Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus.

Antibodies to the foot-and-mouth disease virus (FMDV) capsid induced by infection or vaccination can provide serotype-specific protection and be measured using virus neutralization tests and viral structural-protein (SP-)ELISAs. Separate tests are needed for each serotype, but cross-serotype reactions complicate serotyping. In this study, inter-serotypic responses were quantified for five SP-ELISA formats by testing 294 monovalent mainly bovine sera collected following infection, vaccination, or vaccination and infection with one of five serotypes of FMDV. Over half of the samples, representing all three immunization categories, scored positive for at least one heterologous serotype and some scored positive for all serotypes tested. A comparative approach to identifying the strongest reaction amongst serotypes O, A and Asia 1 improved the accuracy of serotyping to 73-100% depending on the serotype and test system, but this method will be undermined where animals have been infected and/or vaccinated with multiple FMDV serotypes. Preliminary studies with stabilized recombinant capsid antigens of serotypes O and A that do not expose internal epitopes showed reduced cross-reactivity, supporting the hypothesis that capsid integrity can affect the serotype-specificity of the SP-ELISAs. The residual cross-reactivity associated with capsid surface epitopes was consistent with the evidence of cross-serotype virus neutralization.

Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry.

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus.

Combining a Universal Capture Ligand and Pan-Serotype Monoclonal Antibody to Develop a Pan-Serotype Lateral Flow Strip Test for Foot-and-Mouth Disease Virus Detection.

Foot-and-mouth disease virus (FMDV) causes FMD, a highly contagious disease of cloven-hoofed animals including cattle, goats, pigs and sheep. Rapid detection of FMDV is critical to limit the devastating economic losses due to FMD. Current laboratory methods for FMDV detection such as virus isolation, real-time reverse transcription PCR and antigen detection enzyme-linked immunosorbent assay (AgELISA) are labor-intensive, requiring trained personnel and specialized equipment. We present the development and validation of a pan-serotype lateral flow strip test (LFST) that uses recombinant bovine integrin αvß6 as a universal capture ligand and a pan-serotype monoclonal antibody (mAb) to detect FMDV. The LFST detected all seven FMDV serotypes, where the diagnostic sensitivity was comparable to the AgELISA, and the diagnostic specificity was 100% without cross-reactivity to other viruses causing vesicular disease in livestock. This rapid test will be useful for on-site FMDV detection, as well as in laboratories in endemic countries where laboratory resources are limited.

Novel Capsid-Specific Single-Domain Antibodies with Broad Foot-and-Mouth Disease Strain Recognition Reveal Differences in Antigenicity of Virions, Empty Capsids, and Virus-Like Particles.

Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs.

Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold.

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19.

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays.

Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.

Correction: A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles.

[This corrects the article DOI: 10.1371/journal.pone.0157435.].

Truncated Bovine Integrin Alpha-v/Beta-6 as a Universal Capture Ligand for FMD Diagnosis.

Foot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV) serotype-specific antibodies in small animals (rabbits and guinea pigs) that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvß6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvß6 and FMDV empty capsids in FMD diagnostic assays.

A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles.

Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvß6 (a cellular receptor for FMDV). In cattle vaccinated with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge, they were protected against disease and no FMDV RNA was detected in their sera post-challenge. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed. This prime-boost system, using reagents that can be generated outside of high-containment facilities, offers significant advantages to achieve control of FMD by vaccination.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design.

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

A role for endoplasmic reticulum exit sites in foot-and-mouth disease virus infection.

Picornaviruses replicate their genomes in association with cellular membranes. While enteroviruses are believed to utilize membranes of the early secretory pathway, the origin of the membranes used by foot-and-mouth disease virus (FMDV) for replication are unknown. Secretory-vesicle traffic through the early secretory pathway is mediated by the sequential acquisition of two distinct membrane coat complexes, COPII and COPI, and requires the coordinated actions of Sar1, Arf1 and Rab proteins. Sar1 is essential for generating COPII vesicles at endoplasmic reticulum (ER) exit sites (ERESs), while Arf1 and Rab1 are required for subsequent vesicle transport by COPI vesicles. In the present study, we have provided evidence that FMDV requires pre-Golgi membranes of the early secretory pathway for infection. Small interfering RNA depletion of Sar1 or expression of a dominant-negative (DN) mutant of Sar1a inhibited FMDV infection. In contrast, a dominant-active mutant of Sar1a, which allowed COPII vesicle formation but inhibited the secretory pathway by stabilizing COPII coats, caused major disruption to the ER-Golgi intermediate compartment (ERGIC) but did not inhibit infection. Treatment of cells with brefeldin A, or expression of DN mutants of Arf1 and Rab1a, disrupted the Golgi and enhanced FMDV infection. These results show that reagents that block the early secretory pathway at ERESs have an inhibitory effect on FMDV infection, while reagents that block the early secretory pathway immediately after ER exit but before the ERGIC and Golgi make infection more favourable. Together, these observations argue for a role for Sar1 in FMDV infection and that initial virus replication takes place on membranes that are formed at ERESs.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen.

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity.

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.

Foot-and-mouth disease virus induces autophagosomes during cell entry via a class III phosphatidylinositol 3-kinase-independent pathway.

Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ∼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.

Integrin sub-unit expression in cell cultures used for the diagnosis of foot-and-mouth disease.

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 αVß6 and αVß8. In contrast, cell surface expression of αVß3 or mRNA for the ß1, ß3 or ß5 subunits did not appear to contribute to sensitivity of cells to FMDV. These findings confirm the requirement for αV6 and αVß8 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.

Foot-and-mouth disease virus forms a highly stable, EDTA-resistant complex with its principal receptor, integrin alphavbeta6: implications for infectiousness.

The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin alphavbeta6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to alphavbeta6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin alphavbeta6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.

Guinea pig-adapted foot-and-mouth disease virus with altered receptor recognition can productively infect a natural host.

We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I(248)-->T in 2C, Q(44)-->R in 3A, and L(147)-->P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L(147)-->P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L(147)-->P, and this infection was inhibited by antibodies to alphavbeta6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin alphavbeta6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T(248)-->N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species.

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins.

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.