Foot-and-mouth Disease Virus O1 Research Articles

Optimized Adenoviral Vector That Enhances the Assembly of FMDV O1 Virus-Like Particles in situ Increases Its Potential as Vaccine for Serotype O Viruses.

Although replication-defective human adenovirus type 5 (Ad5) vectors that express in situ the capsid-encoding region of foot-and-mouth disease virus (FMDV) have been proven to be effective as vaccines in relevant species for several viral strains, the same result was not consistently achieved for the O1/Campos/Brazil/58 strain. In the present study, an optimization of the Ad5 system was explored and was proven to enhance the expression of FMDV capsid proteins and their association into virus-like particles (VLPs). Particularly, we engineered a novel Ad5 vector (Ad5[PVP2]OP) which harbors the foreign transcription unit in a leftward orientation relative to the Ad5 genome, and drives the expression of the FMDV sequences from an optimized cytomegalovirus (CMV) enhancer-promoter as well. The Ad5[PVP2]OP vaccine candidate also contains the amino acid substitutions S93F/Y98F in the VP2 protein coding sequence, predicted to stabilize FMD virus particles. Cells infected with the optimized vector showed an ∼14-fold increase in protein expression as compared to cells infected with an unmodified Ad5 vector tested in previous works. Furthermore, amino acid substitutions in VP2 protein allowed the assembly of FMDV O1/Campos/Brazil/58 VLPs. Evaluation of several serological parameters in inoculated mice with the optimized Ad5[PVP2]OP candidate revealed an enhanced vaccine performance, characterized by significant higher titers of neutralizing antibodies, as compared to our previous unmodified Ad5 vector. Moreover, 94% of the mice vaccinated with the Ad5[PVP2]OP candidate were protected from homologous challenge. These results indicate that both the optimized protein expression and the stabilization of the in situ generated VLPs improved the performance of Ad5-vectored vaccines against the FMDV O1/Campos/Brazil/58 strain and open optimistic expectations to be tested in target animals.

Efficacy of a high potency O1 Manisa monovalent vaccine against heterologous challenge with foot-and-mouth disease virus of O/SEA/Mya-98 lineage in sheep

Potency tests for commercial oil-adjuvanted foot-and-mouth disease (FMD) vaccines are usually carried out in cattle, using a full dose (2 ml) of vaccine and homologous virus challenge. However, in sheep the recommended vaccine dose is half of the cattle dose (1 ml) and most vaccines have not been potency tested for this species, especially with heterologous viruses. To determine the efficacy of a high potency (>6PD50) FMD virus (FMDV) O1Manisa vaccine in sheep, we carried out a study using a heterologous FMDV (FMDV O/SKR/2010 - Mya-98 strain) challenge. Groups of seven animals each were vaccinated with 2×, 1×, 1/2× or 1/4× dose (2 ml, 1 ml, 0.5 ml or 0.25 ml respectively) and challenged at 7 days post vaccination (dpv). Only 3 of the 7 sheep in the group vaccinated with 2 ml were protected. With 2 additional groups, receiving double or single doses and challenged at 14 dpv, 4 of 7 sheep were protected in each group. None of the sheep had measurable neutralising antibodies against the vaccine or challenge virus at 7 dpv. However, all vaccinated animals challenged at 14 dpv had a homologous neutralising response against FMDV O1 Manisa on the day of challenge and all but one animal also had a heterologous response to FMDV O/SKR/2010. Infectious FMDV and viral RNA could be found in nasal swabs between 1 and 6 days post challenge (dpc) in most vaccinated sheep, but those vaccinated with higher doses or challenged at 14 dpv showed significant decreases in the level of FMDV detection. Intermittent virus shedding was noticed between 1 and 35 dpc in all vaccinated groups, but persistent infection could be demonstrated only in 4 sheep (20%). This study showed that at the recommended dose, a high potency (>6 PD50) FMDV O1Manisa vaccine does not protect sheep against a heterologous challenge at 7 dpv. However, partial protection was observed when a double dose was used at 7 dpv or when double or single dose vaccinated sheep were challenged at 14 dpv.

Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine

A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 107 pfu/animal while a dose of 4×107pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine.

A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle

The role of non-structural protein 3A of foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10–20 amino acid deletion has been implicated as responsible for virus attenuation in cattle: a 10 amino acid deletion in the naturally occurring, porcinophilic FMDV O1 Taiwanese strain, and an approximately 20 amino acid deletion found in egg-adapted derivatives of FMDV serotypes O1 and C3. Previous reports using chimeric viruses linked the presence of these deletions to an attenuated phenotype in cattle although results were not conclusive. We report here the construction of a FMDV O1Campos variant differing exclusively from the highly virulent parental virus in a 20 amino acid deletion between 3A residues 87–106, and its characterization in vitro and in vivo. We describe a direct link between a deletion in the FMDV 3A protein and disease attenuation in cattle.

