Development of rapid neutralization assay of viral hemorrhagic septicemia virus (VHSV) based on chimeric rhabdovirus expressing heterologous glycoprotein.

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV.

A Novel Specific Single-Chain Variable Fragment Diagnostic System for Viral Hemorrhagic Septicemia Virus.

Viral hemorrhagic septicemia virus (VHSV), one of the most important viral marine pathogens worldwide, has a broad range of hosts, such as members of the families Salmonidae and Paralichthyidae. In addition to being highly contagious, VHSV causes high lethality. The transmission of VHSV can be both vertical and horizontal. In fish, the resolution of VHSV infection is challenging. Thus, early diagnosis of VHSV infections is critical, especially in fish farms that have a high population of juvenile fish. Serological methods are commonly used to detect viral antigens. However, limited serological methods are available for marine viruses. In this study, a VHSV-specific single-chain variable fragment (scFv), E5, was selected using the yeast surface display and phage display systems. scFv, a type of recombinant antibody, comprises a variable heavy chain ([Formula: see text]) and a variable light chain ([Formula: see text]) connected by a polypeptide linker. An scFv clone was selected from the VHSV glycoprotein-expressing yeast cells using the bio-panning method. The scFv-encoding gene was subcloned and expressed in the Escherichia coli expression system. The binding affinity of the expressed and purified scFv protein was determined using an enzyme-linked immunosorbent assay and western blotting. Thus, this study reported a method to identify VHSV-specific scFv using bio-panning that can be utilized to develop a diagnostic system for other viruses.

Genome based quantification of VHSV in multiple organs of infected olive flounder (Paralichthys olivaceus) using real-time PCR.

BACKGROUND: Viral hemorrhagic septicemia (VHS) is a serious viral disease that infects the olive flounder in South Korea. The Korean aquaculture industry experienced an economic loss caused by the high infectivity and mortality. OBJECTIVE: This study aimed to evaluate the infection density of VHSV in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm and injected subcutaneously with 106 PFU/fish. METHODS: Each 15 fish were sampled at 0, 3, and 7 days post challenge (dpc), respectively, to perform quantitative analysis of VHSV using SYBR-green based real-time PCR in various tissues including spleen, liver, head-kidney, body-kidney, muscle, esophagus, stomach, intestine, gill, and brain. RESULTS: Organs infected with VHSV were obtained after 3 and 7 days. Each organs were examined for viral infection using real-time PCR. The data obtained from this experiment revealed copy numbers higher than 10 copies per 100 ng cDNA in the spleen (15.26 ± 3.11 copies/100 ng of cDNA), muscle (11.24 ± 2.25 copies), and gill (14.23 ± 6.26 copies), but lower in liver, head-kidney, body-kidney, esophagus, brain and stomach. CONCLUSION: The present study, together with previous data, demonstrated that the gill, spleen, and muscle are the major target organs of VHSV in olive flounder. Therefore, central monitoring of spleen, gill and muscle should be considered and might be necessary if anti-VHSV treatment is to be successful in infected olive flounder.

Assessment of a Serologic Diagnostic Test and Kinetics of Antibody Development in Northern Pike Experimentally Infected with Viral Hemorrhagic Septicemia Virus.

Viral hemorrhagic septicemia virus (VHSV) is an ongoing cause of disease and mortality in freshwater fishes across the Great Lakes region of the Midwestern United States. Antibody detection assays such as enzyme-linked immunosorbent assay (ELISA) are nonlethal serological methods that can have significantly shorter turnaround times than the current validated viral detection diagnostic methodology for VHSV: cell culture with confirmation by polymerase chain reaction (PCR). This study evaluated an ELISA that detects nonneutralizing antinucleocapsid antibodies to VHSV in Northern Pike Esox lucius. Juvenile Northern Pike were experimentally infected with VHSV by intraperitoneal injection. The infected fish were monitored for 12 weeks for signs of disease, and weekly serum samples were obtained. An analysis of the survival data showed that mortality occurred significantly more quickly in inoculated fish than in control fish. Fish that were infected by injection showed a significant increase in antibody response by 2 weeks postinfection. However, variation in the rate and pattern of antibody response among the infected fish was high at any given point. The optimum window for detecting antibodies in Northern Pike is 2-12 weeks postinfection, which generally follows the median time to appearance of clinical signs (21 d postinfection). The receiver-operating characteristic curve analysis showed the ELISA to have a sensitivity of 80.5% and a specificity of 63.2% in Northern Pike, but these values can be adjusted by choosing different percent inhibition cutoffs, which may facilitate the use of the test for specific management goals. The results of this study offer insights into the disease progression and immune kinetics of VHSV, including interindividual variation, which will aid in the management of this economically important virus.

