Foot and mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals. This disease is one of the most important in animal health due to its significant socio-economic impact, especially in case of an outbreak. One important challenge associated with this disease is the ability of the FMD virus (FMDV) to persist in its hosts through still unresolved underlying mechanisms. The absence of relevant in vitro models is one factor preventing advancement in our understanding of FMDV persistence. While a primary bovine cell model has been established using cells from FMDV primary and persistence site in cattle, it appeared interesting to develop a similar model based on ovine anatomical sites of interest to compare host-pathogen interactions. Thus, epithelial cells derived from the palatine tonsils and the dorsal soft palate were isolated and cultured. Their epithelial nature was confirmed using immunofluorescence. Following monolayer infection with FMDV O/FRA/1/2001 Clone 2.2, the FMDV-sensitivity of these cells was evaluated. Dorsal soft palate (DSP) cells were also expanded in multilayers at the air-liquid interface to mimic a stratified epithelium sensitive to FMDV infection. Our investigation revealed the presence of infectious virus, as well as viral antigens and viral RNA, up to 35 days after infection of the cell multilayers. Further experiment with DSP cells from different individuals needs to be reproduced to confirm the robustness of the new model of persistence in multilayer DSP. The establishment of such primary cells creates new opportunities for FMDV research and analysis in sheep cells.

Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is an important pathogen affecting cloven-hoof livestock. However, neither effective vaccines covering all serotypes nor specific antivirals against FMDV infections are currently available. In this study, we employed virtual screening to screen for secondary metabolite terpenoids targeting the RNA-dependent RNA polymerase (RdRp), or 3Dpol, of FMDV. Subsequently, we identified the potential antiviral activity of the 32 top-ranked terpenoids, revealing that continentalic acid, dehydroabietic acid (abietic diterpenoids), brusatol, bruceine D, and bruceine E (tetracyclic triterpenoids) significantly reduced cytopathic effects and viral infection in the terpenoid-treated, FMDV-infected BHK-21 cells in a dose-dependent manner, with nanomolar to low micromolar levels. The FMDV minigenome assay demonstrated that brusatol and bruceine D, in particular, effectively blocked FMDV 3Dpol activity, exhibiting IC50 values in the range of 0.37-0.39 µM and surpassing the efficacy of the antiviral drug control, ribavirin. Continentalic acid and bruceine E exhibited moderate inhibition of FMDV 3Dpol. The predicted protein-ligand interaction confirmed that these potential terpenoids interacted with the main catalytic and bystander residues of FMDV 3Dpol. Additionally, brusatol and bruceine D exhibited additive effects when combined with ribavirin. In conclusion, terpenoids from natural resources show promise for the development of anti-FMD agents.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140-158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines.
RESULTS: This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs.
CONCLUSIONS: The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

The internal ribosome entry site (IRES) element constitutes a cis-acting RNA regulatory sequence that recruits the ribosomal initiation complex in a cap-independent manner, assisted by various RNA-binding proteins and IRES trans-acting factors. Foot-and-mouth disease virus (FMDV) contains a functional IRES element and takes advantage of this element to subvert host translation machinery. Our study identified a novel mechanism wherein RALY, a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family belonging to RNA-binding proteins, binds to the domain 3 of FMDV IRES via its RNA recognition motif residue. This interaction results in the downregulation of FMDV replication by inhibiting IRES-driven translation. Furthermore, our findings reveal that the inhibitory effect exerted by RALY on FMDV replication is not attributed to the FMDV IRES-mediated assembly of translation initiation complexes but rather to the impediment of 80S ribosome complex formation after binding with 40S ribosomes. Conversely, 3Cpro of FMDV counteracts RALY-mediated inhibition by the ubiquitin-proteasome pathway. Therefore, these results indicate that RALY, as a novel critical IRES-binding protein, inhibits FMDV replication by blocking the formation of 80S ribosome, providing a deeper understanding of how viruses recruit and manipulate host factors.
OBJECTIVE: The translation of FMDV genomic RNA driven by IRES element is a crucial step for virus infections. Many host proteins are hijacked to regulate FMDV IRES-dependent translation, but the regulatory mechanism remains unknown. Here, we report for the first time that cellular RALY specifically interacts with the IRES of FMDV and negatively regulates viral replication by blocking 80S ribosome assembly on FMDV IRES. Conversely, RALY-mediated inhibition is antagonized by the viral 3C protease by the ubiquitin-proteasome pathway. These results would facilitate further understanding of virus-host interactions and translational control during viral infection.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals that has a significant socio-economic impact. One concern associated with this disease is the ability of its etiological agent, the FMD virus (FMDV), to persist in its hosts through underlying mechanisms that remain to be elucidated. While persistence has been described in cattle and small ruminants, it is unlikely to occur in pigs. One of the factors limiting the progress in understanding FMDV persistence and, in particular, differential persistence is the lack of suitable in vitro models. A primary bovine cell model derived from the dorsal soft palate, which is the primary site of replication and persistence of FMDV in cattle, has been developed, and it seemed relevant to develop a similar porcine model. Cells from two sites of FMDV replication in pigs, namely, the dorsal soft palate and the oropharyngeal tonsils, were isolated and cultured. The epithelial character of the cells from the dorsal soft palate was then assessed by immunofluorescence. The FMDV-sensitivity of these cells was assessed after monolayer infection with FMDV O/FRA/1/2001 Clone 2.2. These cells were also grown in multilayers at the air-liquid interface to mimic a stratified epithelium susceptible to FMDV infection. Consistent with what has been shown in vivo in pigs, our study showed no evidence of persistence of FMDV in either the monolayer or multilayer model, with no infectious virus detected 28 days after infection. The development of such a model opens up new possibilities for the study and diagnosis of FMDV in porcine cells.