Novel Antiviral Therapeutics to Control Foot-and-Mouth Disease

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.

Role of arginine-56 within the structural protein VP3 of foot-and-mouth disease virus (FMDV) O1 Campos in virus virulence

FMDV O1 subtype undergoes antigenic variation under diverse growth conditions. Of particular interest is the amino acid variation observed at position 56 within the structural protein VP3. Selective pressures influence whether histidine (H) or arginine (R) is present at this position, ultimately influencing in vitro plaque morphology and in vivo pathogenesis in cattle. Using reverse genetics techniques, we have constructed FMDV type O1 Campos variants differing only at VP3 position 56, possessing either an H or R (O1Ca-VP3-56H and O1Ca-VP3-56R, respectively), and characterized their in vitro phenotype and virulence in the natural host. Both viruses showed similar growth kinetics in vitro. Conversely, they had distinct temperature-sensitivity (ts) and displayed significantly different pathogenic profiles in cattle and swine. O1Ca-VP3-56H was thermo stable and induced typical clinical signs of FMD, whereas O1Ca-VP3-56R presented a ts phenotype and was nonpathogenic unless VP3 position 56 reverted in vivo to either H or cysteine (C).

Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response

We previously demonstrated that an adenovirus-based foot-and-mouth disease virus (FMDV) serotype A24 capsid subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but in a similar approach using swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1C, the animals were only partially protected when challenged at 21 days post-vaccination (dpv). Recently, we demonstrated that inclusion of the complete coding region of nonstructural protein 2B in the Ad5-A24 vector resulted in improved immune responses in pigs. We also found that inclusion of a modified CMV promoter (pCI), Ad5-CI-A24-2B, enhanced the efficacy of the vector. To address the limited immunogenicity of Ad5-O1C, we have produced a new set of Ad5 vectors with the complete 2B coding region under the control of either the original or the modified version of the CMV promoter, Ad5-O1C-2B, or Ad5-CI-O1C-2B, respectively. To evaluate the potency and efficacy of the new vectors we performed 2 sets of experiments in cattle. In the first experiment we compared the original vector with vectors containing the pCI promoter and partial or full-length 2B. All groups were challenged, intradermally in the tongue, at 21 dpv with FMDV O1C. We found that in all vaccinated groups 2 of 4 animals were protected from clinical disease. In the second experiment we directly compared the efficacy of vectors with a partial or full-length 2B under the control of the original CMV promoter. While all animals in the control group developed clinical disease, 2 of 4 animals in the group receiving Ad5-O1C vaccine and 3 of 4 animals in the group receiving Ad5-O1C-2B vaccine were completely protected after challenge. We also observed a 100-fold reduction of virus shedding in Ad5-O1C vaccinated animals and the group receiving Ad5-O1C-2B had an additional 10-fold reduction compared with the Ad5-O1C vaccinated group. There was no difference in the level of neutralizing antibodies in the vaccinated groups. However, we detected a significant antigen specific-CD4(+) and CD8(+) T cell response as early as 1 day post-challenge (dpc) in both Ad5-O1C and Ad5-O1C-2B groups. Interestingly, the group receiving Ad5-O1C-2B had a statistically significant higher antigen specific-CD4(+) and CD8(+) T cell response at 5 dpc and 3 and 5 dpc, respectively, as compared to the Ad5-O1C inoculated group. These results indicate that inclusion of the complete 2B coding region improves the efficacy of Ad5 vaccines against FMDV serotype O and induces specific-CD4(+) and CD8(+) T cell responses that correlate with protection.

Pressure-inactivated FMDV: a potential vaccine

Foot-and-mouth disease virus (FMDV) is the causative agent of the foot-and-mouth disease (FMD). Alternative FMD vaccines have been pursued due to important disadvantages of the one currently in use. High hydrostatic pressure (HP) has been observed to inactivate some viruses. Here, we investigated the effects of HP on FMDV O1 Campos-Vallée (CVa) infectivity. A treatment consisting of 2.5 kbar at −15 °C and 1 M urea, completely abolished FMDV infectivity, maintaining the integrity of its capsid structure. Moreover, its ability to elicit neutralizing antibody production in rabbits was preserved. Taken together, our results suggest that HP could be a safe, simple, cheap and reproducible way for viral vaccine production.