Improvement of a diagnostic procedure in surveillance of the listed fish diseases IHN and VHS.

Infectious haematopoietic necrosis (IHN) and viral haemorrhagic septicaemia (VHS) are OIE-listed and notifiable viral fish diseases which are controlled by eradication and surveillance programmes globally. The present study provides improved RT-qPCR procedures based on recently described OIE protocols. Improvements comprise the design of a new TaqMan® probe, replacing a TaqMan® MGB probe that turned out to show impaired binding. Reason for this is SNPs detected in the nucleoprotein N gene sequences of IHNV strains targeted by the RT-qPCR. Furthermore, the IHNV and VHSV RT-qPCR assays were realized as one-step and one-run procedures supplemented by an endogenous control system. The IHNV and VHSV RT-qPCR assays are characterized by a technical sensitivity of 19 and 190 gene equivalents (cRNA) and an analytical sensitivity of 2-7 and 13 TCID50 /ml, respectively. For verification purposes, 105 IHNV and 165 VHSV isolates and several non-targeted viral and bacterial pathogens were included and returned adequate results. However, in field samples divergent results left 14 samples of 154 undetected for IHNV and one sample of 127 for VHSV using cell culture. The study shows that RT-qPCR assays ensure facilitated and reliable testing on IHNV and VHSV in eradication and surveillance programmes.

Viral haemorrhagic septicaemia virus (VHSV Id) infections are detected more consistently using syndromic vs. active surveillance.

The eradication of viral haemorrhagic septicaemia virus (VHSV Id) from Finnish brackish-water rainbow trout Oncorhynchus mykiss farms located in the restriction zone in the Province of Åland, Baltic Sea, failed several times in the 2000s. The official surveillance programme was often unable to find VHSV-positive populations, leading to the misbelief in the fish farming industry that virus eradication could be achieved. The ability of 3 other surveillance programmes to detect infected fish populations was compared with the official programme. One programme involved syndromic surveillance based on the observation of clinical disease signs by fish farmers, while 2 programmes comprised active surveillance similar to the official programme, but included increased sampling frequencies and 2 additional tests. The syndromic surveillance concentrated on sending in samples for analysis when any sign of a possible infectious disease at water temperatures below 15°C was noticed. This programme clearly outperformed active surveillance. A real-time reverse transcriptase-polymerase chain reaction method proved to be at least as sensitive as virus isolation in cell culture in detecting acute VHSV infections. An ELISA method was used to test fish serum for antibodies against VHSV. The ELISA method may be a useful tool in VHSV eradication for screening populations during the follow-up period, before declaring an area free of infection.

Development of a Liquid Chip Technique to Simultaneously Detect Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, and Viral Hemorrhagic Septicemia of Salmonids.

A liquid chip technique was developed to detect spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) of salmonids simultaneously. Sequences of the G gene of SVCV, N gene of IHNV, and G gene of VHSV were used to design SVCV-, IHNV-, and VHSV-specific primers, which were labeled with biotin and subjected to amination modification. They were then coupled with fluorescence-coded microspheres and used for hybridization with reverse-transcription PCR products of SVCV, IHNV, and VHSV. A BD FACSArray was used to detect fluorescence signal in the reaction system. This assay system had a high sensitivity to SVCV, VHSV, and IHNV, with LODs of 10, 10, and 100 pg/µL, respectively. Moreover, the assay was specific for the detection of SVCV, IHNV, and VHSV and was not susceptible to cross-detection of other viruses, including pike fry rhabdovirus, hirame rhabdovirus, infectious pancreatic necrosis virus, viral nervous necrosis virus, yellowtail ascites virus, grass carp reovirus, red sea bream iridovirus, and koi herpesvirus. The liquid chip assay technique established in this study provides a novel, convenient, and rapid approach for the detection of SVCV, IHNV, and VHSV.

Optimization of a Plaque Neutralization Test (PNT) to Identify the Exposure History of Pacific Herring to Viral Hemorrhagic Septicemia Virus (VHSV).

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations. Received November 7, 2016; accepted January 14, 2017.

