Antigenic analysis of serotype O foot-and-mouth disease virus isolates from the Middle East, 1981 to 1988.

During the period 1981-88 foot-and-mouth disease virus (FMDV) serotype O continued to be isolated from outbreaks in the Middle East. Field isolates submitted to the World Reference Laboratory have been examined in relation to reference strains by either complement fixation, virus neutralization or enzyme-linked immunosorbent assays. Most isolates were related to the European type O1 reference strains although strains emerging in late 1987 and 1988 were more closely related to O1/Manisa. In addition, FMDV isolates from Libya in 1981 and Syria in 1987 have shown very little relationship to these reference strains, although evidence of their persistence and spread has not been demonstrated.

Serological prospects for peptide vaccines against foot-and-mouth disease virus.

Antibodies to a synthetic peptide corresponding to the 141 to 160 amino acid sequence of the protein VP1 of type O foot-and-mouth disease virus (FMDV) neutralize a wider range of type O isolates than anti-virion serum. Extending this peptide at the amino terminus reduced the number of strains neutralized by the antipeptide sera. Reactions with antisera to peptides representing non-contiguous native sequences showed that it was also possible to increase the number of strains effectively neutralized. Selected substitutions of a single amino acid at position 148 markedly altered the neutralizing specificity of antibodies elicited by the 141 to 160 peptide. In particular, a peptide with an L----S substitution at this position induced antibodies which neutralized a type O and a type A virus equally, and guinea-pigs inoculated with it were protected from challenge with either virus. Attempts to isolate variant viruses resistant to neutralization with anti-peptide antibody indicated that these occurred at low frequency, and there was some evidence that resistance may be partially conferred by mutations outside the peptide sequence.

Neutralizing epitopes of type O foot-and-mouth disease virus. I. Identification and characterization of three functionally independent, conformational sites.

Eleven neutralizing monoclonal antibodies (MAbs) were produced to the O1BFS 1860/67 strain of foot-and-mouth disease virus (FMDV), and were characterized for their ability to bind viral and subviral antigens in different ELISA tests and to neutralize heterologous type O isolates. Neutralization escape variants of the homologous virus, isolated under pressure from five of these MAbs, were used in cross-neutralization tests with all of the 11 antibodies. These studies identified three functionally independent, conformational, neutralizing sites. The most conformationally dependent site bound antibody which neutralized a range of type O virus isolates. A second site was less dependent on conformation and was recognized by antibody that was strain-specific. The least conformational site bound MAbs which showed limited cross-neutralization of other type O strains. This latter site appeared to be immunodominant and contained several overlapping epitopes which showed some differences in their specificities. Isoelectrofocusing and sequencing studies of the variants strongly suggested that polypeptide VP2 contributes to the immunodominant site.

Neutralizing epitopes of type O foot-and-mouth disease virus. II. Mapping three conformational sites with synthetic peptide reagents.

Four neutralizing monoclonal antibodies (MAbs), recognizing three functionally independent, conformational sites on type O foot-and-mouth disease virus (FMDV) failed to react with immobilized structural proteins or synthetic peptides but bound to the isolated capsid protein VP1 and peptides in solution. Inhibition ELISA techniques were, therefore, applied using peptide antigens and anti-peptide sera to block MAb binding to virus particles, permitting the identification of those portions of the VP1 protein contributing to the epitopes. The binding site of one MAb, which neutralized a range of type O FMDV isolates, was shown to have components within regions 146 to 150 and 200 to 213 of VP1 with a critical involvement of the amino acids at positions 146 and 206 or 207. The determinants recognized by two other MAbs which were directed at similar, but not identical, epitopes from a second site included components from the 200 to 213 and 143 to 146 regions with amino acids 143 and 144, respectively, appearing critical for the inhibition of the virus binding of the two antibodies. These results demonstrate that the two previously identified immunogenic tracts of VP1 are brought into proximity in the quaternary structure of the virion to form an antigenic domain containing several conformational epitopes, some of which are functionally independent. A fourth, strain-specific MAb was effectively blocked from reacting with virus by peptides corresponding to residues 161 to 180 and 200 to 213.