Recombinant adenovirus co-expressing capsid proteins of two serotypes of foot-and-mouth disease virus (FMDV): in vitro characterization and induction of neutralizing antibodies against FMDV in swine

Human adenovirus type 5 (Ad5) has been evaluated as a novel gene delivery vector for the development of live-viral vaccines for foot-and-mouth disease (FMD). In this study, we constructed an Ad5 vector co-expressing the capsid precursor proteins, P1, of FMD virus (FMDV) field strains A24 Cruzeiro and O1 Campos and examined the neutralizing antibody responses in swine after inoculation with the vector. To construct the Ad5 vector, a bicistronic expression cassette containing a cytomegalovirus promoter, the P1 coding sequence of FMDV A24, the internal ribosomal entry site (IRES) of FMDV A12, the P1 coding sequence of FMDV O1 Campos and the coding region of A12 3C protease was inserted into the E1 region of an E1/E3-deleted Ad5. The recombinant adenovirus, Ad5A24+O1, was generated by transfection of 293 cells with full-length pAd5A24+O1 recombinant plasmid DNA. The recombinant Ad5 co-expressed P1 of both A24 and O1 in infected 293 cells and P1 of both serotypes was processed to produce VP0, VP3, and VP1. We further demonstrated the formation of capsid protein complexes by co-precipitation of VP0, VP3, and VP1 with monoclonal antibodies against viral capsid proteins. Swine inoculated with Ad5A24+O1 generated neutralizing antibodies against both A24 and O1. However, the overall neutralizing antibody response was considerably lower than that induced by a commercial FMD vaccine or a monovalent Ad5-A24 vaccine.

Effective synthetic peptide vaccine for foot-and-mouth disease in swine

We have designed a peptide-based vaccine for foot-and-mouth disease (FMD) effective in swine. The peptide immunogen has a G–H loop domain from the VP1 capsid protein of foot-and-mouth disease virus (FMDV) and a novel promiscuous T helper (Th) site for broad immunogenicity in multiple species. The G–H loop VP1 site was optimised for cross-reactivity to FMDV by the inclusion into the peptide of cyclic constraint and adjoining sequences. The incorporation of consensus residues into the hypervariable positions of the VP1 site provided for broad immunogenicity. The vaccine protected 20 out of 21 immunised pigs from infectious challenge by FMDV O1 Taiwan using peptide doses as low as 12.5 μg, and a mild adjuvant that caused no lesions. A safe chemically-defined product would have considerable advantages for vaccination against FMD.

Detection and Characterization of Functional T-Cell Epitopes on the Structural Proteins VP2, VP3, and VP4 of Foot and Mouth Disease Virus O1 Campos

Foot and mouth disease virus (FMDV) is the cause of a widespread infectious disease affecting cloven-hoofed animals. It is controlled by vaccination with immune-inactivated virus grown in tissue culture. However, peptide vaccines represent a safer alternative to the current virus-inactivated immunogens. Their design requires the identification and evaluation of the sequences recognized by T- and B-lymphocytes. Four structural proteins, VP1, VP2, VP3, and VP4, comprise the viral capsid of the FMDV, but only VP1 has been extensively studied regarding the existence of relevant T-cell epitopes. Here, we utilize a murine model to present a functional T-cell epitope mapping on the complete sequences of VP2, VP3, and VP4 of FMDV O1 Campos. We used two in vitro assays to describe 13 amino acid sequences, each one of them including at least one T-cell epitope. The in vivo T-cell helper function of these sequences was studied in an adoptive cell-transfer assay in mice. Immunization experiments with a fusion peptide containing one of the sequences characterized were also done comparing the helper activity of this sequence with other T-cell epitopes included in the major immunogenic region of VP1.

Improvement of the immune response to foot and mouth disease virus vaccine in calves by using Avridine as adjuvant

