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Detection of foot-and-mouth disease virus in biological samples - Recent diagnostic possibilities
Abstract
Primary bovine thyroid cell cultures and IB-RS-2 continuous cell line were used for foot-and-mouth disease virus (FMDV) isolation. In both cell culture systems, all tested samples gave positive results and the specificity of isolated virus was confirmed by the Ag-ELISA. Results of virus isolation test agreed with those obtained by RT-PCR and rRT-PCR, which enabled detection of the genetic material of FMDV. This indicates a high and comparable sensitivity of the applied diagnostic assays, which permit a reliable detection of FMDV in biological material.
... The results observed in the present study are consistent with previously published case series where it is stated that the demyelinating form (AIDP) is the most prevalent in the country. 3,4,8,9 The results obtained in our study documented that 69% of cases correspond to demyelinating forms, 17% to axonal variants and 13% to other forms such as Miller-Fisher syndrome. These findings are notable because in the analyzed population the Miller-Fisher syndrome was a frequent variant (above the Colombian population average) that was lower than 1%. ...
... being more frequent among the men. Additionally, in our analysis, the frequency of previous infections (44%) is much lower than that reported in world and national literature, ranging between 50% and 77%.7,8,10,15 Similarly, there are Colombian studies that observe a frequency of up to 97% of previous viral infections, which is much greater than the one found in our work.4 ...
Guillain - Barré Syndrome is the most common acute peripheral polyneuropathy in the world. The estimated incidence in Colombia is 1.2-1.7 cases per 100 000 inhabitants, although during 2016 an increase in the incidence of the disease was documented, apparently associated with an epidemiological peak of the Zika virus. We conducted in order to describe the clinical and neurophysiological characteristics of adult patients with Guillain - Barré Syndrome treated at Hospital Universitario San Ignacio, Bogota, Colombia, between 2009 and 2017. An observational, descriptive, cross-sectional study was designed. This article is protected by copyright. All rights reserved.
... In recent years, with the progress made in molecular biology, various reverse transcription polymerase chain reaction (RT-PCR) methods are used to detect and identity FMDV isolations (9)(10)(11). Although RT-PCR is used to determine genetics conditions of existing strains in the field and determining the origin of virus infection in epidemics regions (12), its limitation is that only a few number of samples in each reaction series can not be used (13). Reverse transcription polymerase chain method (RT-PCR( is also useful for typing FMDV isolates based on sequencing (14) but this method does not have optimal results in terms of specificity and sensitivity (1). ...
... One of aims of this study detecting of RNA of this virus in epithelium sample with negative ELISA/VI result that it was done by RT-PCR. It is interesting that the results of ELISA/VI, conventional RT-PCR and RT-PCR are not compatible in some cases and it was seen in the results of other research (13). Briefly, the current study indicates that onestep RT-PCR introduced recently in our laboratory is a high sensitivity, specificity and effectiveness of diagnosis of RT-PCR in comparison with conventional RT-PCR powerful technique for the reliable detection of FMDV in biological samples (25). ...
Abstract Background and Aims: Accurate and rapid diagnosis is necessary for effective control and prevention of foot-and-mouth disease (FMD). In present study, was evaluated real time reverse transcription-polymerase chain reaction (rRT-PCR) assay along with diagnostic routine methods for the detection of all seven serotypes of FMD virus (FMDV), namely O, C, A, SAT1, 2, 3 and Asia 1 in biological samples at the reference laboratory for FMD, Iran. Materials and Methods: Two different RT-PCR assays targeting two different regions 5´ untranslated region (5´-UTR) and RNA polymerase (3D) of the FMDV genome were used to confirm the presence of FMDV in epithelial suspensions. Results: In the two methods the viral RNA in all tested archival serotypes of FMDV were detected. Specificity of this reaction was confirmed by the use of swine vesicular disease virus and blue-tongue. The amount of cycle threshold (CT) value of all seven serotypes was different and the lowest and highest of CT value achieved for SAT3, A, O types and SAT2, C types, respectively. Conclusion: The results showed that RT-PCR was more sensitive and effective than routine diagnostic methods. Furthermore, RT-PCR as a strong and valuable tool concomitant with diagnostic routine methods facilitate monitoring the fields FMDV strains and suggested that the use of the multiple diagnostic targets could enhance the sensitivity of the molecular methods for the detection of FMDV.