Host cell selection of antigenic variants of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) A22 Iraq 24/64 adapted to grow in BHK monolayer cells induced antibodies which neutralized many isolates belonging to the A serotype. Plaque-purified virus isolated from this stock also induced broadly reactive antibodies, showing that this property is not due to the combined response to a mixture of variants in the original stock virus. However, viruses obtained by passage in suspension BHK cells of either the monolayer cell-adapted virus or a virus cloned from this stock resulted in the selection of virus which induced antibodies with highly specific neutralizing activity. In addition to their antigenic properties the monolayer and suspension cell-adapted viruses could be distinguished by plaque morphology, tendency to aggregate and ability to attach to BHK cells. Monoclonal antibodies (MAbs) induced with the plaque-purified monolayer-adapted virus had neutralizing activity almost as broad as polyclonal serum, showing that this property can be represented by a single epitope on the virus. These neutralizing MAbs recognize a trypsin-sensitive epitope on the virus. Surprisingly, sequence analysis of the structural protein-coding regions of the genomic RNAs of monolayer and suspension cell-adapted viruses showed no amino acid differences in VP1, the protein known to contain the major neutralization epitope in FMDV and to be the only protein susceptible to cleavage by trypsin in the virus particle. Although three coding differences were found in the capsid protein these were all located in VP2.

Epitope mapping of foot-and-mouth disease virus with neutralizing monoclonal antibodies.

Epitopes of strain A22 Iraq 24/64 of foot-and-mouth disease virus have been mapped with monoclonal antibodies (MAbs). Three methods were used: (i) an indirect ELISA using an overlapping set of peptides, (ii) production of neutralization escape variants against each MAb and (iii) sequencing of neutralization escape variants. The study has shown that the virus has at least three overlapping liner neutralizing epitopes within a major antigenic site on VP1. The presence of a second, conformational site was demonstrated but its position on the virus particle was not located. Synthetic peptides with sequences representing the major site elicit antibodies which have similar broad cross-neutralizing activity to polyclonal serum or neutralizing MAbs produced with the virus against a range of field isolates.

The use of a logistic model for the quantitative interpretation of indirect sandwich enzyme labelled immunosorbent assays (ELISA) for antibodies and antigens in foot and mouth disease.

A three parameter logistic model is described for the analysis of profiles of optical density vs log dose for indirect sandwich ELISA tests in foot and mouth disease. The model describes the observed phenomenon of saturability with increasing dose, and its parameters can be interpreted in terms of molecular binding events. A computer program to fit the model is described. An approximate statistical test is developed which can be used to test for departures from equivalence for replicate profiles. It is found that correlation of the optical density values to a standard reference reaction considerably improves reproducibility.

An indirect sandwich enzyme labelled immunosorbent assay for the detection of foot and mouth disease virus immunizing antigen in tissue culture harvests.

The optimum conditions for an indirect sandwich enzyme-linked immunosorbent assay for foot and mouth disease virus 140S antigen assay are described. Factors which could contribute to the variation in the test were investigated and a calibration coefficient for the conversion of ELISA values to antigen concentration in micrograms of 140S antigen per millilitre was calculated. Antigen mass in nine tissue culture harvests was estimated and these correlated well with estimates made by sucrose density gradients (r = 0.95).

The differentiation of foot and mouth disease virus strains using an indirect sandwich enzyme-linked immunosorbent assay saturation model.

Sigmoid saturation curves were fitted to the results of titrations of antiserum to foot and mouth disease virus against homologous and heterologous virus strains. Differentiation of strains was readily evident from the different levels of the homologous and heterologous curves. These differences could be quantified by comparison of the saturation curve parameters K and PRmax. Factors which affect variations in K and PRmax and their biological significance were investigated by varying the first phase antibody and the antigen used in the test. PRmax was found to represent an overall combining potential of the antigen with both sera used in the sandwich test. K, which was theoretically a measure of affinity, also reflected antibody titre. Relationships measured using this model were found to correlate with the reference test system--two-dimensional microneutralization.

The effect of antiserum quality on strain specificity assessment of foot and mouth disease virus by the neutralization reaction.

The factors affecting the virus strain specificity of antibody to foot an mouth disease virus prepared by a variety of protocols in several species were evaluated by neutralization tests. The time at which the serum was taken, the antigen dose given, whether or not revaccination had occurred and the animal species in which the sera were prepared, did not appear to affect the strain specificity of serum prepared to inactivated antigens when measured in neutralization tests, probably because of the restricted nature of the antigenic site involved. However, variation was observed with convalescent animal sera or sera from animals which had received trypsin cleaved virus were used. For these reasons banks of reference antisera are prepared as pooled sera using one or two inoculations of inactivated antigen.

Evaluation of the antigenic variation within type-A foot and mouth disease virus isolates from Asia.

