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Highlights ► Efficient expression of FMDV empty capsids in insect cells after moderation of 3C protease action. ► Expression cassette productive in multiple insect cell lines. ► Empty capsids visualised by transmission electron microscopy. ► Empty capsids react with wide range of positive sera as well as authentic virus. ► Efficient empty capsid synthesis may allow development as a vaccine., Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3 of subtype 3 [VSV-IND-3]) and 189 positive for VSV-NJ (162 vesicular epithelial suspensions and 27 cell culture antigens) from suspected cases of vesicular disease in cattle and horses collected from 11 countries between 1937 and 2008 or else were derived from experimental infection and 777 samples that were either shown to be positive or negative for foot-and-mouth disease (FMD) virus (FMDV) and swine vesicular disease virus (SVDV) or else collected from healthy cattle or pigs and collected from 68 countries between 1965 and 2011. The diagnostic sensitivity of the VSV-IND (for reaction with VSV-IND-1) and VSV-NJ LFDs was either similar or identical at 94.6% (VSV-IND) and 97.4% (VSV-NJ) compared to 92.5% and 97.4% obtained by the reference method of antigen ELISA. The VSV-IND LFD failed to react with viruses of VSV-IND-2 and 3, while the VSV-NJ device recognized all VSV-NJ virus strains. The diagnostic specificities of the VSV-IND and VSV-NJ LFDs were 99.1% and 100, respectively, compared to 99.6% and 99.8% for the ELISA. Reactions with FMDV which can produce indistinguishable syndromes clinically in cattle, pigs and sheep and SVDV (vesicular disease in pigs) did not occur. These data illustrate the potential for the LFDs to be used next to the animal for providing rapid and objective support to veterinarians in their clinical judgment of vesicular disease and for the subtype (VSV-IND-1) and type-specific (VSV-NJ) pen-side diagnosis of VS and differential diagnosis from FMD.

The ability to propagate foot-and-mouth disease virus (FMDV) plays an important role in laboratory diagnosis and the production of vaccines to control the spread of the disease. Many established cell lines suffer from poor sensitivity for isolating virus from field samples. One possible factor that limits sensitivity to FMDV is the lack of expression of surface integrins, the primary class of cell receptor used by FMDV to initiate infection. In this study we have sequenced cDNAs encoding these molecules for pigs and subsequently developed quantitative real-time reverse transcription (RT)-PCR assays to quantify underlying mRNA transcription of integrin molecules. These novel assays were used together with flow-cytometry to determine cell surface expression and of 4 different cell culture systems. These studies have identified a clear correlation of sensitivity to FMDV with expression of integrins αVβ6 and αVβ8. In contrast, cell surface expression of αVβ3 or mRNA for the β1, β3 or β5 subunits did not appear to contribute to sensitivity of cells to FMDV. These findings confirm the requirement for αV6 and αVβ8 as receptors for isolating FMDV from clinical samples and provide important tools and information for the rational design of recombinant cell lines containing these ligands for improved FMDV diagnosis and vaccine production.

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.

Virus diversity indicates a virus reservoir in this country., Foot-and-mouth disease (FMD) is endemic to sub-Saharan Africa. To further understand its complex epidemiology, which involves multiple virus serotypes and host species, we characterized the viruses recovered from FMD outbreaks in Ethiopia during 1981–2007. We detected 5 of the 7 FMDV serotypes (O, A, C, Southern African Territories [SAT] 1, and SAT 2). Serotype O predominated, followed by serotype A; type C was not recognized after 1983. Phylogenetic analysis of virus protein 1 sequences indicated emergence of a new topotype within serotype O, East Africa 4. In 2007, serotype SAT 1 was detected in Ethiopia and formed a new distinct topotype (IX), and serotype SAT 2 reappeared after an apparent gap of 16 years. The diversity of viruses highlights the role of this region as a reservoir for FMD virus, and their continuing emergence in Ethiopia will greatly affect spread and consequent control strategy of the disease on this continent.