... In recent years, by the progress made in molecular biology, various reverse transcription polymerase chain reaction (RT-PCR) methods are used to determine the identity of FMDV isolations (9)(10)(11). Although RT-PCR is used to determine genetics conditions of existing strains in the field and determining the origin of virus infection in epidemics regions (12), its limitation is investigating a few number of samples in each reaction series and rather non-sensitive (13). Reverse transcription polymerase chain method (RT-PCR( is also useful for typing FMDV isolates based on sequencing (14) but this method does not have optimal results in terms of specificity and sensitivity (1). ...
... One of aims of this study detecting of RNA of this virus in epithelium sample with negative ELISA/VI result that it was done by rRT-PCR. It is interesting that the results of ELISA/VI, conventional RT-PCR and rRT-PCR are not compatible in some cases and it was seen in the results of other research (13). Briefly, the current study indicates that onestep rRT-PCR introduced recently in our laboratory is a high sensitivity, specificity and effectiveness of diagnosis of rRT-PCR in comparison with conventional RT-PCR powerful technique for the reliable detection of FMDV in biological samples (25). ...
Background and Aims: Accurate and rapid diagnosis is necessary for central to effective control and prevention of foot-and-mouth disease (FMD). In present study, was evaluated real time reverse transcription-polymerase chain reaction (rRT-PCR) assay along with diagnostic routine methods for the detection of all seven serotypes of FMD virus (FMDV), namely O, C, A, SAT1, 2, 3 and Asia 1 in biological samples at the reference laboratory for FMD, Iran. Materials and Methods: Two different rRT-PCR assays targeting two different regions 5úntranslated5úntranslated region (5´-UTR) and RNA polymerase (3D) of the FMDV genome was used to confirm the presence of FMDV in epithelial suspensions. Results: In the two methods the viral RNA in all tested archival serotypes of FMDV were detected. Specificity of this reaction was confirmed by the use of swine vesicular disease virus and blue-tongue. The amount of cycle threshold (C T) value of all seven serotypes was different and the lowest and highest of C T value achieved for SAT3, A, O types and SAT2, C types, respectively. Conclusion: The results showed that rRT-PCR is more sensitive and effective than diagnostic routine methods. Furthermore, rRT-PCR as a strong and valuable tool concomitant with diagnostic routine methods facilitate monitoring of the fields FMDV strains and suggested the use of the multiple diagnostic targets could future enhanced the sensitivity of molecular methods for the detection of FMDV.
... The methods involving the use of sensitive cells are among those with the highest sensitivity and specificity (7,8). In the case of viral infections, studies involving cell cultures are considered the gold standard in diagnosis. ...
A swine vesicular disease virus (SVDV) replication assay in IB-RS-2, SK-6, and PK-15 cell cultures was performed using the xCELLigence system. The cell status was monitored by impedance measurement, expressed as cell index (CI). Proliferation of particular cells was examined at the beginning of the study. The cells exhibited the ability to form a monolayer, and the CI values increased with the cell culture growth. After about 23 h and while still in the growth phase, the cells were infected with decimal virus dilutions (10
A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler
Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses
causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis.
Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic
to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where
FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes
of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell
lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient
tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.
Background
Foot-and-mouth disease (FMD) is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. Virus isolation and enzyme-linked immunosorbent assay (ELISA) are the gold standard tests for diagnosis of FMD. As these methods are time consuming, assays based on viral nucleic acid amplification have been developed.
Results
A previously described real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. To verify the applicability of this assay under field conditions in Brazil, 460 oral swabs from cattle were collected in areas free of FMD (n = 200) and from areas with outbreaks of FMD (n = 260). Three samples from areas with outbreaks of FMD were positive by real-time RT-PCR, and 2 of those samples were positive by virus isolation and ELISA. Four other samples were considered inconclusive by real-time RT-PCR (threshold cycle [Ct] > 40); whereas all 200 samples from an area free of FMD were real-time RT-PCR negative.
Conclusion
real-time RT-PCR is a powerful technique for reliable detection of FMDV in a fraction of the time required for virus isolation and ELISA. However, it is noteworthy that lack of infrastructure in certain areas with high risk of FMD may be a limiting factor for using real-time RT-PCR as a routine diagnostic tool.
