ABSTRACT Foot-and-mouth disease (FMD) causes severe global economic loss, necessitating rapid viral characterization. Nanopore sequencing provides a simple, real-time workflow suitable for on-site outbreak response, addressing the limitations of conventional methods. In this study, we optimized a previously published amplicon-based protocol and used this method to characterize a diverse range of samples (vesicular fluid, epithelium, serum, nasal/oral swabs, and environmental samples) collected during FMD outbreaks in 2025 in the Republic of Korea. Of the 129 samples collected, we successfully recovered complete genomes from 37 samples and VP1 sequences from 85 samples. Amplifying the S-fragment in isolation and separately barcoding each pool of PCR amplicons markedly improved sequence recovery. Furthermore, sequencing success depended on viral load and sample type. Based on comparisons with real-time RT-PCR results, whole-genome sequence (WGS) recovery exceeded 77.3% at cycle threshold (Ct) values ≤25 across all clinical samples. In the Ct > 30 category, serum samples yielded the highest WGS recovery rates (44.4%). This rate was markedly higher than the success rates observed for epithelium (20.0%) and nasal swabs (9.1%), whereas oral swabs and environmental samples failed to yield any sequences (0%). However, VP1 recovery from environmental samples reached 80% at Ct ≤ 30 (8/10), providing an approach to enable non-invasive monitoring. These findings demonstrate that amplicon-based nanopore sequencing is a practical method for the rapid generation of genomic data during FMD outbreaks. IMPORTANCE Although rapid detection and genomic data analysis are crucial for effective foot-and-mouth disease (FMD) control, the collection of these data can be challenging for certain sample types and impacted by reduced viral loads that result from nationwide FMD vaccination. This study provides a practical solution through large-scale evaluation of an optimized amplicon-based nanopore sequencing protocol to enhance the sequencing success rates for both clinical and environmental samples. Using a modified protocol to enhance genome recovery, we demonstrated that sequence data could be retrieved from diverse sample types (even with high real-time RT-PCR cycle threshold values). We identified serum as the most suitable sample, with environmental sample sequencing allowing for non-invasive monitoring during outbreaks. These results support the use of nanopore sequencing for rapid genomic analysis, particularly in outbreak responses, such as rapid surveillance, emergency vaccine selection, and epidemiological monitoring.

Background/Objectives: Foot-and-mouth disease (FMD) remains a significant threat to livestock, particularly in the pool 1 region (East Asia), where serotype A is prevalent. Vaccination is the most effective control measure, and the selection of the appropriate vaccine strain is critical for ensuring effective protection. The A/ASIA/Sea-97 lineage (and its G1 and G2 sublineages) has been reported in this region, necessitating the development of an appropriate vaccine. This study aimed to develop a potent candidate vaccine strain capable of providing effective protection against the G1 and G2 sublineages of the A/ASIA/Sea-97 lineage. Methods: Chimeric vaccine development was achieved by replacing and inserting antigenic sites derived from the A/ASIA/Sea-97 G1 (VP4, VP2, and VP3) and G2 sublineage (VP1 and GH loop) strains. The candidate strains were evaluated for protective efficacy in mice and pigs. Results: In mice, the two candidate vaccines provided strong protection against challenge with a G1 sublineage virus (A/POC/2010) and A22 Iraq and two G2 sublineage viruses (A/YC/2017 and A/GP/2018). Subsequently, the most effective candidate was selected for testing in pigs. One month after vaccination, the pigs were protected against two A/ASIA/Sea-97 viruses (A/POC/2010 and A/GP/2018) prevalent in East Asia. Conclusions: These results demonstrate that the developed strain has significant potential as a vaccine against the type A FMD viruses circulating in East Asia and that vaccination with this strain could be an effective strategy for regional FMD control.

Small interfering RNAs are typically used to block gene expression via the sequence-specific degradation of mRNA. We previously developed a recombinant adenovirus expressing three short hairpin RNAs, targeting foot-and-mouth disease virus (FMDV) driven by the U6 promoter (Ad-3siRNA). These exhibited rapid antiviral effects against FMDV. Herein, we investigated the immunostimulatory effects of Ad-3siRNA in combination with an FMD inactivated vaccine. Ad-3siRNA induced various cytokines, including type I interferon, and upregulated interferon-stimulated genes in swine cells. Using the combination of Ad-3siRNA and a foot-and-mouth disease (FMD) inactivated vaccine, we observed enhanced protection efficacy until 1 week post-vaccination and enhanced neutralizing antibody levels from 1 week post-vaccination in mice. Furthermore, humoral immunity was significantly enhanced in pigs injected with a vaccine and a combination of Ad-3siRNA for 7 weeks post-vaccination. Ad-3siRNA induced various cytokines, such as IFN-α, IFN-γ, IL-12, IL-6, IL-15 and IL-18, in mice. In conclusion, we demonstrated that Ad-3siRNA, a novel immunostimulatory RNA delivered by a human adenoviral vector, could enhance protection and humoral immunity in combination with an FMD-inactivated vaccine. Exploring the applications of Ad-3siRNAs against other viral diseases and further detailed mechanistic studies are warranted.

