Interfering factors in the diagnosis of Senecavirus A.

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Validation of a competitive enzyme-linked immunosorbent assay to improve the serological diagnosis of swine vesicular disease.

Swine vesicular disease (SVD) is an infectious viral disease of pigs. The clinical symptoms of SVD are indistinguishable from other vesicular diseases. In countries free of vesicular diseases, rapid SVD diagnosis and differentiation from other vesicular diseases are essential. In this report, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and validated to improve the current SVD serological diagnosis. In this cELISA, an anti-SVD monoclonal antibody (mAb) captures the recombinant SVD virus-like particle (SVD-VLP) antigen, and 5B7 mAb is used as a competitor to compete with polyclonal antibodies in SVD-positive sera. The cut-off value of the SVD-VLP based cELISA (SVD-VLP cELISA) is ≥ 65% inhibition (%). The determined diagnostic specificity was 99.2%. SVD-VLP cELISA successfully detected SVD antibodies in the sera of SVD-infected animals and produced a diagnostic sensitivity of 100%. A panel of SVD positive sere including outbreak samples (n = 11) and samples (n = 5) from experimentally inoculated pigs, were correctly identified as positive by the SVD-VLP cELISA. In terms of reducing false positives detected by the currently used cELISA (5B7 cELISA), the performance of SVD-VLP cELISA is comparable to the gold standard virus neutralization test.

La maladie vésiculeuse du porc (SVD) est une maladie virale infectieuse des porcs. Les symptômes cliniques de la SVD sont indiscernables des autres maladies vésiculaires. Dans les pays exempts de maladies vésiculaires, un diagnostic rapide de la SVD et une différenciation avec les autres maladies vésiculaires sont essentiels. Dans ce rapport, un test immuno-enzymatique compétitif (cELISA) a été développé et validé pour améliorer le diagnostic sérologique actuel de la SVD. Dans ce cELISA, un anticorps monoclonal anti-SVD (mAb) capture l'antigène recombinant de particules de type virus SVD (SVD-VLP), et le mAb 5B7 est utilisé comme compétiteur pour concurrencer les anticorps polyclonaux dans les sérums positifs pour la SVD. La valeur seuil du cELISA basé sur SVD-VLP (cELISA SVD-VLP) est ≥ 65 % d'inhibition (%). La spécificité diagnostique déterminée était de 99,2 %. SVD-VLP cELISA a détecté avec succès des anticorps SVD dans les sérums d'animaux infectés par SVD et a produit une sensibilité diagnostique de 100 %. Un panel de sérums positifs pour la SVD, comprenant des échantillons d'épidémie (n = 11) et des échantillons (n = 5) de porcs inoculés expérimentalement, a été correctement identifié comme positif par le cELISA SVD-VLP. En termes de réduction des faux positifs détectés par le cELISA actuellement utilisé (5B7 cELISA), les performances du cELISA SVD-VLP sont comparables au test de neutralisation du virus de référence.(Traduit par Docteur Serge Messier).

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs.

Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 × 101 copies/µL, which was significantly more sensitive than that by gel electrophoresis of 6.4 × 103 copes/µL. This assay was specific and had no cross-reaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.

Development and evaluation of swine vesicular disease isotype-specific antibody ELISAs based on recombinant virus-like particles.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform.

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6 hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

Generation, characterization, and application in serodiagnosis of recombinant swine vesicular disease virus-like particles.

Swine vesicular disease (SVD) is a highly contagious viral disease that causes vesicular disease in pigs. The importance of the disease is due to its indistinguishable clinical signs from those of foot-and-mouth disease, which prevents international trade of swine and related products. SVD-specific antibody detection via an enzyme-linked immunosorbent assay (ELISA) is the most versatile and commonly used method for SVD surveillance and export certification. Inactivated SVD virus is the commonly used antigen in SVD-related ELISA. A recombinant SVD virus-like particle (VLP) was generated by using a Bac-to-Bac baculovirus expression system. Results of SVD-VLP analyses from electron microscopy, western blotting, immunofluorescent assay, and mass spectrometry showed that the recombinant SVD-VLP morphologically resemble authentic SVD viruses. The SVD-VLP was evaluated as a replacement for inactivated whole SVD virus in competitive and isotype-specific ELISAs for the detection of antibodies against SVD virus. The recombinant SVD-VLP assay produced results similar to those from inactivated whole virus antigen ELISA. The VLP-based ELISA results were comparable to those from the virus neutralization test for antibody detection in pigs experimentally inoculated with SVD virus. Use of the recombinant SVD-VLP is a safe and valuable alternative to using SVD virus antigen in diagnostic assays.