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.

Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses.

The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 × 10 −5 per site per day (95% confidence interval [CI], 1.75 × 10 −5 to 2.80 × 10 −5 ) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.

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.

Two indirect, sandwich ELISAs are described for use in African swine fever (ASF) diagnosis. One assay uses polyclonal serum raised in rabbits and guinea pigs against the cytoplasmic soluble ASF virus protein and the second, a combination of monoclonal antibody raised against the VP73 protein and rabbit polyclonal serum. Both assays have been shown to detect antigen of representative field strains of phylogenetically distinct groupings of ASF virus but the ELISA, which utilises polyclonal antisera was slightly more sensitive than that using the monoclonal antibody.

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

A solid-phase competition ELISA has been developed to measure antibodies to foot-and-mouth disease (FMD) virus and has been validated using an extensive range of sera from cattle. The assay uses polyclonal antisera and inactivated purified 146S antigens of FMD virus and was compared with the liquid-phase blocking ELISA and the virus neutralisation test on a range of serum sets. When examining test sera at a 1:5 dilution with a cut-off point of 30% inhibition of reaction, the solid-phase competition ELISA was as sensitive as the liquid-phase blocking ELISA for sera from infected or vaccinated animals. The limit of detection of the solid-phase ELISA was similar to that of the liquid-phase assay and both tests had lower limit of detection (i.e. were able to detect lower amounts of antibody) than the virus neutralisation test. The specificity of the solid-phase ELISA was considerably higher than that of the liquid-phase blocking ELISA and almost equivalent to that of the virus neutralisation test. The assay thus retains the sensitivity of the liquid-phase blocking ELISA whilst being easier to use, more robust and specific, and therefore offers an improvement for FMD virus antibody detection.

Foot-and-mouth disease (FMD) is the most contagious animal virus disease of cloven-hoofed livestock and requires reliable and accurate diagnosis for the implementation of measures to control effectively its spread. Routine diagnosis of FMD is carried out at the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright by the combined use of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase chain reaction (RT-PCR) methods. These techniques require skilled personnel and dedicated laboratory facilities which are expensive. The development of a rapid and simple test for the detection of FMD virus antigen using Clearview chromatographic strip test technology for field application is described. This device detected FMD viral antigen in nasal swabs, epithelial suspensions and probangs from clinical samples submitted from the field, from animals infected experimentally and in supernatant fluids resulting from their passage in cell culture. The test system was more sensitive than ELISA for the diagnosis of all seven serotypes of FMD virus in the epithelial suspensions and nasal swabs and had equivalent sensitivity to the ELISA for the detection of contemporary virus strains in cell culture supernatant fluids. The study demonstrated the potential for this device to confirm a clinical diagnosis at the site of a suspected FMD outbreak, thereby offering the possibility of implementing control procedures more rapidly. Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult. In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.

A lateral flow device (LFD) for the detection of foot-and-mouth disease virus (FMDV) of the SAT 2 serotype was developed using a monoclonal antibody (Mab 2H6). The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia: 305 positive for FMDV type SAT 2 from suspected cases of vesicular disease collected from 30 countries and 1002 samples shown to be negative for FMDV type SAT 2 collected from 67 countries between 1968 and 2008. The diagnostic sensitivity of the LFD for FMDV type SAT 2 was higher at 88% compared to 79% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 100% for the ELISA. The device recognized FMDV strains of wide diversity within the FMDV SAT 2 serotype and gave a superior performance for their detection compared to the 1F10 LFD which had been developed previously and shown to perform less well for the detection of FMDVs of this particular serotype. Reactions in the SAT 2 2H6 LFD with the viruses of other FMDV serotypes and swine vesicular disease (which produces a clinically indistinguishable syndrome in pigs), did not occur. These data illustrate the potential for the LFD to be employed to complement the 1F10 device next to the animal in the pen-side diagnosis of FMD, for providing rapid and objective support to veterinarians in their clinical judgment of the disease and for specific confirmation of a FMDV type SAT 2 infection.