Foot-and-mouth disease (FMD) is a highly contagious disease in cloven-hoofed animals, caused by the foot-and-mouth disease virus (FMDV). It is endemic in Asia and Africa but spreads sporadically throughout the world, resulting in significant losses in the livestock industry. Effective anti-FMDV therapeutics could be a supportive control strategy. Herein, we utilized computer-aided, structure-based virtual screening to filter lead compounds from the National Cancer Institute (NCI) diversity and mechanical libraries using FMDV 3C protease (3Cpro) as the target. Seven hit compounds were further examined via cell-based antiviral and intracellular protease assays, in which two compounds (NSC116640 and NSC332670) strongly inhibited FMDV, with EC50 values at the micromolar level of 2.88 µM (SI = 73.15) and 5.92 µM (SI = 11.11), respectively. These compounds could inactivate extracellular virus directly in a virucidal assay by reducing 1.00 to 2.27 log TCID50 of the viral titers in 0-60 min. In addition, the time-of-addition assay revealed that NSC116640 inhibited FMDV at the early stage of infection (0-8 h), while NSC332670 diminished virus titers when added simultaneously at infection (0 h). Both compounds showed good FMDV 3Cpro inhibition with IC50 values of 10.85 µM (NSC116640) and 4.21 µM (NSC332670). The molecular docking of the compounds on FMDV 3Cpro showed their specific interactions with amino acids in the catalytic triad of FMDV 3Cpro. Both preferentially reacted with enzymes and proteases in physicochemical and ADME analysis studies. The results revealed two novel small molecules with antiviral activities against FMDV and probably related picornaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants.

Foot-and-mouth disease (FMD) is a highly contagious viral disease affecting cloven-hoofed animals. One of the issues related to this disease is the persistence of its causative agent, foot-and-mouth disease virus (FMDV). While the mechanisms of FMDV persistence remain unclear, there are clues that it may be related to protein-protein interactions (PPI) between viral proteins and cellular proteins involved in the interferon (IFN) response. Since FMDV persistence has been described in cattle, sheep and goats but not in swine, we screened PPI involving FMDV proteins and sixteen major type-I IFN pathway proteins from these four species by nanoluciferase-2-hybrid complementation assay, in order to identify new PPI and determine their host specificity. As the results concerning the 3Dpol were the most interesting in view of the limited data concerning its role in immune escape, we decided to focus particularly on this protein. The identified PPI were confirmed by GST pull-down. We identified PPI between 3Dpol and seven IFN pathway proteins, namely, IKKα, IKKε, IRF3, IRF7, NEMO, MDA5 and MAVS. These PPI are conserved among the four studied species, with the exception of the one between 3Dpol and MAVS, which was only found with the swine protein. We also showed, using luciferase reporter assays, that 3Dpol could inhibit the induction phase of the IFN pathway. These results demonstrate, for the first time, a putative role for 3Dpol in FMDV innate immune escape.

Foot-and-mouth disease virus (FMDV) has developed various strategies to antagonize the host innate immunity. FMDV Lpro and 3Cpro interfere with type I IFNs through different mechanisms. The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γ signaling transduction. Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown. In this study, it was shown that FMDV replication was resistant to IFN-γ treatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes (ISGs). We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation. 3Cpro expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences (GAS) promoter activity, without affecting the protein level, tyrosine phosphorylation, and homodimerization of STAT1. Finally, we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities. Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.