A mortality event in wrasse species (Labridae) associated with the presence of viral haemorrhagic septicaemia virus.

Viral haemorrhagic septicaemia (VHS) is an infectious disease of farmed and wild fish and has an extensive host range in both freshwater and marine environments. In December 2012, a wrasse population consisting of ballan, Labrus bergylta (Ascanius), corkwing, Symphodus melops (L.), cuckoo, Labrus mixtus L., goldsinny, Ctenolabrus rupestris (L.), and rock cook, Centrolabrus exoletus (L.), held at a marine hatchery in the Shetland Isles, Scotland, experienced a mortality event. Approximately 10 000 wrasse were being held at the facility on behalf of an Atlantic salmon, Salmo salar L., aquaculture company prior to being deployed for the biological control of parasites on marine pen Atlantic salmon, aquaculture sites. Fish Health Inspectors from Marine Scotland Science initiated a diagnostic investigation, and subsequent diagnostic testing confirmed the site to be VHSV positive by qRT-PCR and virus isolation followed by ELISA. A VHSV genotype-specific qRT-PCR assay revealed that the isolates belonged to genotype III, the European marine strain of the virus. The virus genotype was further confirmed by nucleic acid sequencing of the partial nucleoprotein (N) and glycoprotein (G) genes followed by BLAST nucleotide searches. This study reports for the first time the detection of VHSV within multiple wrasse species and highlights the need for a comprehensive risk-based approach to the use of wrasse and other finfish species as biological controls within the aquaculture industry.

Development of a multiplex assay to measure the effects of shipping and storage conditions on the quality of RNA used in molecular assays for detection of viral haemorrhagic septicemia virus.

Abstract In routine diagnostics, real-time reverse transcriptase quantitative PCR (RT-qPCR) has become a powerful method for fish health screening. Collection, transportation, and storage conditions of specimens could dramatically affect their integrity and could consequently affect RT-qPCR test results. In this study, to assess the expression profile of elongation factor 1 alpha (ELF-1α) gene, head kidney (HK) tissues from Atlantic Salmon Salmo salar were exposed at room temperature, 4°C, -20°C, and -80°C as well as in 70% ethanol for 6, 12, 24, 48, and 72 h. Data showed a significant increase of RT-qPCR cycle threshold (Ct) values for ELF-1α ranging from 14.7 to 26.5 cycles for tissues exposed to room temperature. In order to mimic the sample transportation conditions, different temperatures of storage were used and tissue quality was evaluated using ELF-1α gene expression. Data showed that Ct values for ELF-1α increased significantly when the tissues were transported on ice for 2 h, stored at -20°C, thawed on ice for 6 h, and stored again at -80°C. The HK tissues collected from Atlantic Salmon challenged with viral hemorrhagic septicemia virus (VHSV) through intraperitoneal injection were exposed at room temperature for 0, 6, 12, 24, 48, 72, and 96 h. Data showed a good correlation of values for ELF-1α and VHSV Ct although the ELF-1α mRNA of the host degraded faster than the RNA of VHSV. Based on these data, HK tissues could be transported on ice or ice packs without the quality of the tissue being affected when stored at -80°C upon arrival at the laboratory. In addition, 70% ethanol could be used as a preservative for long-distance transportation. For an efficient diagnostic test, a duplex VHSV-ELF-1α was developed and optimized. Data showed that the sensitivity of the duplex assay for VHSV was similar to the singleplex. Received November 25, 2013; accepted February 14, 2014.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols.

Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

Genotype-specific Taqman® assays for the detection and rapid characterisation of European strains of viral haemorrhagic septicaemia virus.

Viral haemorrhagic septicaemia virus (VHSV) is the agent of a disease that causes mortality events in marine and freshwater fish. It is one of the most important pathogens in European rainbow trout (Oncorhynchus mykiss) aquaculture. Four major genotypes of the virus are recognised reflecting different geographic and host ranges. Genotyping of VHS isolates is important for disease management enabling monitoring of disease spread into new geographical regions or susceptible species. This study sought to develop molecular tools for rapid and efficient classification of European VHSV genotypes. Specificity of genotype-specific real-time reverse transcription polymerase chain reaction (RT-qPCR) assays targeting the viral nucleoprotein (N) gene was tested using 66 viral isolates. All designed Taqman(®) RT-qPCR assays were genotype specific, displayed a high sensitivity and together constituted a diagnostic method for the rapid discrimination of European VHSV genotypes. Practical diagnostic applications of such assays demonstrated in this study include: (1) rapid genotype determination of isolates; and (2) identification of mixed-genotype isolates originating from pooled samples in areas where genotype distribution is known to overlap. However, the most important application will be supporting international VHSV surveillance programmes through the provision of a rapid specific and sensitive isolate characterisation method.