A reverse transcription polymerase chain reaction (RT-PCR) procedure is described for the detection of marine caliciviruses including vesicular exanthema of swine virus (VESV), San Miguel sea lion virus (SMSV), bovine Tillamook virus (BCV Bos-1) and caliciviruses (CV) isolated from dolphin (Cetacean CV), gorilla (Primate CV) and rattlesnake (Reptile CV) using primers (1F and 1R) designed from the capsid-coding region of the viral genome. These primers were compared with those described by Neill, J.D. and Seal, B.S., 1995: Development of PCR primers for specific amplification of two distinct regions of the genomes of San Miguel sea lion and vesicular exanthema of swine viruses, Mol. Cell. Probes 9, 33-38 (Hel1/Hel2), which had been designed from the 2C-like region of the calicivirus genome. Both sets proved to be extremely useful diagnostic tools for all of the known marine calicivirus serotypes with the exception of three: SMSV-8 and -12 and mink CV suggesting that these three caliciviruses may belong to a different group. Neither of the two primer sets reacted with strains of the vesicular disease viruses of foot-and-mouth disease (FMD), swine vesicular disease (SVD) or vesicular stomatitis (VS) nor with two feline caliciviruses (FCV). The 1F/1R primer set has the advantage over the Hel1/Hel2 set in that it generates a larger PCR product for nucleotide sequence investigations and so provides greater opportunity for identifying molecular differences between the viruses.

Foot-and-mouth disease (FMD) is endemic in Tanzania. Since the first reports in 1954, FMD has caused significant economic losses in the country due to mortality and morbidity of livestock and costs associated with controlling the disease. The aim of this study was to review the serotype and genetic relationships of the FMD virus (FMDV) recovered from outbreaks in Tanzania, and compare them with viruses detected from elsewhere in the sub-Saharan region. At the World Reference Laboratory for foot-and-mouth disease (WRLFMD), a total of 106 FMD viruses have been isolated from samples collected between 1967 and 2009 from northern, southern, eastern and central parts of Tanzania. The presence of FMDV was determined by laboratory methods such as VI, CF, antigen ELISA and RT-PCR. Phylogenies of VP1 sequences were determined by the Neighbour-joining method. Foot-and-mouth disease virus SAT1 was the most frequent serotype (46.2%; n = 49) isolated in Tanzania followed by O (26.4%; n = 27), A (14.1%; n = 15) and SAT 2 (11.3%; n = 13). Genotyping showed that type O viruses fell into either the EAST AFRICA 1 (EA-1) or EA-2 topotypes, type A’s into the AFRICA topotype (genotype I), type SAT 1’s into topotype I and type SAT 2’s into topotype IV. This study reveals that serotypes A, O, SAT1 and SAT2 cause FMD outbreaks in Tanzania. Recent samples from outbreaks in 2008, 2009 and 2010 have been typed as serotypes A, O, SAT1 and SAT2. Phylogenetic analysis of FMDV isolates from Tanzania showed that they are genetically related to lineages and topotypes from West and East Africa. In Tanzania, lack of comprehensive animal movement records and inconsistent vaccination programs make it difficult to determine the exact source of FMD outbreaks or to trace the transmission of the disease over time. Therefore, further collection and analysis of samples from domestic and wild animals, together with improved local epidemiological investigation of FMD outbreaks is required to elucidate the complex epidemiology of FMD in the sub-Saharan region.

The solid-phase competition ELISA (SPCE) has been evaluated in both screening and titration assay formats for detecting antibodies against foot-and-mouth disease virus (FMDV) for the six non-O serotypes A, C, SAT 1, SAT 2, SAT 3 and Asia 1. Cut-off values were determined as a percentage inhibition of 40 for the SAT serotypes and 50 for serotypes A, C and Asia 1, which gave rise to specificity values ranging from 99.41% to 99.9% for the different serotypes. The relative sensitivity between the SPCE and LPBE/virus neutralisation test was 100%/109%. Antiserum titres derived by the SPCE for samples of serotypes O, A(22) and Asia 1 were more than 11, 1 and 5 times of those determined by virus neutralisation test, respectively. This study indicated that the non-type O SPCEs have sufficient sensitivities and specificities for use as serological diagnostic tests for the qualitative and quantitative detection of antibodies against FMDV.

