper international guidelines, extend a professional courtesy to the supplier of the disputed shrimp sample, nor take a critical approach in interpreting their own data. It is unfortunate that the authors did not follow through with additional testing, or seek a second opinion from an independent laboratory, before implicating shrimp imported from Hawaii as the source of TSV in Korea.

Taura syndrome virus in Penaeus vannamei shrimp originating from Hawaii

Taura syndrome virus in Penaeus vannamei shrimp originating from Hawaii

Reduced replication of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei held in warm water

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a serious pathogen, which for the last 24 years has been responsible for negatively affecting the growth and survival of some species of cultured penaeid shrimp. The Pacific white shrimp Litopenaeus vannamei is particularly affected by IHHNV as the virus can cause poor growth and physical deformities in infected populations. There are no known treatments for shrimp affected with IHHNV and management tools for preventing this disease are limited to the exclusion of the virus from cultured shrimp populations. Previous studies have determined that warm-water culture conditions inhibit the replication rate of two other important shrimp viruses, White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) in L. vannamei. The purpose of this study was to compare the effect of water temperature on the replication rate of IHHNV in shrimp held in warm water (32.8 ± 1.0 °C), compared to the replication rate of IHHNV in shrimp held in cool water (24.4 ± 0.5 °C). A total of 72 specific pathogen-free (SPF) L. vannamei were used for this study. They were divided into four groups, namely 1) saline-injected shrimp held in warm water (31.1 ± 1.0 °C), 2) saline-injected shrimp held in cool water (26.3 ± 0.6 °C), 3) IHHNV-injected shrimp held in warm water (32.8 ± 1.0 °C), and 4) IHHNV-injected shrimp held in cool water (24.4 ± 0.5 °C). The concentration of IHHNV in shrimp collected from day 1 through day 17 post-exposure was measured with real time polymerase chain reaction (PCR) using the TaqMan® technique. The results from samples collected on days 6, 12, and 17 post-exposure showed a statistically significant difference ( p < 0.05) between the viral load of the IHHNV-injected shrimp held in warm water versus the IHHNV-injected shrimp held in cool water. The IHHNV-injected shrimp held in the warm-water conditions averaged 1.44 × 10 3, 1.77 × 10 3, and 1.20 × 10 5 virus copies/50 ng of shrimp tissue DNA on days 6, 12, and 17, respectively. The IHHNV-injected shrimp in the cool-water conditions averaged 2.25 × 10 5, 1.8 × 10 6, and 4.6 × 10 6 virus /50 ng DNA on days 6, 12, and 17, respectively. These results clearly indicate that under these conditions water temperature had a profound effect on the replication rate of IHHNV in L. vannamei. The results support the findings of the previous studies and further point to the potential application of elevated environmental temperature as a management strategy for certain viral diseases within the shrimp farming industry.

Effects of inbreeding on survival and growth of Pacific white shrimp Penaeus (Litopenaeus) vannamei

The objective of this study was to investigate the effects of inbreeding on the performance of Pacific white shrimp, Penaeus (Litopenaeus) vannamei, under various culture conditions through a retrospective analysis of family performance data. Fourteen years of pedigree and eight years of performance data from Oceanic Institute's selective breeding program were used in the analysis. During this period, shrimp performance was evaluated in growout trials (in pond and raceway systems), laboratory challenges to three isolates of Taura syndrome virus (TSV), and a laboratory challenge to White spot syndrome virus (WSSV). The effects of inbreeding on growth and survival were estimated by regressing family phenotypic means (adjusted for contemporary group effects) on inbreeding coefficients. During growout, inbreeding had a small but significant effect on growth (2.6 to 3.9% reduction per 10% inbreeding) but had no effect on survival. The effects of inbreeding on survival after exposure to viral pathogens ranged from moderate (8.3% reduction per 10% inbreeding) to severe (38.7% reduction), although not all effects were significant. Furthermore, the effects of inbreeding on survival appeared to be sensitive to environmental quality, as inbreeding depression was more severe in more stressful environments (smallest effect during growout trials and largest effect during exposure to WSSV). These results suggest that moderate to high levels of inbreeding (>10%) should be avoided in shrimp breeding programs, especially when shrimp are reared under stressful conditions. In addition, the effects of inbreeding on survival appear to be significant enough to justify the use of inbreeding as a germplasm protection strategy (under certain scenarios) for genetic improvement programs.

Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells

Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV.

Real-time reverse transcription polymerase chain reaction assay using TaqMan probe for detection and quantification of Infectious myonecrosis virus (IMNV)

Infectious myonecrosis, caused by Infectious myonecrosis virus (IMNV), is an important emerging disease of shrimp that has affected the production of cultured Litopenaeus vannamei in Northeast Brazil. In this study we report the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe to detect this virus in shrimp. The real-time RT-PCR showed a strong linear correlation ( r 2 = 0.986) between threshold cycles ( C T) and RNA quantities. The assay gave negative results for other viruses, including Yellow head virus (YHV), Taura syndrome virus (TSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), and White spot syndrome virus (WSSV) and the necrotizing hepatopancreatic bacterium (NHPB). This real-time RT-PCR assay can detect as few as 10 IMNV copy numbers/μl RNA, while the nested RT-PCR can detect no fewer than 1000 IMNV copy numbers/μl RNA. Specific-pathogen-free L. vannamei were used in the infectivity assay. There were one control group (Group 1) and one viral challenged group (Group 2), from which shrimp were sampled for RT-PCR and histological analysis. The RNA from dead shrimp was extracted and tested for IMNV by nested and real-time RT-PCR. The shrimp in Group 1 showed 100% survival, while those in Group 2 showed a 0% survival. The first mortality in the Group 2 was observed at Day 8 post-inoculation (p.i.); and the mortalities dramatically increased after Day 40 p.i. Histological sections from Group 2 shrimp taken at intervals throughout the study exhibited acute to chronic phase lesions of IMNV infection, and consecutive tissue sections reacted to the IMNV-specific cDNA probes by in situ hybridization. The real-time RT-PCR detected the presence of IMNV in all 30 of the challenged specimens in Group 2. In contrast, the nested RT-PCR detected the presence of IMNV in 23 of the 30 specimens. The real-time RT-PCR revealed that the 7 specimens not detected by nested RT-PCR contained relatively low IMNV copy numbers compared to the other 23 specimens. These results demonstrate that the real-time RT-PCR developed in this study is a sensitive diagnostic method for IMNV.

Rapid and sensitive detection of Taura syndrome virus using nucleic acid-based amplification

Requiring only simple heating devices, isothermal nucleic acid-based amplification (NASBA) is a potential detection platform to be developed for on-site diagnosis of aquaculture pathogens. In this report, an NASBA assay has been developed for the Taura syndrome virus (TSV), one of the most devastating RNA virus pathogens for several penaeid shrimp species. The NASBA amplicons were detected by agarose gel electrophoresis and confirmed by Northern-blotting and dot-blotting analysis, using a biotinylated TSV-specific primer. The sensitivity of the TSV NASBA coupled with dot-blotting detection was approximately 5-fold less sensitive than that of the commercially available RT-nested, PCR-based IQ2000 TSV Detection and Prevention System that was also confirmed to be more sensitive than the RT-PCR-based TSV detection protocol recommended by the OIE (Office International des Epizooties). The specificity of the TSV NASBA reaction was substantiated by the results that RNA of non-target viruses did not generate any signals. Furthermore, a simple colorimetric microtiter plate assay employing TSV-specific capture and detection primers was developed as a simple alternative approach for the detection of NASBA amplicons. Taken together, the combination of the isothermal NASBA and colorimetric solid phase-based assays should allow sensitive, straightforward, and speedy on-site detection of TSV.