Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA).

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.

Serological and biochemical analysis of some recent type A foot-and-mouth disease virus isolates from the Middle East.

In 1986 and 1987 foot-and-mouth disease virus (FMDV) serotype A was isolated from outbreaks of disease in Saudi Arabia and Iran. Selected virus isolates were antigenically distinct from the prototype A22 virus strain (A22/Iraq/64), but were serologically related to each other. However, polyacrylamide gel electrophoresis showed that whilst the respective Saudi Arabian structural polypeptides were homogeneous, those from an Iran isolate were distinct. Direct sequencing of part of the P-1D (VP1) gene demonstrated considerable difference in nucleotide homology between the two groups of viruses; the Saudi Arabian viruses were closely related to each other but only distantly related to both the A22 prototype virus strain and the Iranian virus isolate. The latter viruses were only slightly more closely related to each other. Thus there appeared to be at least two distinct FMDV type A variants co-circulating in the Middle East, both of which differed considerably from the classical A22 subtype.

Presence of a 43-kDa host-cell polypeptide in purified aphthovirions.

A 43kDa cellular polypeptide (P43), which comigrates with host-cell actin in both SDS-PAGE and isoelectrofocusing slab gels, was found associated to 140 S aphthoviral particles purified from BHK 21 cells labeled with [35S]methionine prior to infection. Ultracentrifugation analysis of disrupted virions demonstrates that polypeptide P43 is not associated to VP1-3 containing 12 S subunits but remains, like viral polypeptide VP4, at the top of the sucrose gradients. In addition, in vitro iodination or trypsin treatment show that P43 is protected from the action of both procedures and therefore supports the hypothesis that host-cell polypeptide P43 is located within the viral particles.

Serological and biochemical analysis of foot-and-mouth disease virus (serotype C3) isolated in Argentina between 1981 and 1986.

The evolution in the field is described for foot-and-mouth disease viruses belonging to serotype C3 in Argentina between 1981 and 1986. During 1981 and 1982 only three isolations of this serotype took place, which showed minor serological and biochemical variations from the prototype strain C3 Resende-Brasil/55. At the beginning of 1983 an outbreak was detected in a restricted geographical region caused by strains which had important serological and biochemical differences from the prototype strain. However, revaccination of the animals with the available commercial vaccine was sufficient to control the disease. During 1984 and up to 1986, a new outbreak of the same serotype took place in an extended geographical region of Argentina. During this outbreak, two field strains were isolated, studied and later included in the vaccine. One of them, strain C84, was a serological variant of the prototype strain and its inclusion in the vaccine had only a limited effect in controlling the disease. Inclusion of the other representative strain, C85, together with virus C84 finally reduced the number of outbreaks to the usual number reported.

Detection of foot-and-mouth disease virus with DNA probes in bovine esophageal-pharyngeal fluids.

Infectivity and dot-blot hybridization techniques were compared for the detection of FMDV in esophageal-pharyngeal fluids from experimentally infected cows. The probe used includes the viral polymerase sequence which allows the detection of the three types of virus (A, O, and C) with equivalent sensitivity. Virus was detected by dot-blot hybridization as well as by infectivity, according to sample analysis of esophageal-pharyngeal fluids extracted seven days post-infection. It was not possible to recover infective virus from some samples extracted at 180 and 560 days post-infection, although specific viral RNA was detected by dot-blot hybridization. This could indicate the presence of a high ratio of non-infective viral mutants in FMDV carrier cattle. These results emphasize the usefulness of molecular hybridization techniques for FMDV carrier-state detection.

Surface structure and RNA-protein interactions of foot-and-mouth disease virus.

The surface structure of foot-and-mouth disease virus (FMDV) and the interaction of the individual capsid proteins with the virus RNA have been examined using modification reagents. By measuring the extent of modification of the lysine residues of intact and disrupted virus particles and the 12S protein subunit with Bolton & Hunter reagent it was found that 54% of the residues of VP1, 15% of the residues of VP2 and 37% of the residues of VP3, equivalent to five, two and four lysine residues respectively, are on the surface of the intact virus particle. Polypeptide VP4 was not modified in intact virus particles, indicating that it has no lysine residues on the surface of the virus. Modification with sodium metabisulphite, which causes a specific transamination reaction between cytidylic acid residues in ssRNA and closely associated basic amino acids, cross-linked all four structural proteins to the virus RNA. Both fragments of VP1, produced by treatment of the virus particle with trypsin, are also cross-linked to the RNA. These observations have been combined with the evidence that the immunogenic activity of VP1 may be contained in two discontinuous sites, at amino acids 141 to 160 and 200 to 213, in proposing a model for the arrangement of this polypeptide in the virus particle.

Antigenic comparison of the polypeptides of foot-and-mouth disease virus serotypes and other picornaviruses.