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality.

We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5' untranslated region (5'-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.

Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates.

Development of a Luminex assay for the detection of swine antibodies to non-structural proteins of foot-and-mouth disease virus.

Foot-and mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS) are highly contagious vesicular diseases of swine but are not easy to differentiate clinically. For the purpose of instant detecting of FMD and differentiating it from the other vesicular diseases, a Luminex assay was developed. Sera from 64 infected, 307 vaccinated, and 280 naïve pigs were tested by the Luminex assay. Diagnostic sensitivity of the assay was 100%. Diagnostic specificity of the assay was 98.7% in vaccinated pigs and 97.5% to 100% in naïve pigs. Agreement between the results from the Luminex assay and those from a 3ABC polypeptide blocking ELISA was 96.3% with kappa statistics of 0.92. The Luminex assay can detect the immune response to NSP-3ABC in swine as early as eight days post-infection. Moreover, all of the 15 vaccinated but unprotected pigs were all detected by the Luminex assay. The results indicated that the Luminex assay has potential with specificity in detecting antibodies to FMDV 3ABC NSP and in distinguishing FMDV-infected pigs from with either SVDV or VSV.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

[Development of an indirect ELISA for the detection of antibodies to swine vesicular disease virus during monitoring studies].

An indirect ELISA (I-ELISA) has been developed for swine vesicular disease virus-specific antibody detection. The analytic sensitivity of I-ELISA testing of serum samples from experimentally infected pigs with the known VN titer was 2 log2. Its diagnostic specificity was demonstrated as 100% in 4485 swine serum samples from different regions of the Russian Federation.

[Comparison of different ELISAs for detection of antibodies to swine vesicular disease virus in sera from experimentally infected animals].

The study has shown the efficiency of a competitive ELISA (C-ELISA) variant or an indirect ELISA (I-ELUSA) in the detection of antibodies to swine vesicular disease virus (SVDV) versus traditional assays, such as a microneutralization test, a blocking ELIDA test, and a the reference test Ceditest SVDV (Cedi-Diagnostics B.V., Netherlands). Specific antibodies in the pig sera could be detected by C-ELISA on days 4-5 and by I-ELISA on day 6 after experimental SVDV infection. Specific antibodies were detected in a contact pig 11 days after the beginning of the experiment.

Swine vesicular disease in northern Italy: diffusion through densely populated pig areas.

At the end of 2006, a recrudescence of swine vesicular disease (SVD) was recorded in Italy and the disease spread widely throughout the northern regions. Lombardy, a densely populated pig area, was most affected and the presence of the disease caused heavy economic losses to the entire pig industry. Although SVD is considered only moderately contagious, the epidemic in the north was characterised by a rapid spread of the condition. Numerous difficulties were encountered in eradicating it. Over the past decade, there has been a significant increase in the population of pigs in Lombardy, concentrated mainly in a few areas which were the most severely affected during the 2006 to 2007 SVD epidemic. Increases in both the pig population and animal movements, combined with weak biosecurity measures, increased the spread rate of the disease and hampered eradication activities.

Development and laboratory evaluation of a lateral flow device for the detection of swine vesicular disease virus in clinical samples.

A lateral flow device (LFD) for the detection of swine vesicular disease (SVD) virus (SVDV) and differential diagnosis from foot-and-mouth disease (FMD) was developed using a monoclonal antibody (Mab C70). The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of SVDV and porcine teschovirus (enterovirus; PEV). The collection of test samples included 157 which were positive for SVDV (84 vesicular epithelial suspensions and 73 cell culture antigens) from suspected cases of vesicular disease in pigs collected from 14 countries between 1966 and 2008 and 663 samples which were either shown to be negative for SVDV and FMD virus (FMDV) or else collected from healthy pigs or demonstrated to be positive for FMDV, PEV or vesicular exanthema (VEV) and collected from 16 countries between 1965 and 2008 or else were derived from experimental animals. Three further samples containing vesicular stomatitis virus (VSV) were also tested. The diagnostic sensitivity of the LFD for SVDV was similar at 82% compared to 86% obtained by the reference method of antigen ELISA, and the diagnostic specificity was 100% compared to 99.7% for the ELISA. The device recognized virus strains of each of the known genotypes of the sole SVDV serotype. Reactions with FMDV, VEV, VSV and PEV which can produce clinically indistinguishable syndromes in pigs, did not occur. These data illustrate the potential for the LFD to be used next to the animal for providing rapid and objective support to veterinarians in their clinical judgment of vesicular disease in pigs and for the specific pen-side diagnosis of SVD and differential diagnosis from FMD.