A case of foot-and-mouth disease (fmd) on a cattle farm in Normandy, Surrey, was confirmed on Friday August 3, 2007, the first case in the uk since 2001. The infection was detected nearby on a second farm on August 6. On September 12, fmd was confirmed on a farm approximately 20 km from Normandy in Egham, and this was followed by cases on five more farms in that area in the next three weeks. The majority of the infected farms consisted of multiple beef cattle holdings in semi-urban areas. In total, 1578 animals were culled on the infected farms, and fmd virus infection was confirmed in 278 of them by the detection of viral antigen, genome or antibodies to the virus, or by clinical signs. This paper describes the findings from animal inspections on the infected farms, including the estimated ages of the fmd lesions and the numbers of animals infected. It also summarises the test results from samples taken for investigation, including the detection of preclinically viraemic animals by using real-time reverse transcriptase-pcr.
To evaluate a portable real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay designed to detect all 7 viral serotypes of foot-and-mouth disease virus (FMDV).
Laboratory and animal studies.
Viruses grown in tissue culture and animals experimentally infected with FMDV.
1 steer, pig, and sheep were infected with serotype O FMDV. Twenty-four hours later, animals were placed in separate rooms that contained 4 FMDV-free, healthy animals of the same species. Oral and nasal swab specimens, oropharyngeal specimens obtained with a probang, and blood samples were obtained at frequent intervals, and animals were observed for fever and clinical signs of foot-and-mouth disease (FMD). Samples from animals and tissue cultures were assayed for infectious virus and viral RNA.
The assay detected viral RNA representing all 7 FMDV serotypes grown in tissue culture but did not amplify a panel of selected viruses that included those that cause vesicular diseases similar to FMD; thus, the assay had a specificity of 100%, depending on the panel selected. The assay also met or exceeded sensitivity of viral culture on samples from experimentally infected animals. In many instances, the assay detected viral RNA in the mouth and nose 24 to 96 hours before the onset of clinical disease.
The assay reagents are produced in a vitrified form, which permits storage and transportation at ambient temperatures. The test can be performed in 2 hours or less on a portable instrument, thus providing a rapid, portable, sensitive, and specific method for detection of FMDV.
Foot and mouth disease (FMD) is a limiting factor for the economic progress of the animal industry in South America. The presence of the disease results in the imposition of national and international sanitary barriers to animals and animal products, and, most especially, a reduction in the availability of protein from animal origin and in income. Rapid and accurate identification of infected animals, those with either clinical or subclinical disease as well as with persistent infection, is essential for maintaining an efficient eradication programme. The polymerase chain reaction was used to rapidly identify infected animals. With a primer set that corresponds to a conserved region of the 3D sequence of the viral genome, it was possible to amplify, regardless of the serotype, 116 strains of FMD virus, of which 109 were strains collected from outbreaks of FMD throughout South America from 1945 to the most recent outbreaks in 2000/2001. The PCR technique should be of considerable value in facilitating the diagnosis of FMD in South America. where laboratory resources are limited and a rapid response is needed, particularly in areas where national programmes for controlling or eradicating the disease are being implemented.
The performance of an automated real-time reverse transcription polymerase chain reaction (RT-PCR) was compared to virus isolation (VI) in cell culture and antigen detection enzyme-linked immunosorbent assay (ELISA) for the laboratory diagnosis of foot and mouth disease (FMD). The World Reference Laboratory for FMD in Woking, the United Kingdom, examined a collection of 334 epithelia received from eighteen countries between August 2002 and January 2004. The results showed that all VI positive (n = 195) and VI and ELISA positive samples combined (n = 204) were also positive by RT-PCR. Depending on the cut-off used, FMD virus genome was detected in a minimum of an additional 60 samples (18% of all samples tested). Furthermore, the RT-PCR generated results in less than one day from test commencement in contrast to up to 4 days to define some positive and all negative samples by VI. The study demonstrates that real-time RT-PCR provides an extremely sensitive and rapid procedure for improved laboratory diagnosis of FMD.
Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5' untranslated region (5'UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1% (347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5'UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV.