The cross-reactivity of proteins coded for by the seven serotypes of foot-and-mouth disease virus (FMDV) was assessed by reaction of infected cell lysates with polyclonal and monospecific antisera against the structural and nonstructural proteins of FMDV type A12 strain 119ab. It was shown that the homologous polypeptides from most serotypes are antigenically related. The least cross-reactivity occurred between VP1, VP3, and the protease (3C) of type A12 and South African Territories types 1 and 3. There was also a reduced degree of reactivity of A12 VP1 serum with VP1 from some A subtypes and the other serotypes. Comparison of FMDV proteins with polypeptides from other picornaviruses by a radioimmune binding assay revealed a low level of reactivity of antisera against some A12 polypeptides with encephalomyocarditis virus (EMCV) infected cell lysates but no reactivity with bovine enterovirus type 1 and swine vesicular disease virus infected cells. The same EMCV proteins were immunoprecipitated by the various reactive A12 antisera, but the reaction was abolished if the lysate from EMCV infected cells was denatured prior to immunoprecipitation.

Biochemical characterization of an aphthovirus type C3 strain Resende attenuated for cattle by serial passages in chicken embryos.

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Cytoskeletal association of an aphthovirus-induced polypeptide derived from the P3ABC region of the viral polyprotein.

Monolayers of BHK cells infected with aphthovirus (FMDV) were labeled for short times with [35S]methionine at 2 hr p.i. and fractionated by detergent treatment and low speed centrifugation. Polyacrylamide gel analysis showed an asymmetric distribution of the FMDV-induced polypeptides among the three different subcellular fractions obtained. Polypeptide P88-1, the viral capsid protein precursor, is mainly found in the soluble cytoplasmic extract while polypeptides P100-3, P52-2AC, P34-2C, and P14-2A are the major viral components of a detergent soluble extract of the crude nuclear pellet. Analysis of the detergent resistant fraction (DRF), which is mainly composed of cell nuclear chromatin and insoluble cytoskeletal elements, shows a clear enrichment in an incompletely characterized polypeptide which is tentatively designated P54. Variable amounts of polypeptides P100-3 and those of the P72 complex are also detected in this fraction. The preferential location of P54 in an equivalent subcellular fraction obtained by mild detergent treatment of infected monolayers in situ, and also in a high-salt resistant subfraction of the DRF, strongly suggests a close association of this polypeptide with vimentin-actin containing components of the cell. Polypeptide P54 is immunoprecipitated by viral specific antiserum from convalescent guinea pigs but not by serum against FMDV capsid proteins, indicating that it does not share common antigenic determinants with polypeptides processed from the viral capsid precursors. On the other hand, protease V8 mapping of polypeptides P100-3, P54, P88-1, and VP1-3 shows that P54 derived from the 3' end coding region of the viral genome. Further analysis by limited protease digestion also demonstrates that P54 has partial overlap with P72-3CD while it does not share any common peptide with P56a-3D, indicating that P54 contains the sequences coded in the 3ABC region of the FMDV RNA. This assumption is reinforced by the basic behavior shown by P54 in two-dimensional gels. The results support the hypothesis of a close intracellular interaction of a short-lived polypeptide, containing the viral protease and VPg sequences, with the host cytoskeleton, during infection of BHK cells with FMDV.

Potential for the transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle.

Foot-and-mouth disease viruses of types SAT 1 and SAT 2 isolated from diseased cattle and carrier buffalo, either on the same farm or in the same ecological area within a short time of each other, were compared by T1 oligonucleotide mapping. No similarity was observed between the maps obtained, indicating that the different populations of virus were unique to each species and that no interspecies transmission had occurred.

Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins.

Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambda PL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in 35S-labeled extracts from foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and-mouth disease virus gene products not previously identified in vivo or in vitro.

Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos.

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed.

An international collaborative study on foot and mouth disease virus assay methods. 2. Quantification of 146S particles.

Workers in 11 laboratories in Europe and one in North America participated in a collaborative study to assess the variability of a sucrose gradient procedure used for the quantification of foot and mouth disease virus (FMDV). To this end, a range of standards was distributed from one of the participating laboratories. A series of adenine preparations were used to assess the various spectrophotometers/UV monitors and it showed most to be accurate and linear in their responses. The FMDV and MS2 ribophage standards were used to assess the sucrose gradient procedure and indicated low levels of within laboratory variation whereas between laboratory variation was greater. Recalculation of results from the unprocessed data in the coordinating laboratory permitted the identification and reduction of one source of between laboratory variation. Despite the observed variations, the results indicated that meaningful comparisons could be made between the data of different laboratories provided that a procedure similar to the one described in this report is used.

Analysis of the secondary structure of the poly(C) tract in foot-and-mouth disease virus RNAs.