Diagnostic capacity for viral haemorrhagic septicaemia virus (VHSV) infection in rainbow trout (Oncorhynchus mykiss) is greatly increased by combining viral isolation with specific antibody detection.

Detection of disease specific antibodies in farmed rainbow trout (Oncorhynchus mykiss) has been proposed as an alternative or supplement to the currently approved procedures for diagnosis and surveillance in this species. In samples from natural outbreaks of the disease viral haemorrhagic septicaemia (VHS) at two freshwater farms in southern Denmark serologic testing was used to broaden the diagnostic window from outbreak to diagnosis in the laboratory as compared to traditional procedures of isolation and identification of the virus. The serologic assay clearly increased the chance of detecting present or previous infections where the pathogen could not be isolated by standard methods (indicating older infections where the virus had been cleared). Our data allowed us to monitor the levels of neutralising antibodies in relation to the presence of the virus in fish experiencing two different types of outbreaks at two different farms. By sequence analysis of the viral glycoprotein from selected isolates we found no evidence for escape mutants having developed in the fish showing high titres of neutralising antibodies.

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

QCM DNA biosensor for the diagnosis of a fish pathogenic virus VHSV.

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases damaging both fresh and marine fish species. VHS caused by VHSV and diagnosis of VHSV has been dependent on the conventional methods, such as cell culture and RT-PCR, which takes a few days or several hours. This study demonstrates a rapid and sensitive QCM biosensor for diagnosis of VHSV infection in fish. The QCM biosensor was developed to detect a main viral RNA encoding G protein in VHSV using the specific DNA probe. To maximize the sensitivity of the biosensor, we prepared three different DNA probes which modified 3' end of DNA by thiol, amine, or biotin and compared three different immobilisation methods on quartz surface coated with gold: immobilisation of thiol labelled probe DNA on naked gold surface, immobilisation of amino labelled probe DNA on gold surface prepared as carboxyl chip using MPA followed by EDC/NHS activation, and immobilisation of biotin labelled probe DNA on gold surface after immobilising avidin on carboxyl chip prior to biotin. As a result, immobilisation method using avidin-biotin interaction was most efficient to immobilise probe DNA and to detect target DNA. The QCM biosensor system using biotinylated probe DNA was stable enough to withstand 32 times of repeated regenerations and the detection limit was 0.0016muM. Diagnosis using the QCM biosensor system was more sensitive and much faster than a conventional RT-PCR analysis in detecting the viral RNA.

Development of a sensitive and controlled real-time RT-PCR assay for viral haemorrhagic septicaemia virus (VHSV) in marine salmonid aquaculture.

A survey was undertaken to determine the potential distribution of viral haemorrhagic septicaemia virus (VHSV) in marine cage-based salmonid farms in Scotland. A rapid, accurate and sensitive quantitative real-time RT-PCR (qRT-PCR) assay was developed, targeting a conserved region of the nucleoprotein (N) gene of the virus. The qRT-PCR assay was shown to be more sensitive than the conventional VHSV RT-PCR. A validation protocol included several different virus isolates as the target and confirmed that the assay could detect all European VHSV genotypes (I, II and III). Both endogenous and exogenous controls were designed to control for integrity of template and distinguish between true VHSV positives and contamination with the positive control material. In total, the universal European VHSV qRT-PCR assay with exogenous positive control was applied to screen 2040 individual Atlantic salmon Salmo salar and 150 individual rainbow trout Oncorhynchus mykiss. No evidence of the presence of VHSV in association with either salmonid species in Scottish marine farms was detected. However, both marine Atlantic salmon and rainbow trout are still considered possible carriers of VHSV, which remains a potential threat to freshwater farming. Therefore, a continued surveillance of these species in marine environment is recommended.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS).

A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes (He, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63 degrees C for only 1h, and required only a simple water bath or heating block to provide a constant temperature of 63 degrees C. RT-LAMP amplification products were detected by visual inspection using SYBR Green I stain and had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the RT-LAMP assay was found to be similar to the commonly used RT-PCR method: both methods detected VHS RNA at a dilution of 10(6). The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for VHS virus.