Summary In 2007, serological evidence for foot-and-mouth disease (FMD) infection was found as a result of differential diagnostic testing of Cypriot sheep suspected to be infected with bluetongue or contagious ecthyma. Seropositive sheep and goats were subsequently uncovered on ten geographically clustered flocks, while cattle and pigs in neighbouring herds were all seronegative. These antibodies were specific for serotype-O FMD virus, reacting with both structural and non-structural (NS) FMD viral proteins. However, no FMD virus could be recovered from the seropositive flocks. FMD had not been recorded in Cyprus since 1964 and there has been no vaccination programme since 1984. Since all the seropositive animals were at least 3 years old and home-bred, it was concluded that infection had occurred approximately 3 years previously had passed un-noticed and died out spontaneously. It therefore appears that antibodies to FMD virus NS proteins can still be detected around 3 years after infection of small ruminants, but that virus carriers cannot be detected at this time. This unusual situation of finding evidence of historical infection in a FMD-free country caused considerable disruption and alarm and posed questions about the definition of what constitutes a FMD outbreak.

A simple lateral-flow assay (LFA) based on a monoclonal antibody (MAb 70-17) was developed for the detection of foot-and-mouth disease virus (FMDV) under nonlaboratory conditions. The LFA was evaluated with epithelial suspensions ( n = 704) prepared from current and historical field samples which had been submitted to the Pirbright Laboratory (United Kingdom) and from negative samples ( n = 100) collected from naïve animals in Korea. Four FMDV serotypes (type O, A, Asia 1, and C) were detected in the LFA, but not the remaining three FMDV serotypes (SAT 1, SAT 2, and SAT 3). The diagnostic sensitivity of the LFA for FMDV types O, A, C, and Asia 1 was similar, at approximately 87.3%, to that of 87.7% obtained with antigen enzyme-linked immunosorbent assay (Ag-ELISA). The diagnostic specificity of the LFA was 98.8%, compared to 100% for the Ag-ELISA. These results demonstrate that the LFA using the FMDV MAb 70-17 to detect FMDV is a supportive method for taking rapid measurements at the site of a suspected foot-and-mouth disease outbreak in Asia before diagnosing the disease in the laboratory, thereby offering the possibility of implementing control procedures more rapidly.

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.

A lateral flow device (LFD) for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV) was developed using a monoclonal antibody (Mab 1F10) shown to be pan-reactive to FMDV strains of each serotype by ELISA. The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia (304 positive and 1003 negative samples) from suspected cases of vesicular disease collected from 86 countries between 1965 and 2008 and negative samples collected from healthy animals. The diagnostic sensitivity of the LFD for FMDV was similar at 84% compared to 85% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 99.9% for the ELISA. The device recognized FMDV strains of wide diversity of all seven serotypes but weaker reactions were often evident with those of type SAT 2, several viruses of which were not detected. Reactions with the viruses of swine vesicular disease and vesicular stomatitis that produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The test procedure was simple and rapid, and typically provided a result within 1-10min of sample addition. Simple homogenizers that could be used in field conditions for preparing epithelial suspensions were demonstrated to be effective for LFD application. These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease.

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O1 BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes., Author Summary Foot-and-mouth disease (FMD) outbreaks in the United Kingdom during August and September 2007 have caused severe disruption to the farming sector and cost hundreds of millions of pounds. Investigating and determining the source of these outbreaks is imperative for their effective management and future prevention. Foot-and-mouth disease virus (FMDV) has a high mutation rate, resulting in rapid evolution. We show how complete genome sequences (acquired within 24–48 h of sample receipt) can be used to track FMDV movement from farm to farm in real time. This helped to determine the most likely source of the outbreak, assisted ongoing epidemiological investigations as to whether these field cases were linked to single or multiple releases from the source, and predicted the existence of undetected intermediate infected premises.