There were 2030 designated cases of foot-and-mouth disease (FMD) during the course of the epidemic in the UK in 2001 (including four from Northern Ireland). Samples from 1720 of the infected premises (IPs) were received in the laboratory and examined for either the presence of FMD virus (virological samples from 1421 IPs) or both FMD virus and antibody (virological and serological samples from 255 IPs) or antibody alone (from 44 IPs). The time taken to issue final diagnostic results ranged from a few hours in cases in which positive results were obtained by ELISA on epithelia containing sufficient virus to be detected, to several days for samples containing small amounts of virus requiring amplification through cell culture, negative samples or samples tested for antibody. Two subsets of samples were analysed retrospectively by real-time reverse transcriptase-PCR (RT-PCR); first, epithelia that were negative by both ELISA and virus isolation (VI) in cell culture, and secondly, samples that were negative by ELISA on epithelial suspension but positive by VI. There was broad agreement between the RT-PCR and VI/ELISA combined, except that the RT-PCR procedure did not detect a group of related virus isolates from Wales. These viruses had evidently evolved during the epidemic and had a nucleotide substitution in the RT-PCR probe site, which prevented them from being detected by the routine diagnostic probe. No evidence of FMD virus, antibody or nucleic acid was found in approximately 23 per cent (390 of 1730) of IPs from which samples were received, suggesting that the incidence of FMD during the outbreak may have been over-reported.
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.
RAPID and accurate diagnosis is necessary for the effective control of foot-and-mouth disease (FMD). Some laboratorybased methods are rapid, generating a result within a few hours, but the time taken to transport suspect clinical material to the laboratory can be lengthy, and this delay can preclude laboratory confirmation of disease. Notably, the implementation of the control strategy adopted by the UK government in the 2001 FMD epizootic – to slaughter animals on infected premises within 24 hours – meant that in the majority of cases, diagnosis based on clinical signs was not substantiated by laboratory investigation. Retrospective analyses have indicated that the reliance upon clinical diagnosis alone resulted in over-reporting of FMD, since the presence of FMD virus (FMDV) on 23 per cent of farms designated as infected premises was not confirmed by laboratory methods (Ferris and others 2006). These factors have given rise to the idea that the reliability of diagnosis may be improved by using rapid and sensitive tests that can be used in situ or close to a suspect premises, without transferring the samples to a central laboratory. The use of rapid diagnostic assays was recommended in two major reports considering the 2001 FMD outbreak (Anderson 2002, Royal Society 2002). Previous studies (Callahan and others 2002, Hearps and others 2002), have developed rapid reverse transcriptase-PCR (RT-PCR) assays for detecting FMDV; however, limitations in the hardware and some aspects of the protocols used have restricted the adoption of these assays for field detection of FMDV. In light of the general interest in this issue, this short communication describes recent progress that has been made towards the development of a mobile RT-PCR assay for FMDV detection. This study describes the evaluation of a portable real-time
Foot-and-mouth disease (FMD) is the most contagious disease of mammals and has great potential for causing severe economic losses in susceptible cloven-hoofed animals. FMD is caused by a virus of the genus Aphthovirus, family Picornaviridae. Serological tests in laboratories have identified seven different serotypes as O, A, C, SAT 1, SAT 2, SAT 3 and Asia 1. FMD is diagnosed by the virus isolation or demonstration of FMD viral antigen or nucleic acid in samples of biological specimens. The purpose of the study was to apply the isolation test in cell culture and a RT-PCR assay for the detection of foot-and-mouth disease virus in biological materials. Out of the total of 14 examined samples, 6 (42.8%) were found positive using these methods. The antigen ELISA was used for the confirmation of specificity of the isolation assay. Primary bovine thyroid cells were found the most sensitive cell culture system for the detection of foot-and-mouth disease virus, followed by secondary lamb kidney and certain IB-RS-2 cells.
An indirect, sandwich enzyme-linked immunosorbent assay (ELISA) has been compared in parallel with the standard complement-fixation typing test (CFT) for the diagnosis of foot-and-mouth and swine vesicular diseases. The superior sensitivity, reproducibility, economical use of reagents and ease of performance of the ELISA confirm the hypothesis that it should replace the CFT as the routine test of choice.
A rapid double sandwich enzyme-linked immunosorbent assay (ELISA) has been used for the identification and type differentiation of foot-and-mouth disease (FMD) viruses in epithelial tissue samples submitted for diagnosis from the field. No difficulty was experienced in the direct typing of freshly harvested epithelium from recently ruptured vesicles by the complement fixation (CF) test or ELISA. The ELISA was more sensitive and specific, but proved no more efficient than the traditional CF test in the direct typing of samples of poorer quality from many countries overseas where communications are often difficult. However, when both tests were used concurrently, FMD virus typings were confirmed in 27 more samples. Some possible reasons for the failure of ELISA to detect virus in certain cases are discussed.