[Serological examinations for swine vesicular disease (SVD) in a closed pig breeding herd using ELISA]. / Serologische Untersuchungen mit einem ELISA auf vesikuläre Schweinekrankheit (swine vesicular disease, SVD) in einem geschlossenen Schweinezuchtbestand.

In a closed pig establishment housing about 18,000 pigs, 2895 gilts were tested pre-export for SVD (swine vesicular disease) antibodies using Ceditest/PrioCHECK SVDV-AB ELISA. 130 gilts (4.5%) tested positive. In addition, 561 animals of this farm were sampled per random for SVD serology. One in 241 weaners (0.4%), eight in 150 gilts (5.3%) and 18 in 170 (10.6%) pregnant sows tested ELISA SVD-antibody positive. Of the ELISA positive samples, 23 tested positive in VNT (virus neutralization test). Of these, 20 VNT-positive animals were re-sampled two weeks later and re-tested via ELISA and VNT in different laboratories, displaying falling titres with one to two animals remaining VNT-positive. Epidemiological investigations and clinical examinations on site did not yield any evidence for SVD. 745 faecal samples taken from individual pigs and collected from pens tested negative in SVDV-RNA-PCR. 40 of these samples tested negative in virus isolation on cell culture. Pathological examinations on fallen pigs did not reveal any evidence for SVD either. After comparing our ELISA results with data recorded in the ELISA validation by Chenard et al. (1998), we propose that the published test performance is perhaps not currently applicable for the commercial test. Provided that SVD-antibody negative pigs were tested, a specificity of 99.6% in weaners, 95.5% in gilts and 89.4% in pregnant sows would appear to be more appropriate for the Ceditest/PrioCHECK SVDV-AB ELISA. Details are provided for all examined pigs regarding husbandry, breed, age, weeks pregnant and previous vaccinations. The results of other serological tests on the same sera are given. Possible clusterings of false-positive SVD-ELISA results are discussed.

Advances in viral disease diagnostic and molecular epidemiological technologies.

The early and rapid detection and characterization of specific nucleic acids of medico-veterinary pathogens have proven invaluable for diagnostic purposes. The integration of amplification and signal detection systems, including online real-time devices, have increased speed and sensitivity and greatly facilitated the quantification of target nucleic acids. They have also allowed for sequence characterization using melting or hybridization curves. The newer-generation molecular diagnostic technologies offer, hitherto, unparalleled detection and discrimination methodologies, which are vital for the positive detection and identification of pathogenic agents, as well as the effects of the pathogens on the production of antibodies. The development phase of the novel technologies entails a thorough understanding of accurate diagnosis and discrimination of present and emerging diseases. The development of novel technologies can only be successful if they are transferred and used in the field with a sustainable quality-assured application to allow for the optimal detection and effective control of diseases. The aim of these new tools is to detect the presence of a pathogen agent before the onset of disease. This manuscript focuses mainly on the experiences of two World Organisation for Animal Health collaborating centers in context to molecular diagnosis and molecular epidemiology of transboundary and endemic animal diseases of viral origin, food safety and zoonoses.

Application of real-time reverse transcription polymerase chain reaction for the detection of SVDV.

Application of real-time RT-PCR (rRT-PCR) for detection of swine vesicular disease virus (SVDV) in samples of archival SVDV isolates and clinical samples collected from SVDV infected pigs was described. A primer set that targets the IRES region of the SVDV genome and TaqMan probe specific for a highly conserved region in SVDV RNA IRES region were used. The assay detected viral RNA in all tested archival strains of SVDV isolated in Europe during years 1972-73 and 1992 as well as in clinical samples collected from experimentally infected pigs. The rRT-PCR can provide quantitative and qualitative information and is more sensitive and faster to perform than the conventional RT-PCR.

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals.

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.

Diagnostic evaluation of multiplexed reverse transcription-PCR microsphere array assay for detection of foot-and-mouth and look-alike disease viruses.

A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.