The serological interrelationships among 17 type A FMD virus strains from eight Asian countries were studied by the two-dimensional microneutralization test. Complex direct and indirect relationships were observed. Overall, however, the virus strains studied could be classified as belonging to the A22 group on the basis of r value differentiation at P less than 0.01.

Demonstration of neutralizing and non-neutralizing epitopes on the trypsin-sensitive site of foot-and-mouth disease virus.

The isolation of monoclonal antibodies directed against the trypsin-sensitive site on the 140S particle of foot-and-mouth disease virus (FMDV) has enabled the demonstration of at least three distinct epitopes within this site. Reaction with two of these resulted in neutralization of virus infectivity. None of the epitopes appeared to be present on the 12S particles, and one of the neutralizing epitopes was sensitive to even milder configurational changes of the particle.

Computerization of foot and mouth disease virus strain relationship data.

Since 1976 the two dimensional microneutralization test has been adopted in our laboratory as the reference test for foot and mouth disease virus (FMDV) strain differentiation. Large numbers of homologous-heterologous comparisons have been performed and manual storage and retrieval of the data had become cumbersome. The objective of computerization was to provide a databank with fast easy access to enable accurate statistical calculations to be performed on the raw data entries and to simplify the evaluation of statistical and biological significance. The computer system is written in Fortran for an IBM 4341 computer running under the VM/CMS operating system.

A serological evaluation of 1979-1982 Kenyan foot-and-mouth disease type SAT 2 viruses.

Serological evaluations of foot-and-mouth disease type SAT 2 viruses isolated in Kenya between 1979 and 1982 were performed using the two-dimensional microneutralization test. Nine field isolates of epizootiological significance were compared with four vaccine viruses. The results obtained identified Tan 5/68 as the most appropriate reference vaccine virus strain since it had the broadest serological spectrum. Potent Tan 5/68 vaccines would be expected to provide adequate protection against the contemporary SAT 2 field viruses. In the case of K183/74, which also was shown to have a broad spectrum with viruses isolated in Kenya, the results show that the 1982 isolate from central Kenya was significantly divergent (r less than 1.00 at P = 0.01) and warranted tactical revaccination for its control. The study highlighted the fact that strain R1215 which had been isolated from the oesophageal-pharyngeal swabs of asymptomatic carrier cattle had a narrow serological spectrum suggesting that such viruses could be unsuitable as vaccine for the national campaign.

Antibody response in bovine pharyngeal fluid following foot-and-mouth disease vaccination and, or, exposure to live virus.

Samples of pharyngeal fluid and serum were collected from cattle after exposure to live foot-and-mouth disease (FMD) virus (with or without prior vaccination) or after subcutaneous vaccination with inactivated virus. The pharyngeal fluid samples were examined for FMD neutralising activity and specific anti-FMD IgG, IgM and IgA antibodies. The neutralising activity of the serum was also monitored. A peak of neutralising activity which occurred in the pharyngeal fluid of unvaccinated cattle seven days after virus exposure corresponded to a rise in specific IgM and IgA antibodies. This peak appeared to be due to serum and tissue fluid escaping from the damaged mucosa during the acute inflammatory phase of infection. At later stages (20 to 60 days after virus exposure) the pharyngeal fluid neutralising activity corresponded to a rise in specific IgA antibodies, suggesting that active local antibody production was taking place. The pharyngeal fluid neutralising activity detected after revaccination with oil emulsion or aqueous vaccines, without exposure to live virus, corresponded to a rise in specific IgG and IgM antibody levels and this may have been due to serum transudation.

Antibody response of pigs to foot-and-mouth disease oil emulsion vaccine: the antibody classes involved.

Groups of three pigs were vaccinated with water-in-oil emulsion vaccine and revaccinated either 21 and 148 or 106 days later. Sera were taken periodically for six months and fractionated into heavy and light elements on sucrose density gradients. The heavier fraction contained IgM and the lighter fraction IgG and IgA. Neutralising antibodies were first detectable eight days after initial vaccination (dpiv), rose to a peak between 14 and 21 dpiv and persisted at relatively high titres until the time of revaccination. Neutralising antibody at eight dpiv was attributed to IgM but by 10 to 14 dpiv both IgM and IgG were involved. Thirty-five days (and later) after a single vaccination all the neutralising activity in the sera was due to IgG. The revaccinations produced an increment in the whole serum neutralising titres and in each case both IgM and IgG class antibodies were involved.