Detection Of Taura Syndrome Virus Research Articles

Rapid and sensitive detection of Taura syndrome virus (TSV) in shrimp based on an isothermal enzymatic recombinase amplification (ERA) assay

Rapid and sensitive detection of Taura syndrome virus (TSV) in shrimp based on an isothermal enzymatic recombinase amplification (ERA) assay

Amplification-free CRISPR-Cas13a assay for detection of Taura syndrome virus

Taura syndrome virus (TSV), which causes Taura syndrome, poses serious threats to the aquaculture industry. To date, early detection is still an important way to control its epidemic and spread. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) 13a (CRISPR-Cas13a) system, has been used to detect virus RNA. In this study, we developed an amplification-free CRISPR-Cas13a assay for directly detection of TSV from shrimp RNA. This assay could detect 107 copies/μL (16.6 pM) TSV in vitro transcribed (IVT) RNA in 5 min and 106 copies/μL (1.66 pM) within 15 min at 37 °C. It could distinguish TSV RNA specifically without any cross reaction with other shrimp pathogens. 56 Penaeus vannamei clinical samples were validated using this newly established assay, revealing 100 % consistency with the typical real-time RT-PCR (RT-qPCR) assay. These results demonstrate that this new assay for TSV is accurate, rapid and cost-effective, thus, it is promising for early diagnosis of RNA virus in aquaculture.

Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson's-Fixed Paraffin-Embedded Shrimp Tissue.

Taura syndrome is a World Organization for Animal Health (OIE)-listed disease of marine shrimp that is caused by Taura syndrome virus (TSV), a single-stranded RNA virus. Here we demonstrate the utility of using 15-year-old archived Davidson’s-fixed paraffin-embedded (DFPE) shrimp tissues for TSV detection and phylogenetic analyses. Total RNA was isolated from known TSV-infected DFPE tissues using three commercially available kits and the purity and ability to detect TSV in the isolated RNA were compared. TSV was successfully detected through RT-qPCR in all the tested samples. Among the TSV-specific primers screened through RT-PCR, primer pair TSV-20 for the RNA-dependent RNA polymerase (RdRp), primers TSV-15 and TSV-16 for the capsid protein gene VP2 and primers TSV-5 for the capsid protein gene VP1 amplified the highest number of samples. To assess the phylogenetic relation among different TSV isolates, the VP1 gene was amplified and sequenced in overlapping segments. Concatenated sequences from smaller fragments were taken for phylogenetic analyses. The results showed that the TSV isolates from this study generally clustered with homologous isolates from the corresponding geographical regions indicating RNA derived from DFPE tissues can be used for pathogen detection and retrospective analyses. The ability to perform genomic characterization from archived tissue will expedite pathogen discovery, development of diagnostic tools and prevent disease spread in shrimp and potentially other aquaculture species worldwide.

Detection of Taura Syndrome Virus (TSV) in Litopenaeus vannamei in the Philippines

Detection of Taura Syndrome Virus (TSV) in Litopenaeus vannamei in the Philippines

Prokaryotic expression and immunodot-blot detection of Taura syndrome virus capsid proteins VP2 and VP3

Capsid protein genes vp2 and vp3 of Taura syndrome virus (TSV) were cloned into pet-16b-1 and pGEX-4t-3 expression vector respectively, and transformed intoEscherichia coli BL21 or DH5α for protein expression and purification. After induction with IPTG, recombinant VP2 (rVP2) and recombinant VP3 (rVP3) were produced in E. coli, purified by SDS-PAGE and used to immunize Balb/c mice forthe production of polyclonal antisera: anti-rVP2 and anti-rVP3. Two antigenic peptides originating from TSV capsid protein VP2 and VP3 respectively were synthesized as antigen for the production of monoclonal antibodies (Mabs). Mab specific to VP2, VP3, anti-rVP2 and anti-rVP3 antisera all showed specific immunoreactivities to corresponding recombinant viral proteins by Western blot assay. The Mabs as well as the polyclonal antisera could detect VP2 and VP3 in homogenates from gills of TSV-infected shrimp by immunodot-blot. This is the first step towards our target of preparing TSV capsid proteins as oral vaccine and developing simple immuno-diagnostic test kits for TSV detection in Penaeus vannamei shrimp. Key words: Taura syndrome virus (TSV), Penaeus vannamei, Mabs, VP2 and VP3 protein, Western blot.

