Detection of Bovine Antibodies against a Conserved Capsid Epitope as the Basis of a Novel Universal Serological Test for Foot-and-Mouth Disease.

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents. As a consequence, FMD reference laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and postvaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterized using a panel of intertype-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide enzyme-linked immunosorbent assay (VP2-ELISA) was optimized using experimental and reference antisera from immunized, convalescent, and naïve animals (n = 172). The VP2-ELISA is universal and simple and provided sensitive (99%) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for serosurveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in countries where the disease is endemic.

Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs.

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.

Validation of a foot-and-mouth disease antibody screening solid-phase competition ELISA (SPCE).

This paper describes the validation of a solid-phase competition enzyme-linked immunosorbent assay (SPCE) for the serological detection of antibody to serotype O foot-and-mouth disease (FMD) in sheep, cattle and pigs. The specificity of the SPCE was calculated from the results of testing known negative sera from sheep, cattle and pigs (n=3030, 1418 and 1495, respectively). The mean percentage inhibition (PI) for known negative sheep, cattle and pig sera were 19.3, 24.1 and 20.8%, respectively. The specificity of the SPCE at a cut-off point (COP) of 60 PI was 99.50% for sheep sera, 99.44% for cattle sera and 100% for pig sera. The analytical sensitivity of the SPCE was examined by testing sera from sheep, cattle and pigs. Based on the testing of serial bleeds from experimentally infected animals, seroconversion at the 60 PI COP occurred between 4 and 9 days post-infection or -exposure, similar to that observed using the virus neutralisation test (VNT) with a COP of 1/45. When applied to 267 sheep and 143 pig samples, that were obtained in Great Britain (GB) during the 2001 FMD UK outbreak, the SPCE identified more positive samples than did the VNT. Estimates of the accuracy, repeatability and reproducibility of the SPCE were verified during the large-scale serosurveillance necessitated by the 2001 outbreak. Results from field and experimental sera showed that when compared against the VNT, the sensitivity of the SPCE was less affected by the choice of virus strain used in the test. Using the O(1) UKG 2001 FMD virus in the VNT with samples representative of the uninfected GB sheep population, the test specificity was 100% at a COP of 1/45.

Virulence of swine vesicular disease virus is determined at two amino acids in capsid protein VP1 and 2A protease.

To identify the genetic determinants of virulence for swine vesicular disease virus, a panel of recombinant and site-directed mutant viruses were constructed from cDNA clones of a virulent J1'73 strain and an avirulent H/3'76 strain. Initial studies mapped the genetic determinants of virulence to either or both of the two sites at nucleotide (nt) 2842, encoding VP1-132, and nt 3355, encoding 2A-20. To determine their relative importance with regard to virulence, viruses mutated at either of these two sites from the avirulent to the virulent genotype and vice versa were tested in pigs. Viruses, mutated at nt 2842 to the virulent genotype (vSVLS104MJ1) or mutated at nt 3355 to the virulent genotype (vSVLS201MJ1), slightly recovered virulence but were very weak compared with viruses with site-directed mutations at both sites (vSVLS104/201MJ1). On the other hand, viruses, mutated at nt 2842 to the avirulent genotype (vSVLS104M00) or mutated at nt 3355 to the avirulent genotype (vSVLS201M00), did not have attenuated virulence. Sequence analysis of viruses recovered from inoculated pigs revealed that reversion at nt 3355 to the virulent genotype occurred in pigs which had been inoculated with vSVLS201M00. These results suggested that both amino acids determined the virulent phenotype, but that the 2A-20 site might be the major determinant for virulence.

Mapping the genetic determinants of pathogenicity and plaque phenotype in swine vesicular disease virus.

A series of recombinant viruses were constructed using infectious cDNA clones of the virulent J1'73 (large plaque phenotype) and the avirulent H/3'76 (small plaque phenotype) strains of swine vesicular disease virus to identify the genetic determinants of pathogenicity and plaque phenotype. Both traits could be mapped to the region between nucleotides (nt) 2233 and 3368 corresponding to the C terminus of VP3, the whole of VP1, and the N terminus of 2A. In this region, there are eight nucleotide differences leading to amino acid changes between the J1'73 and the H/3'76 strains. Site-directed mutagenesis of individual nucleotides from the virulent to the avirulent genotype and vice versa indicated that A at nt 2832, encoding glycine at VP1-132, and G at nt 3355, encoding arginine at 2APRO-20, correlated with a large-plaque phenotype and virulence in pigs, irrespective of the origin of the remainder of the genome. Of these two sites, 2APRO-20 appeared to be the dominant determinant for the large-plaque phenotype but further studies are required to elucidate their relative importance for virulence in pigs.

Molecular analysis of encephalomyocarditis viruses isolated from pigs and rodents in Italy.

Partial nucleotide sequences of encephalomyocarditis (EMC) viruses isolated from five, apparently independent, outbreaks of fatal myocarditis in pigs in Italy were compared with three EMC viruses isolated from wild rodents from a different geographic region in the same country. These viruses were also compared with EMC viruses isolated from pigs in other European countries and three historical strains. All the Italian EMC viruses were closely related (> 94.6% nucleotide identity), but were distinct from viruses occurring in Belgium in 1991 (< 80.5% nucleotide identity), Greece in 1990 (< 83.3% nucleotide identity) and the three older viruses (< 82.9% nucleotide identity). An EMC virus isolated from pigs in the Netherlands in 1988, was closely related to the Italian viruses (95.3-99.3% nucleotide identity). It is suggested that pigs may play a role in the movement of EMC viruses between different geographic regions.

Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-94.

Viruses from the recent epidemic of swine vesicular disease (SVD) in Europe have been isolated and characterized by antigenic and genetic methods to examine the likely epidemiological origins of the disease. Antigenic analysis was performed on 77 SVD viruses (SVDV) isolated in Europe between 1966 and 1994 using two panels of monoclonal antibodies (MAb) in a trapping ELISA. Genetic analysis of 33 of the SVD viruses by reverse transcription-polymerase chain-reaction (RT-PCR) amplification and nucleotide sequencing of the ID (VP1) coding region was also performed. Comparison of the nucleotide sequences with each other and with three other previously published SVDV sequences revealed four distinct groups which correlated exactly with the results of the pattern of reactivity with MAbs. The first group consisted solely of the earliest SVD virus isolated (ITL/1/66) while the second group comprised viruses present in Europe and Japan between 1972 and 1981. The third group consisted of viruses isolated from outbreaks of SVD in Italy between December 1988 and June 1992. Viruses isolated between 1987 and 1994 from Romania, the Netherlands, Italy and Spain formed a fourth group. The genetic and antigenic similarity of the most recent virus isolates from Western Europe to a virus isolated in Romania 5 years previously suggests that the possible origin of the recent epidemic of swine vesicular disease in Western Europe was in Eastern Europe.

Complete nucleotide sequence of a coxsackie B5 virus and its relationship to swine vesicular disease virus.

We report the first complete nucleotide sequence of the picornavirus coxsackievirus B5 (CB5), strain 1954/UK/85, an isolate from a case of hand-foot-and-mouth disease. We have compared the sequence with those of other coxsackie B viruses, coxsackievirus A9, poliovirus and swine vesicular disease virus (SVDV). The genes encoding the three major capsid proteins are most closely related to those of SVDV but the 5' and 3' noncoding regions and the P3 gene are more similar to the corresponding regions in the other coxsackie B viruses than to those of SVDV. These observations are considered in the light of the antigenic and biochemical relationships between SVDV and CB5.