Sodium bisulphite modification of foot-and-mouth disease virus (FMDV) RNA in solution indicates that the majority of the poly(C) tract in the RNA is single-stranded in concordance with previous results with encephalomyocarditis virus RNA. The reaction kinetics are biphasic; 60% of the cytidylic acid in the poly(C) tract reacts like synthetic poly(C), and the remainder with the kinetics of the cytidylic acid in the rest of the RNA. The reactivity of the poly(C) tract with poly(I) indicates that it is looped out and exposed in the RNA. The deamination reaction has also been used to investigate the structure of the replicative form (RF) and replicative intermediate (RI) isolated from infected cells. Analysis by gel electrophoresis of the long RNase A- and T1-resistant oligonucleotides of RI suggests that it has five single-stranded poly(C) tracts to every one which is base-paired. Bisulphite reactivity of the poly(C) tract and gel electrophoresis of the ribonuclease-resistant oligonucleotides of RF indicate that the poly(C) is base-paired to a poly(G) tract in this molecule. The presence of a poly(G) tract in RF and RI provides unequivocal evidence that the poly(C) is replicated via poly(G) in the negative strand.

Isolation and biochemical characterization of intertypic recombinants of foot-and-mouth disease virus.

Recombinants were isolated between two European serotypes (O and A) and between two of the most distantly related serotypes (O from Europe and SAT2 from Africa) using appropriate ts mutants in an infectious centre assay. The recombinants were characterised by electrofocusing of their induced proteins and by RNase-T1 fingerprinting of their RNA. The approximate location of the cross-over event in each recombinant was determined by sequencing the unique distinguishable O or A oligonucleotides and locating them within the known genome sequence. Nine different types of recombinant were identified from the two types of cross (O X A and O X SAT) and all had a single cross-over in the middle or 3' half of the genome, i.e. in the nonstructural coding region. Recombination between the most distantly related viruses (O X SAT2) appeared to occur at a lower frequency than recombination between serotypes of the same group (O X A). A higher incidence of recombinant proteins with unique pI was also observed in the O X SAT2 crosses.

A study of antigenic variants of foot-and-mouth disease virus by polyacrylamide gel electrophoresis of their structural polypeptides.

Twenty-nine foot-and-mouth disease (FMD) type A virus strains, previously classified serologically as distinct subtypes were analysed by polyacrylamide gel electrophoresis (PAGE) to determine the extent of variation in the pattern of the structural polypeptides and to evaluate the technique as an aid to existing subtyping techniques. The majority of the subtypes examined had distinct polypeptide patterns, however, some variation also occurred between strains within a subtype. The position of VP2(1B) and VP3(1C) was often unchanged in different strains within a subtype and between geographically related subtypes over long periods of time. Changes in the position of VP1(1D) were also observed within a subtype. The technique was considered to be of value for the screening of isolates prior to conventional serological subtyping procedures and in the tracing of the possible origin of FMD outbreaks.

Localization of a neutralization epitope of foot-and-mouth disease virus using neutralizing monoclonal antibodies.

Neutralizing monoclonal antibodies raised against intact foot-and-mouth disease virus reacted with intact particles and with isolated VP1 from different strains from the same subtype. Prior treatment of the virus with either trypsin or with arginine-specific protease abolished recognition of both the virus and of VP1, suggesting the presence of a neutralization epitope in the central region of VP1 cleaved by these two enzymes. A synthetic peptide analogue of part of this region showed poor reactivity, however, with neutralizing antibody.

Isolation of capsid proteins of foot-and-mouth disease virus by chromatofocusing.

A method for the isolation of foot-and-mouth disease virus (FMDV) capsid proteins was developed. The FMDV capsid proteins VP1, VP2, VP3 and VP0 were isolated from sucrose gradient purified virus by chromatofocusing in a pH 7.4-4.0 gradient on Polybuffer exchanger PBE 94. Under the conditions used the proteins eluted in the sequence VP1, VP2, VP0 (when present) and VP3. Capsid protein VP4 did not elute and could not be isolated by this method. Protein concentration in the eluate was monitored by the use of a radiolabelled marker and recoveries of approximately 50% of the input marker could be achieved when using up to 15 mg of virus and a 30-ml column. The high capacity and relative simplicity of chromatofocusing make it a useful alternative to other methods of purifying proteins.

Identification of an exposed region of the immunogenic capsid polypeptide VP1 on foot-and-mouth disease virus.

Iodination of intact foot-and-mouth disease virus results in the selective labeling of VP1, substantiating its exposed location on the virion. A comparison of tryptic peptides revealed that a single tyrosine-containing peptide was labeled with iodine on intact or protease-cleaved virus. The labeled peptide from intact and protease-cleaved virus was characterized by molecular weight sizing and sequence analysis. Carboxypeptidase digestion of intact VP1, limited trypsin-cleaved VP1, and VP1 purified from bacterially contaminated tissue cultures yielded carboxyterminal residues of leucine, valine-arginine, and serine-alanine, respectively. The correlation of these findings with previous data on the amino acid sequence derived from nucleotide sequencing of serotypes A12 and O1 of foot-and-mouth disease virus VP1 places the probable exposed antigenic region of VP1 in a serotype-variable region including residues 136 through 144.