The epidemiological analysis of the cattle population during the eradication plan of foot and mouth disease (FMD) in Argentina clearly indicated a higher incidence of the disease in animals within their first year of age. It is important to improve the efficacy of the vaccination in those animals. In a previous report, we have shown the effect of an immunomodulator, Avridine (Avr), in the enhancement of the immune response elicited by FMD virus (FMDV) vaccines in experimental hosts [Berinstein, A., Pérez Filgueira, M., Schudel, A., Zamorano, P., Borca, M., Sadir, A.M., 1993. Avridine and LPS from Brucella ovis: effect on the memory induced by foot-and-mouth disease virus vaccination in mice. Vaccine 11, 1295–1301]. In this report, we analyze the effect of Avr in the improvement of the anti-FMDV immune response elicited in young animals immunized with a tetravalent vaccine. The anti-FMDV antibody response was evaluated using a liquid-phase blocking sandwich ELISA (LPBE) [Smitsaart, E.N., Zanelli, M., Rivera, I., Fondevila, N., Compaired, D., Maradei, E., Bianchi, T., O'Donnell, V., Schudel, A.A., 1998. Assessment using ELISA of the herd immunity levels induced in cattle by foot and mouth disease oil vaccines. Prev. Vet. Med 33, 283–296] while the cellular response was detected using an antigen specific lymphoproliferative test [Zamorano, P., Wigdorovitz, A., Chaher, M., Fernández, F., Sadir, A., Borca, M., 1994. Localization of B and T cell epitopes on a synthetic peptide containing the major immunogenic site of FMDV O1 Campos. Virology 201, 383–387]. The results show that, while no differences were detected in the cellular response, the anti-FMDV antibody reaction was significantly (<0.05) higher in animals immunized with the immunogen containing Avr. At 90 days post vaccination, 89–100% of the animals immunized with Avr presented predicted protection (PP) higher than 82% while just 50–61% of the animals immunized with vaccine without immunomodulator presented that characteristic. Also, it is shown that the increase in the anti-FMDV antibody titre in animals immunized with the vaccine containing Avr was mediated by an increase in the levels of both IgG1 and IgG2 which presented a significative correlation with LPELISA antibodies titres. It is concluded that the addition of Avr in the FMDV vaccines improve the immune status of the calves, the cattle population that suffers the highest epidemiological risk.

Nucleotide sequence of the P1 region of foot-and-mouth disease virus strain O1 Caseros.

It has been shown that variation of antigenic site I in VP1 of foot-and-mouth disease virus (FMDV) plays an important role in the antigenic diversification of this virus. However, the O1 Campos strain is able to efficiently cross-protect cattle against the O1 Caseros strain, despite having a different sequence in the site I. In this paper we report and compare the P1 coding region for the capsid proteins of FMDV O1 Caseros and O1 Campos. The deduced amino acid sequence showed a total of 31 amino acid differences. Eight of them are located in surface-exposed loops that have been implicated in antigenic sites. This study should help to identify additional sites to be considered in the development of a new generation of FMDV vaccines.

A 10-Amino-Acid Linear Sequence of VP1 of Foot and Mouth Disease Virus Containing B- and T-Cell Epitopes Induces Protection in Mice

The area of foot and mouth disease virus (FMDV) comprising residues 140 and 160 of capsid protein VP1 has been used extensively as an immunogen in natural and experimental hosts. A detailed epitope mapping of this region, however, has not been reported. For this purpose a synthetic peptide containing the residues 135 to 160 (p135-160) of VP1 of FMDV O1 Campos was analyzed for its T- and B-cell epitopes. The p135-160 is highly immunogenic, either by itself or coupled to a carrier protein (BSA), elicits a long-lasting neutralizing antibody response in mice, and provides solid protection against virulent challenge. By using a set of synthetic 10mer overlapping peptides, which cover the entire sequence 135-160 of VP1, we have shown that at feast four discrete B epitopes are regularly distributed along the peptide, Although immunization with each of the 10mers coupled with BSA as a carrier protein induced peptide-specific antibody responses, individually none of the 10mers was able to induce neutralizing antibodies. However, anti-135-160 antibodies sorted by immunoaffinity chromatography using each of the 10mers revealed the existence of at least four discrete neutralizing sites: one spanning residues 135-144, at least two more between residues 140 and 154, and another in the region 150-160. Moreover, T-cell epitopes were identified, both by antigen-dependent proliferation assays and by adoptive cell transfer. By both methods, a T-cell epitope was located in the area comprising residues 135-144; the cell transfer experiment, which seems to be more sensitive, also identified a second T-cell epitope between residues 150 and 160. Interestingly, when the region 135-144, which contains both B- and T-cell epitopes, was in a tandem repeat configuration it induced a strong neutralizing antibody response in mice and solid protection against the challenge.

Chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies against FMDV in guinea pigs

Five poliovirus recombinants containing sequences corresponding to foot-and-mouth disease virus (FMDV) antigenic sites were constructed. Viable virus was recovered from four of these plasmids, in which the VP1 beta B-beta C loop (antigenic site 1) of poliovirus type 1 Sabin had been replaced with sequences derived from the VP1 beta G-beta H loop (antigenic site 1) of FMDV O1 Kaufbeuren (O1K), chimera O1.1 (residues 141 to 154), chimera O1.2 (residues 147 to 156), and chimera O1.3 (residues 140 to 160) or from the beta B-beta C loop of VP1 (antigenic site 3) in chimera O3.1 (residues 40 to 49). One chimera (O1.3) was neutralized by FMDV-specific polyclonal serum and monoclonal antibodies directed against antigenic site 1 of FMDV. Chimeras O1.3 and O3.1 induced site-specific FMDV-neutralizing antibodies in guinea pigs. Chimera O1.3 was capable of inducing a protective response against FMDV challenge in some guinea pigs.