Comparison of Taura syndrome virus (TSV) detection methods during chronic-phase infection in Penaeus vannamei

Methods to detect Taura syndrome virus (TSV) were assessed for their ability to detect the virus during chronic phase infection in the Pacific white shrimp Penaeus vannamei. In situ hybridization (ISH), immunohistochemistry (IHC) using monoclonal antibody 1A1, conventional RT-PCR and real-time quantitative (q)RT-PCR were compared using shrimp sampled over 60 wk following experimental TSV infection. Between Weeks 7 and 60, hematoxylin-eosin histology confirmed the presence of lymphoid organ spheroids (LOS) and an absence of lesions in the cuticular epithelium. ISH detected TSV in LOS over the duration of the study. IHC was generally less sensitive than ISH, and after Week 24, was often unable to confirm TSV infection. Detection of TSV by RT-PCR was highly dependent on sample source after Week 43, where viral RNA was detected in 12 of 14 hemolymph samples but only 5 of 16 pleopod samples. qRT-PCR detected TSV over the 60 wk in both hemolymph and pleopods, although RNA copy numbers in pleopods were consistently lower throughout the study. This study demonstrates that ISH and qRT-PCR are the most reliable methods for detecting TSV during late chronic phase infection. RT-PCR was also reliable if hemolymph was used as the sample source.

Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acid under isothermal conditions using four sets of specially designed primers that recognize six distinct target sequences with high specificity and sensitivity. In this report, a 60-min reverse transcription LAMP (RT-LAMP) method for amplification of Taura syndrome virus (TSV) cDNA using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of TSV-specific amplicons. The LFD process involved a 5-min post RT-LAMP step for specific hybridization of cDNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled TSV amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5 min of sample exposure. Using the combined RT-LAMP and LFD system, the total assay interval was approximately 70 min, excluding RNA extraction time. Detection sensitivity was comparable to other commonly used methods for nested RT-PCR detection of TSV. In addition to reduced assay time when compared to electrophoresis, combination of RT-LAMP with LFD confirms amplicon identity by hybridization and eliminates the need to handle carcinogenic ethidium bromide.

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.

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.

The molecular detection of Taura syndrome virus emerging with White spot syndrome virus in penaeid shrimps of Thailand

This study is to investigate the outbreak of the Taura syndrome (TS) in penaeid shrimps of Thailand. Following the outbreak in 2003, the Taura syndrome virus (TSV) was detected in two closely related penaeids; the Pacific white shrimp, Penaeus vannamei, and the black tiger prawn, Penaeus monodon. Multiple viral infections were detected, we found that the TSV-infected P. vannamei also presented evidence of the White spot syndrome virus (WSSV) as evidenced by histological examination and molecular diagnostic methods. Infections of TSV and WSSV were investigated over a period of ten months between October 2003 and July 2004. TSV infection in shrimps was diagnosed by RT-PCR using the OIE recommended 9195/9992 primers. Positive shrimp gave the expected amplicon of 231 bp. We also amplified and sequenced a fragment of the vp1 gene (354 bp). The pairwise comparisons of vp1 revealed that Thai TSV was phylogenetically related to the Taiwanese strains (98.3% identity).

Polyclonal antibodies specific for VP1 and VP3 capsid proteins of Taura syndrome virus (TSV) produced via gene cloning and expression

Capsid protein genes VP1 and VP3 of Taura syndrome virus (TSV) were cloned into pGEX-6P-1 expression vector and transformed into Escherichia coli BL21. After induction, recombinant VP1 (rVP1) and recombinant VP3 (rVP3) were produced, purified by SDS-PAGE and used for immunization of Swiss mice for antisera production. Anti-rVP1 and anti-rVP3 antisera showed specific immunoreactivities to rVP1 and rVP3 proteins, respectively, by Western blot assay and also yielded good results for detection of TSV in various shrimp tissues by immunohistochemistry. This is the first step towards our target of preparing monoclonal antibodies specific to rVP1 and rVP3 for use in simple immuno-diagnostic test kits for TSV detection and identification.