We describe the characterization of a foot-and-mouth disease (FMD) serotype A virus responsible for recent outbreaks of disease in Egypt. Phylogenetic analysis of VP1 nucleotide sequences demonstrated a close relationship to recent FMD virus isolates from East Africa, rather than to viruses currently circulating in the Middle East.

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.

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen 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 was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect 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 with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR 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 more » (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 using focused single-target rRT-PCR assays. « less

SIR, — We report here the genetic typing of a virus from the first occurrence of swine vesicular disease (svd) in Portugal since 2004. This outbreak was reported to the World Organisation for Animal Health (oie) on June 27, 2007, by the Portuguese Ministry of Agriculture and involved a farm in

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.

During a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (FMD) vaccination and infection appeared to be known with some certainty. Five herds had reported outbreaks of disease one to five months previously but clinical FMD had not been observed in the sixth herd. A trivalent vaccine (South African Territories [SAT] types 1, 2 and 3) had been used in some of the herds at various times either before and/or after the recent outbreaks of FMD. The primary aim of this study was to evaluate the performance of serological tests for the detection of SAT-type FMD virus infection, particularly elisas for antibodies to non-structural proteins (NSPs) of FMD virus and solid phase competition ELISAS (SPCEs) for serotypes SAT1 and SAT2. Secondary aims were to examine NSP seroconversion rates in cattle that had been exposed to infection and to compare virus detection rates by virus isolation and real-time reverse transcriptase-PCR (rtRT-PCR) tests on both oesophagopharyngeal fluids and nasopharyngeal brush swabbings. In addition, the hooves of sampled animals were examined for growth arrest lines as clinical evidence of FMD convalescence. Laboratory tests provided evidence of FMD virus infection in all six herds; SAT2 viruses were isolated from oesophagopharyngeal fluids collected from two herds in northern Zimbabwe, and SAT1 viruses were isolated from three herds in southern Zimbabwe. Optimised rtRT-PCR was more sensitive than virus isolation at detecting FMD virus persistence and when the results of the two methods were combined for oesophagopharyngeal fluids, between 12 and 35 per cent of the cattle sampled in the convalescent herds were deemed to be carriers. In contrast, nasopharyngeal swabs yielded only two virus-positive specimens. The overall seroprevalence in the five affected herds varied with the different NSPS from 56 per cent to 75 per cent, compared with 81 per cent and 91 per cent by homologous SPCE and virus neutralisation tests respectively. However, if serological test results were considered only for the cattle in which persistent infection with FMD virus had been demonstrated, 70 to 90 per cent scored seropositive in the different NSPs.

A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV). Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated. The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n = 43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR. In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance 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.

International audience; Foot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore- and machine milk samples were collected over a 4-week period from two dairy cows infected with FMDV and from two in-contact cows held in the same pen. The whole, skim, cream and cellular debris components of the milks were tested by automated rRT-PCR and results compared to virus isolation (VI) in cell culture. The onset of clinical signs of FMD in all four cows correlated with viraemia, and the presence of FMDV in other clinical samples. rRT-PCR results matched closely with VI in detecting FMDV in all milk components and generally coincided with, but did not consistently precede, the onset of clinical signs. rRT-PCR detected FMDV in milk up to 23 days post inoculation which was longer than VI. Furthermore, the detection limit of FMDV in milk was greater by rRT-PCR than VI and, in contrast to VI, rRT-PCR detected virus genome following heat treatment that simulated pasteurisation. rRT-PCR was also able to detect FMDV in preservative-treated milk. In conclusion, this study showed that automated rRT-PCR is quicker and more sensitive than VI and can be used to detect FMDV in whole milk as well as milk fractions from infected animals.