The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.
Universal and serotype-specific primer sets were used in simple reverse transcription polymerase chain reaction (RT-PCR) assays on field samples of epithelium and vesicular fluid to determine their suitability for primary diagnosis of all seven serotypes of foot-and-mouth disease (FMD). The specificity of reactions was confirmed by using other vesicular disease viruses, namely: swine vesicular disease virus, vesicular stomatitis virus and three vesiviruses. This resulted in the identification of a universal O/A/C/Asia 1 primer set (1F/1R) located in the 5' untranslated region (UTR) of the FMD virus genome for the successful detection of virus of these serotypes in clinical samples although this primer set detected FMD virus of the SAT1/2/3 serotypes less efficiently. The 5' UTR universal primer set could be used for the primary diagnosis of FMD in conjunction with the routine diagnostic methods of virus isolation in cell culture and ELISA, although a more favourable reaction would be expected with FMD viruses of the O/A/C/Asia 1 group than with those of the SAT serotypes. The other examined universal and serotype-specific primer sets, located principally in the P1 capsid-coding region, were generally inferior to the 5' UTR universal primer set. It is envisaged that this evaluation of primers will lead to the development of alternative PCR strategies, for example nested PCR formats, with concomitant improvement in the speed of primary diagnosis of FMD which under present procedures can be lengthy.
A fluorogenic RT-PCR (5'-nuclease probe-based) assay using a primer/probe set designed from the internal ribosomal entry site region of the virus genome was developed for the specific detection of all seven serotypes of foot-and-mouth disease (FMD) virus in epithelial suspensions and cell culture virus preparations. The reverse transcription polymerase chain reaction (RT-PCR) specifically detected FMD virus in sample submissions from the UK 2001 FMD outbreak with greater sensitivity than our conventional RT-PCR procedure and our routine diagnostic procedures of ELISA and virus isolation in cell culture. The fluorogenic RT-PCR provides relatively fast results, enables a quantitative assessment to be made of virus amounts and can handle more samples and/or replicates of samples in a single assay than the conventional RT-PCR procedure. Therefore it is seen as a valuable tool to complement the routine diagnostic procedures for FMD virus diagnosis.
Foot-and-mouth disease (FMD) diagnostic methods are reviewed. As the presence of clinical signs alone is inconclusive, laboratory diagnosis should always be carried out. The presence of FMD virus can be demonstrated by cell culture isolation, complement fixation test, ELISA or the more recent polymerase chain reaction (PCR) method. Serological diagnosis is also a valuable tool. The virus neutralization test has been replaced by ELISA and the antibody response to some viral non-structural proteins allows to discriminate between vaccinated and infected animals on a herd basis. More rapid and accurate tests as well as an earlier detection system in preclinical state are still needed.
Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD.
The detection of foot-and-mouth disease virus (FMDV) in persistently infected carriers among exposed ruminants is of great importance in disease control. For this purpose, a real time, fluorogenic reverse transcription polymerase chain reaction (real-time RT-PCR) assay was evaluated for the identification of FMDV carrier animals. The results indicate that this real time RT-PCR assay may be suitable for detection of FMDV carrier animals.
Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10 vesicular epithelia, which were derived from submissions from suspect cases of FMD or swine vesicular disease (SVD). Sixteen samples were derived from six FMD virus positive epithelia representing four different serotypes (two each of types O and A and one each of types Asia 1 and SAT 2), two from samples which had been found to be negative by antigen ELISA and virus isolation (VI) in cell culture and two from SVD virus positive epithelia. Some of the FMD virus positive samples were prepared from 10-fold serial dilutions of three of the initial suspensions. Each laboratory tested the samples by one or more of its available RT-PCR procedures and inoculated cell cultures that it routinely uses for FMD diagnosis in attempts to isolate virus, the specificity of which was confirmed by antigen ELISA. The best of the RT-PCR assays used in each laboratory gave comparable results while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. This prototype panel of samples would appear suitable for external quality assurance of these tests but would benefit from the inclusion of more negative samples and an extension in the serial dilution range of one or more of the FMD positive sample titration series.
Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon by melting point analysis for added specificity and at the same time allows the detection of mutations in the probe region. As such, the described new method is suitable for the robust real-time detection of index cases caused by any serotype of FMDV.