Taura syndrome virus from Penaeus vannamei shrimp cultured in Korea

Mass mortality occurred among Penaeus vannamei shrimp cultured in Korea in 2004. In an earlier study, we reported white spot syndrome virus (WSSV) as a causative agent of mass mortality of P. monodon shrimp in Korea (Moon et al. 2003; Dis Aquat Org 53:11-13). However, in the present study, we detected Taura syndrome virus (TSV) from the moribund 2004 P. vannamei shrimp by reverse transcription polymerase chain reaction (RT-PCR). In addition, during our regular screening for the TSV in stocks of P. vannamei imported from Hawaii, USA, we also detected TSV by RT-PCR. The nucleotide sequences of the partial capsid protein VP1 of 2 Korean isolates were 99% identical to each other and 96 to 99% identical to those of TSVs isolated from the Americas, Taiwan, and Thailand. Phylogenetic analysis revealed that the 2 Korean isolates were closely related to TSV types from Thailand. This is the first report on the detection of TSV during an epizootic among cultured P. vannamei in Korea, and our results suggests the possibility that TSV has been introduced via the imported stock of P. vannamei.

Taura syndrome virus from Belize represents a unique variant

A Taura syndrome virus (TSV) isolate from cultured Penaeus vannamei grown in Belize, Central America was characterized and shown to be a unique isolate. Mortality rates in laboratory infections of specific pathogen-free (SPF) P. vannamei, reactivity of the virus with monoclonal antibody (MAb) 1A1 and phylogenetic analysis demonstrated that the Belize isolate (BLZ02TSV) is a new valiant of TSV. The Hawaiian 1994 TSV isolate (HI94TSV, GenBank AF277675) was used as the reference isolate for these studies. Laboratory infections of SPF P. vannamei with BLZ02TSV demonstrated higher mortalities and earlier onset of mortalities compared to infections with HI94TSV. Shrimp tissues infected with BLZ02TSV reacted with a TSV-specific gene probe by in situ hybridization and were positive by RT-PCR using TSV diagnostic primers, thus indicating that the isolate was TSV. However, Western blot analysis and immunohistochemistry using MAb 1A1 demonstrated that BLZ02TSV did not react with the antibody, suggestive of changes in the VP1 region of the genome that codes for the polypeptide to which MAb 1A1 binds. Phylogenetic analysis of a 1.3 kbp fragment of the TSV VP1 capsid region revealed that BLZ02TSV represents a distinct group among more than 29 isolates of TSV studied thus far. This research demonstrates that BLZ02TSV is a unique isolate of TSV and reiterates a problem related to the use of MAb 1A1 for detection of TSV in clinical specimens.

Nonsusceptibility of Primate Cells to Taura Syndrome Virus

Taura syndrome virus (TSV), a pathogen of penaeid shrimp and member of the family Dicistroviridae, was recently reported to have the ability to infect primate cells. We independently retested this hypothesis. Three lines of primate cells FRhK-4, MA-104, and BGMK, which are highly susceptible to infection by human picornaviruses, were challenged with TSV. Viral replication was assayed by real-time reverse transcription-polymerase chain reaction using cell media samples collected on days 0, 4, and 7 postchallenge. By day 7, genome copy numbers had decreased 25%-99%. No cytopathic effect was observed after 7 days. An in situ hybridization assay, with gene probes specific for detection of TSV, was negative for TSV in challenged cells. The infectivity of residual virus in the cell culture media at day 7 was confirmed by bioassay using TSV-free indicator shrimp (Litopenaeus vannamei). TSV did not infect the primate cells tested, and no evidence of zoonotic potential was found.

Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT-PCR

Real-time RT-PCR using SYBR Green chemistry uses a green fluorescence dye, SYBR Green I, that binds to double stranded DNA (dsDNA) and exhibits enhancement of fluorescence upon binding to the DNA. The indiscriminate binding ability of SYBR Green I dye to dsDNA often results in non-specific products. We have shown that increasing the amplicon size from ∼50 to ∼75–100 bp increases the specificity due to higher melting temperature of the amplicon and also enhances the sensitivity of detection of real-time RT-PCR using SYBR Green chemistry while detecting two RNA viruses in laboratory-challenged shrimp, the Taura syndrome virus (TSV), and yellow head virus (YHV). The increased sensitivity of the larger amplicon over the smaller amplicon varied from 1.6 to 6.82-fold (with a median value of 4-fold) for the TSV-infected samples, and 1.80–10.27-fold (with a median value of 4-fold) for the YHV-infected samples. The longer amplicon also has a higher Tm value compared with the shorter amplicon (75.6 vs. 72.0 °C for TSV, and 81.3 vs. 72.5 °C for YHV). The increased melting temperature of the longer amplicon compared with the shorter amplicon will enable easier discrimination of a specific product from a primer dimer or other non-specific products. The improved method for the detection of TSV and YHV will be applicable not only to the detection of other viral pathogens but also to the quantitative measurement of cellular gene expression by real-time SYBR Green RT-PCR.

Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry

Taura syndrome virus (TSV) and yellow head virus (YHV) are the two RNA viruses infecting penaeid shrimp ( Penaeus sp.) that have caused major economic losses to shrimp aquaculture. A rapid and highly sensitive detection and quantification method for TSV and YHV was developed using the GeneAmp ® 5700 Sequence Detection System and SYBR Green chemistry. The reverse transcriptase polymerase chain reaction (RT-PCR) mixture contained a fluorescent dye, SYBR Green, which exhibits fluorescence enhancement upon binding to double strand cDNA. The enhancement of fluorescence was found to be proportional to the initial concentration of the template cDNA. A linear relationship was observed between input plasmid DNA and cycle threshold ( C T) values for 10 6 down to a single copy of both viruses. To control for the variation in sample processing and in reverse transcription reaction among samples, shrimp β-actin and elongation factor-1α (EF-1α) genes were amplified in parallel with the viral cDNA. The sensitivity and the efficiency of amplification of EF-1α was greater than β-actin when compared to TSV and YHV amplification efficiency suggesting that EF-1α is a better internal control for the RT-PCR detection of TSV and YHV. In addition, sample to sample variation in EF-1α C T value was lower than the variation in β-actin C T value of the corresponding samples. The specificity of TSV, YHV, EF-1α and β-actin amplifications was confirmed by analyzing the dissociation curves of the target amplicon. The C T values of TSV and YHV samples were normalized against EF-1α C T values for determining the absolute copy number from the standard curve of the corresponding virus. The method described here is highly robust and is amenable to high throughput assays making it a useful tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture.

Production and use of antibodies for the detection of Taura syndrome virus in penaeid shrimp

Production and use of antibodies for the detection of Taura syndrome virus in penaeid shrimp

Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura syndrome virus (TSV) in experimentally infected shrimp.

Taura Syndrome Virus (TSV) has adversely affected the shrimp culture industries of the Americas. First recognized in 1992, this viral agent has spread throughout the shrimp growing regions of South and Central America to become established in North America in the short span of 5 yr. Diagnostic methods for TSV include histopathology, bioassay using susceptible Penaeus vannamei as the indicator species and in situ hybridization with TSV specific complimentary DNA (cDNA) gene probes. An additional method for detecting TSV is through the use of reverse transcription polymerase chain reaction (RT-PCR). Two oligonucleotide primers were selected using the sequence information from a cloned cDNA segment of the TSV genome. The primers, designated 9195 and 9992, used in the RT-PCR procedure amplify a 231 base pair (bp) fragment of the cDNA. Using the RT-PCR technique, TSV has been detected in the hemolymph of P. stylirostris and P. vannamei with experimentally induced TSV infections.