Recombinant integrin alpha v beta6 was evaluated as a capture ligand in a sandwich ELISA for the detection and serotyping of foot-and-mouth disease (FMD) virus. Our routinely applied method employs seven serotype-specific rabbit polyclonal antibodies as capture ligands and seven serotype-specific guinea pig polyclonal antibodies as detecting reagents. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease (SVD). Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific monoclonal antibodies (mabs) were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/mab detector combination was superior to that of our routinely employed rabbit/guinea pig ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the alpha v beta6 recombinant protein also has the potential for application in other test procedures for viral identification (e.g. pen-side chromatographic strip-tests, biosensors, immunocapture RT-PCR, antigenic characterization procedures and monoclonal antibody profiling of emerging field virus strains) and in antibody detection assays employed for the diagnosis of FMD.

Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus isolation, and ELISAs for antigen and antibody) were used in parallel. Each inoculated pig developed a significant viraemia and clinical disease, and excreted virus, which was transmitted to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs.

Automated fluorogenic (5' nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or clotted blood, milk and oesophageal-pharyngeal fluid ('probang') samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak serotype O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12 h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probang and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture. The combined tests of ELISA, virus isolation in cell culture and RT-PCR might, therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then either ELISA plus RT-PCR or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory diagnostic test results.

Primary cells derived from calf thyroid (CTY), calf kidney (CK) and piglet kidney (PK) were immortalised by oncogene transfection and their susceptibility to infection by foot-and-mouth disease (FMD) virus and swine vesicular disease (SVD) virus examined. Eighty-five immortalised cell lines (47 CTY, 20 CK and 18 PK) proved stable upon repeated cell culture passage and many supported the growth of FMD virus and several of the PK cell lines supported SVD virus. However, none of the immortalised lines exhibited either the degree of sensitivity or the specificity for all virus serotypes and strains as shown by primary CTY and IB-RS-2 cell cultures which are routinely employed for vesicular virus diagnosis.

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.

Multiple primers designed from the 1D and 2AB regions of the foot-and-mouth disease (FMD) viral genome were evaluated extensively for the detection of all seven serotypes of the virus by reverse transcription polymerase chain reaction (RT-PCR) at the OIE/FAO World Reference Laboratory for FMD (WRL), Pirbright. The primers had been characterised previously elsewhere on a relatively small number of cell culture grown isolates and epithelial suspensions and had been shown to identify and differentiate all seven serotypes of FMD virus. The extended study evaluated several RT-PCR protocols on epithelial suspensions and supernatant fluids, resulting from their passage in cell culture, derived from clinical samples of diverse molecular characteristics. Each of the serotype-specific primers in selected RT-PCR protocols demonstrated suitable specificity and detected cell culture passaged isolates with some success but were not adequate for the serotyping of suspensions prepared from clinical samples of epithelium. The results showed that the primers can be used in RT-PCR procedures in conjunction with the routine detection methods of virus isolation and ELISA for the diagnosis and serotyping of FMD virus.

A reverse transcription polymerase chain reaction (RT-PCR) method was compared with virus isolation in cell culture and the antigen detection ELISA for the primary diagnosis of foot-and-mouth disease (FMD) on 166 clinical samples from the field. Eighty samples were positive by virus isolation/ELISA and 78 by RT-PCR. The RT-PCR detected FMD viral RNA in 11 of the 86 samples assessed as negative by virus isolation/ELISA but conversely failed to diagnose 13 samples identified as positive by the latter procedures. This RT-PCR is not serotype-specific so a cDNA product is indicative of the presence of FMD viral RNA only. Confirmation of the specificity of the cDNA product and the identification of the serotype requires nucleotide sequence analysis. The value of the RT-PCR is that it can rapidly facilitate the molecular analysis of field isolates and thus provide important epidemiological information regarding the source of outbreaks. However, it is a sophisticated technique requiring specialised equipment, expertise and refined reagents and has to be used in conjunction with current procedures for FMD diagnosis.