Protection conferred by TrpE fusion proteins containing portions of the C-terminal region of capsid protein VP1 of foot-and-mouth disease virus

Major immunogenic sites of foot-and-mouth disease virus (FMDV) have been mapped to the C-terminal third of capsid protein VP1; we studied the immunogenicity of a series of TrpE-FMDV fusion proteins containing this region of FMDV strain O1 Campos. Fusion protein TrpE-dCN, which contains a dimer of VP1 amino acid sequences consisting of amino acids 200 to 213 linked by a diproline spacer to amino acids 141 to 158 (200-213 approximately P-P-G approximately 141-158), induced the best response. A single inoculation of guinea-pigs with 100 micrograms TrpE-dCN elicited high levels of neutralizing antibodies and protected all the animals against challenge infection with homologous virus. Although the closely related FMDV strains O1 Campos and O1 Caseros induced high levels of cross-protection, TrpE-dCN-vaccinated guinea-pigs were poorly protected against challenge infection with heterologous FMDV strain O1 Caseros. Nucleotide sequence analysis revealed that amino acid differences at residues 149 and 152 were critical for the induction of cross-protection and that neutralizing epitopes not present in TrpE-dCN are likely to be responsible for conferring a high level of cross-protection between FMDV strains O1 Campos and O1 Caseros.

A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C II. Application

The complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was evaluated to detect antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. Log 10 serum titres of uninfected, unvaccinated cattle ( n = 100) were less than 1.80 in the CTB-ELISA. Sera from cattle vaccinated with either monovalent or trivalent vaccines were tested in both the CTB-ELISA and the serum neutralisation test (SNT); titres in both tests correlated positively ( P < 0.001). Titres of sera from cattle, sheep, and pigs vaccinated twice with FMDV A10 Holland also correlated positively in both tests. In another experiment, cattle vaccinated with FMDV strain C1 Detmold were intradermolingually challenged 3 weeks after primary vaccination; at the same time two controls were challenged. At 8 days after challenge, serum titres of the controls were distinctly higher in the CTB-ELISA than in the SNT, whereas serum titres of the vaccinated cattle were equally high in both tests. In potency tests for monovalent vaccines against FMDV strains A10 Holland, O1 BFS or C1 Detmold, serum titres correlated strongly in both tests with protection against the homologous FMDV strain. We concluded that the CTB-ELISA is not only sensitive, but easier to perform and more rapid and reproducible than the SNT. The CTB-ELISA may be useful in evaluating the immune response in cattle during FMD vaccine potency tests.

Analysis of Foot-and-Mouth Disease Virus Type O1 Brugge Neutralization Epitopes Using Monoclonal Antibodies

Monoclonal antibodies (MAbs) were elicited with inactivated, purified foot-and-mouth disease virus (FMDV) type O1 strain Brugge (140S) and with 12S protein subunits. Each MAb was tested for its capacity to bind to FMDV O1 Brugge 140S virions, 12S subunits and purified VP1 by radioimmunoassay (RIA) and to neutralize viral infectivity in mouse protection assays. Those MAbs which reacted only with 12S subunits in RIA did not neutralize infectious virus. One MAb, 12FE9.2.1, reacted with 140S, 12S and purified VP1 of FMDV O1 Brugge and neutralized infectious virus. Reactions with different biosynthetic FMDV type O1 Campos VP1 polypeptides localized the binding site of 12FE9.2.1 between amino acid residues 135 and 172. Eight MAbs reacted with both 140S virions and 12S subunits and neutralized infectious virus. Monoclonal antibodies that reacted only with 12S protein subunits or with 140S, 12S and VP1 did not compete in RIA with 140S/12S-reactive neutralizing MAbs for 12S binding sites. The ability of 140S/12S-reactive MAbs to compete for the 12S binding site defined by MAb 10GA4.2.2 was directly related to their capacity to neutralize infectious virus, suggesting that these MAbs were all affecting a single neutralization site. In mouse protection and plaque reduction neutralization assays, 10GA4.2.2 and 12FE9.2.1 were equally effective in neutralizing the homologous FMDV type O1 Brugge, and in cross-neutralization assays both exhibited high titres against four additional strains of type O1 FMDV.