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 (3C^pro) 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 3C^pro inhibition with IC50 values of 10.85 µM (NSC116640) and 4.21 µM (NSC332670). The molecular docking of the compounds on FMDV 3C^pro showed their specific interactions with amino acids in the catalytic triad of FMDV 3C^pro. 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.

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 3D^pol, 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 3D^pol 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 3D^pol. The predicted protein–ligand interaction confirmed that these potential terpenoids interacted with the main catalytic and bystander residues of FMDV 3D^pol. 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.

Foot-and-Mouth Disease (FMD) is a contagious, blistering disease caused by the Foot-and-Mouth Disease virus (FMDV), which affects livestock globally. Currently, no commercial antiviral agent is available for effective disease control. This study investigated the antiviral potential of natural-derived alkaloids against FMDV in BHK-21 cells. Twelve alkaloids were assessed for their antiviral activities at various stages of FMDV infection, including pre-viral entry, post-viral entry, and prophylactic assays, as well as attachment and penetration assays by evaluating cytopathic effect reduction and directed-virucidal effects. The results showed that ipecac alkaloids, cephaeline (CPL) and emetine (EMT), exhibited dual effects with robust antiviral efficacy by reducing cytopathic effect and inhibiting FMDV replication in a dose-dependent manner. Evaluation through immunoperoxidase monolayer assay and RT-PCR indicated effectiveness at post-viral entry stage, with sub-micromolar EC₅₀ values for CPL and EMT at 0.05 and 0.24 µM, respectively, and high selective indices. Prophylactic effects prevented infection with EC₅₀ values of 0.23 and 0.64 µM, respectively. Directed-virucidal effects demonstrated significant reduction of extracellular FMDV, with CPL exhibiting a dose-dependent effect. Furthermore, the replicase (3Dpol) inhibition activity was identified using the FMDV minigenome assay, which revealed strong inhibition with IC₅₀ values of 0.15 µM for CPL and 4.20 µM for EMT, consistent with the decreased negative-stranded RNA production. Molecular docking confirmed the interaction of CPL and EMT with residues in the active site of FMDV 3Dpol. In conclusion, CPL and EMT exhibited promising efficacy through their dual effects and provide an alternative approach for controlling FMD in livestock.

Feline infectious peritonitis virus (FIPV), caused by a mutated form of feline coronavirus, poses a significant threat to feline health worldwide, with limited therapeutic options available. This study investigated the antiviral potential of α-mangostin (α-MG) and its enriched extracts (AMEs), obtained via microwave-assisted extraction, against FIPV. We evaluated their cytotoxicity, direct virucidal activity, and antiviral activity in CRFK cells. Both α-MG and AMEs demonstrated significant antiviral activity, with EC₅₀ values from 2.71 to 2.88 μg/mL and favorable selectivity indices (3.25–3.66). Notably, AMEs exhibited direct virucidal effects, effectively reducing viral titers. Furthermore, treatment with these compounds significantly reduced inflammatory cytokine expression (IFN-β, TNF-α, and IL-6 mRNA levels) and decreased viral loads in FIPV-infected cells. Drug combination studies using the ZIP model revealed enhanced cooperative effects when AMEs and α-MG were combined with GC-376 or GS-441524, with GC-376 combinations showing particularly strong synergistic potential. These findings suggest that α-MG and AMEs are promising candidates for FIPV treatment, either as monotherapy or in combination therapy. This study provides insights into developing novel therapeutic strategies to combat FIPV infections and offers a foundation for future veterinary antiviral drug development.

Foot-and-mouth disease (FMD) is a highly contagious viral infection affecting livestock. Although inactivated vaccines are commonly used, their effectiveness is limited by an immunity gap. Therefore, complementary antiviral strategies are required for effective control and prevention. Lignans, plant-derived compounds, have shown promising antiviral properties, yet their potential against foot-and-mouth disease virus (FMDV) remains underexplored. This study employed virtual screening to identify lignan compounds targeting viral RNA-dependent RNA polymerase (3D^pol). Six lignan compounds were selected for cytotoxicity and antiviral activity evaluation including pre-viral entry, post-viral entry, and protective effect assays. Antiviral activity assay showed that (-)-asarinin and sesamin exhibit potent inhibition effects in the post-viral entry with EC50 of 15.11 μM and 52.98 μM, respectively, using immunoperoxidase monolayer assay. Both compounds exhibited dose-dependent reduction in viral replication with significant suppression of negative-strand RNA production. Lignans’ ability to target FMDV 3D^pol was further confirmed using a cell-based FMDV minigenome assay. Among the tested lignans, (-)-asarinin demonstrated remarkable inhibition of GFP expression (IC50 value at 10.37 μM), while sesamin required a higher concentration for similar effects. In silico prediction revealed that these lignans preferentially bind to FMDV 3D^pol active site. These findings are the first to establish (-)-asarinin and sesamin as promising antiviral candidates against FMDV.

Feline infectious peritonitis virus (FIPV) causes a fatal disease in cats that remains challenging to treat due to the absence of effective vaccines and limited antiviral therapies. Given its central role in viral replication and transcription, RNA-dependent RNA polymerase (RdRp) represents a highly promising and strategic target for antiviral drug development. To enable live-cell monitoring of RdRp activity without external substrates or cell disruption, we developed a biologically validated, cell-based dual-fluorescent reporter assay using green fluorescent protein and red fluorescent protein to evaluate RdRp-dependent reporter activity. Following validation with the well-characterized RdRp inhibitor ribavirin, the dual-reporter platform achieved a Z-factor of 0.54 and a Z′-factor of 0.61. The system was further utilized to assess the antiviral potential of fifteen alkaloid compounds. Among compounds evaluated, lycorine and lycorine hydrochloride exhibited potent inhibition of RdRp-dependent reporter activity and yielded IC₅₀ values of 2.83 ± 0.45 μM and 1.58 ± 0.20 μM, respectively. Further evaluation revealed that both compounds significantly reduced FIPV replication in infected cell. These findings identify lycorine and lycorine hydrochloride as promising anti-FIPV candidates and support further investigation of lycorine-class alkaloids as anti-coronaviral scaffolds. Together, the validated cell-based screening platform and whole-virus antiviral activity provide a foundation for advancing RdRp-targeted antiviral discovery against FIPV.