Detection of white spot syndrome virus in seafood samples using a magnetosome-based impedimetric biosensor.

White spot syndrome virus (WSSV) is a significant threat to the aquaculture sector, causing mortality among crabs and shrimps. Currently available diagnostic tests for WSSV are not rapid or cost-effective, and a new detection method is therefore needed. This study demonstrates the development of a biosensor by functionalization of magnetosomes with VP28-specific antibodies to detect WSSV in seafood. The magnetosomes (1 and 2 mg/ml) were conjugated with VP28 antibody (0.025-10 ng/µl), as confirmed by spectroscopy. The magnetosome-antibody conjugate was used to detect the VP28 antigen. The binding of antigen to the magnetosome-antibody complex resulted in a change in absorbance. The magnetosome-antibody-antigen complex was then concentrated and brought near a screen-printed carbon electrode by applying an external magnetic field, and the antigen concentration was determined using impedance measurements. The VP28 antigen (0.025 ng/µl) bound more efficiently to the magnetosome-VP28 antibody complex (0.025 ng/µl) than to the VP28 antibody (0.1 ng/µl) alone. The same assay was repeated to detect the VP28 antigen (0.01 ng/µl) in WSSV-infected seafood samples using the magnetosome-VP28 antibody complex (0.025 ng/µl). The WSSV in the seafood sample was also drawn toward the electrode due to the action of magnetosomes controlled by the external magnetic field and detected using impedance measurement. The presence of WSSV in seafood samples was verified by Western blot and RT-PCR. Cross-reactivity assays with other viruses confirmed the specificity of the magnetosome-based biosensor. The results indicate that the use of the magnetosome-based biosensor is a sensitive, specific, and rapid way to detect WSSV in seafood samples.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Detection of infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in whiteleg shrimp (Penaeus vannamei) imported from Vietnam to South Korea.

In this study, whiteleg shrimp (Penaeus vannamei) imported from Vietnam were collected from South Korean markets, and examined for 2 viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV, recently classified as decapod penstyldensovirus-1), and white spot syndrome virus (WSSV). Among 58 samples, we detected IHHNV in 23 samples and WSSV in 2 samples, using polymerase chain reaction and sequencing analyses. This is the first report of IHHNV and WSSV detection in imported shrimp, suggesting that greater awareness and stricter quarantine policies regarding viruses infecting shrimp imported to South Korea are required.

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond-reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection.

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow-out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV-positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m-PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m-PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 µg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

BACKGROUND: White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. METHODS/PRINCIPAL FINDINGS: Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR. CONCLUSION: The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Expression profile of bio-defense genes in Penaeus monodon gills in response to formalin inactivated white spot syndrome virus vaccine.

White spot syndrome virus (WSSV) is the most devastating pathogen of penaeid shrimp. While developing technology to vaccinate shrimp against WSSV, it is imperative to look into the immune response of the animal at molecular level. However, very little information has been generated in this direction. The present study is an attempt to understand the expression of bio-defense genes in gill tissues of Penaeus monodon in response to formalin inactivated WSSV. A WSSV vaccine with a viral titer of 1×10(9) DNA copies was prepared and orally administered to P. monodon at a rate of 1.75×10(6) DNA copies of inactivated virus preparation (IVP) day(-1) for 7days. The animals were challenged with WSSV on 1st and 5th day post vaccination, and temporal expression of bio-defense genes in gill tissues was studied. Survival of 100% and 50% were observed respectively on 1st and 5th day post vaccination challenge. The humoral immune genes prophenoloxidase (proPO), alpha 2-macroglobulin (α2M), crustin and PmRACK, and the cell mediated immune genes caspase and Rab7 were up regulated in gill tissue upon vaccination and challenge. The expression of humoral gene crustin and cellular gene Rab7 was related to survival in IVP administered shrimp. Results of the study suggest that these genes have roles in protecting shrimp from WSSV on vaccination.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

BACKGROUND: The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. RESULTS: The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. CONCLUSION: The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Prevalence of viral pathogens WSSV and IHHNV in wild organisms at the Pacific Coast of Mexico.

This study investigated whether white spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus, can survive in wild invertebrates and vertebrates in the environment surrounding shrimp farms along the Pacific coast of Mexico. The evidences imply that both viruses have a potential of persisting in crabs, blue, white and brown shrimps. The most prevalent virus, IHHNV was present in 19.5% (344/1736) followed by WSSV in 3.6% (65/1736). Coinfection of WSSV and IHHNV was also detected in crabs, blue and white shrimps. This is the first prevalence report of WSSV and IHHNV associated with wildlife species in Mexico.

Natural occurrence of White spot syndrome virus and Infectious hypodermal and hematopoietic necrosis virus in Neohelice granulata crab.

White spot syndrome virus (WSSV) and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) are two infectious agents associated to economic losses in shrimp aquaculture. As virus spread occurs through vectors and hosts, this study sought to verify the presence of WSSV and IHHNV in Neohelice granulata crab from Lagoa dos Patos estuary in Brazil and nearby shrimp farms. DNA extractions were performed with phenol/chloroform protocol. Molecular diagnosis was carried out by nested PCR for WSSV and one-step PCR for IHHNV. Results showed the presence of WSSV on crabs of both Lagoa dos Patos and farms, while IHHNV was found only on crabs collected in estuary. This is the first study to report IHHNV presence in N. granulata. Moreover, as analyzed crabs had no clinical symptoms or showed in situ mortality, we suggest its use as a bioindicator for virus occurrence in aquatic environments.

Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions.

Aiming to develop a rapid, low-cost, and user-friendly system for the diagnosis of white spot syndrome virus (WSSV), a PCR assay performed in capillary tubes under insulated isothermal conditions (iiPCR assay) was established on the basis of Rayleigh-Benard convection. WSSV amplicons were generated reproducibly within 30 min from a target sequence-containing plasmid in an iiPCR device, in which a special polycarbonate capillary tube (R-tube™) was heated isothermally by a copper ring attached to its bottom and shielded by a thermal baffle around its upper half. Furthermore, WSSV-specific amplicons were produced from nucleic acid extracts of WSSV-infected Penaeus vannamei in the WSSV iiPCR assay, with sensitivity comparable to that of an OIE-certified commercial nested PCR kit (IQ2000™ WSSV Detection and Prevention System). Specificity of the WSSV iiPCR assay was demonstrated as no amplicons were generated from shrimp genomic DNA, and IHHNV, MBV, and HPV DNA. iiPCR has a potential as a low-cost method for sensitive, specific and rapid detection of pathogens.

Survey of protozoan, helminth and viral infections in shrimp Litopenaeus setiferus and prawn Macrobrachium acanthurus native to the Jamapa River region, Mexico.

We surveyed protozoan and metazoan parasites as well as white spot syndrome virus (WSSV) and infectious hypodermal hematopoietic necrosis virus (IHHNV) in white shrimp Litopenaeus setiferus and the palaemonid prawn Macrobrachium acanthurus native to the lower Jamapa River region of Veracruz, Mexico. The presence of parasites and the infection parameters were evaluated in 113 palaemonid prawns collected during the northwind (n = 45), rainy (n = 38) and dry seasons (n = 30) between October 2007 and July 2008, and in 91 shrimp collected in the rainy season between May and June 2008. In L. setiferus, ciliates of the subclass Apostomatia (Ascophrys sp.) were evident in gills, and third-stage larvae of the nematode Physocephalus sexalatus were evident in the stomach. Cestodes of the genus Prochristianella were evident in the hepatopancreas, while some gregarines of the genus Nematopsis, as well as unidentified larval cestodes, were observed in the intestine. Histology identified Ascophrys sp. in association with gill necrosis and tissue melanization. Slight inflammation was observed in intestinal epithelium near cestode larvae. In M. acanthurus, epibionts of the protozoans Epistylis sp., Acineta sp. and Lagenophrys sp. were observed under uropods, periopods and pleopods. An unidentified ciliate of the Apostomatia was also found in the gills, and Nematopsis was identified in the intestine. No histopathology was observed in association with these parasites. Moreover, neither WSSV nor IHHNV were detected by the polymerase chain reaction (PCR) in any of the L. setiferus or M. acanthurus analysed.

Prevalence of three shrimp viruses in Zhejiang Province in 2008.

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.

Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes.

Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10(2) copies of the target DNA to less than 70min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.

Fast short-fragment PCR for rapid and sensitive detection of shrimp viruses.

This article describes a fast short-fragment PCR method for the detection of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and monodon baculovirus (MBV). Fast two-temperature (95 degrees C denaturation and 60 degrees C annealing/extension) PCRs were performed in 5-10 microl volume samples in miniaturized microplates using a fast Peltier thermal cycler. 40 cycles were completed in 25-30 min. Rapid high-resolution agarose gel electrophoresis of 70-150 bp PCR fragments was performed in 10 min. High sensitivity of PCR product detection (50-100 pg) was obtained using ultra sensitive dyes such as GelStar and a gel documentation system equipped with a blue-light transilluminator. This novel method is faster and more sensitive than its TaqMan real-time PCR counterparts.

Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick.

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other commonly-used methods for nested PCR detection of WSSV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.

Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp.

A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.

Comparison of white spot syndrome virus PCR-detection methods that use electrophoresis or antibody-mediated lateral flow chromatographic strips to visualize PCR amplicons.

White spot syndrome virus (WSSV) PCR-detection methods that used electrophoresis or lateral flow chromatographic strips (LFCS) were to compare and visualize PCR amplicons. Real-time PCR was used to prepare a stock template solution containing 2.85 x 10 (6) copies WSSV/microl from WSSV-infected shrimp. Serial stock dilutions were used as templates for PCR amplification of a WSSV-specific DNA fragment that was detected either in ethidium bromide stained agarose electrophoresis gels or on a chromatographic strip where it interacted with antibody to markers labeled on hybridization complex. PCR amplification employed both 1-step PCR and semi-nested PCR methods. By using 1-step PCR, the LFCS method (100 copies) gave 10 times higher sensitivity than gel electrophoresis (10(3) copies). A combination of a semi-nested PCR with LFCS gave a comparable sensitivity to those with commercial kits for nested PCR (20 copies). In addition, LFCS confirmed amplicon identity, avoided handling of carcinogenic ethidium bromide and could be completed in approximately 20-30 min post-PCR compared with 1h for gel electrophoresis. The costs for the two methods were comparable. In conclusion, semi-nested PCR followed by LFCS is a safe and rapid alternative method for detection of WSSV that provides sensitivity similar to that obtained by standard nested PCR methods.