IntroductionConventional foot-and-mouth disease (FMD) vaccines have been developed to enhance their effectiveness; however, several drawbacks remain, such as slow induction of antibody titers, short-lived immune response, and local side effects at the vaccination site. Therefore, we created a novel FMD vaccine that simultaneously induces cellular and humoral immune responses using the Dectin-2 agonist, D-galacto-D-mannan, as an adjuvant.MethodsWe evaluated the innate and adaptive (cellular and humoral) immune responses elicited by the novel FMD vaccine and elucidated the signaling pathway involved both in vitro and in vivo using mice and pigs, as well as immune cells derived from these animals.ResultsD-galacto-D-mannan elicited early, mid-, and long-term immunity via simultaneous induction of cellular and humoral immune responses by promoting the expression of immunoregulatory molecules. D-galacto-D-mannan also enhanced the immune response and coordinated vaccine-mediated immune response by suppressing genes associated with excessive inflammatory responses, such as nuclear factor kappa B, via Sirtuin 1 expression.ConclusionOur findings elucidated the immunological mechanisms induced by D-galacto-D-mannan, suggesting a background for the robust cellular and humoral immune responses induced by FMD vaccines containing D-galacto-D-mannan. Our study will help to facilitate the improvement of conventional FMD vaccines and the design of next-generation FMD vaccines.

BackgroundCommercial foot-and-mouth disease (FMD) vaccines have limitations, such as local side effects, periodic vaccinations, and weak host defenses. To overcome these limitations, we developed a novel FMD vaccine by combining an inactivated FMD viral antigen with the small molecule isoprinosine, which served as an adjuvant (immunomodulator).MethodWe evaluated the innate and adaptive immune responses elicited by the novel FMD vaccine involved both in vitro and in vivo using mice and pigs. ResultsWe demonstrated isoprinosine-mediated early, mid-term, and long-term immunity through in vitro and in vivo studies and complete host defense against FMD virus (FMDV) infection through challenge experiments in mice and pigs. We also elucidated that isoprinosine induces innate and adaptive (cellular and humoral) immunity via promoting the expression of immunoregulatory gene such as pattern recognition receptors [PRRs; retinoic acid-inducible gene (RIG)-I and toll like receptor (TLR)9], transcription factors [T-box transcription factor (TBX)21, eomesodermin (EOMES), and nuclear factor kappa B (NF-kB)], cytokines [interleukin (IL)-12p40, IL-23p19, IL-23R, and IL-17A)], and immune cell core receptors [cluster of differentiation (CD)80, CD86, CD28, CD19, CD21, and CD81] in pigs. ConclusionThese findings present an attractive strategy for constructing novel FMD vaccines and other difficult-to-control livestock virus vaccine formulations based on isoprinosine induced immunomodulatory functions.

In South Korea, a mandatory nation-wide foot-and-mouth disease (FMD) vaccination policy is in place. However, a major side effect of the current method of intramuscular (IM) administration of oil-adjuvanted FMD vaccines is the formation of granulomas in the muscles of pigs. To address this issue, we assessed the possible application of intradermal (ID) vaccination. Initially, we compared the serological immune response in specific pathogen-free pigs inoculated with FMD vaccines formulated with eight different adjuvants, administered twice at the neck site using a syringe with a needle via the ID route. Among the formulations (water-in-oil-in-water (W/O/W), oil-in-water (O/W), and polymer nanomaterials), ISA 207 of W/O/W was the most effective in inducing immunogenicity followed by ISA 201 of W/O/W. ISA 207 was further tested in formulations of different antigen doses (12 or 1.2 μg) delivered via both IM and ID routes. All four treatments successfully protected the pigs against FMD virus challenges. To assess the feasibility of the field application of the vaccines with ISA 207, we conducted ID vaccination of conventional pigs using a needle-free device, resulting in the detection of significant levels of neutralizing antibodies. ISA 207 was shown to be superior to ISA 201 in inducing immunogenicity via the ID route. In conclusion, ISA 207 could be a suitable adjuvant for ID vaccination in terms of vaccine efficacy for FMD, allowing for alternate use of ID vaccination and subsequent reduction in the incidences of granuloma formation in the field.

Introduction:Many countries use commercial foot-and-mouth disease (FMD) vaccines to prevent FMD pandemics, but these vaccines have disadvantages, such as repeated vaccinations due to the short persistence of antibody (Ab) titers and incomplete host defense despite high Ab titers. To address these shortcomings, we aimed to develop a novel FMD vaccine containing furfurman as an adjuvant. Method:To demonstrate the efficacy of the test vaccine, adaptive immunity was evaluated by measuring Ab and neutralizing Ab titers and host defense against viral infections in experimental and target animals. In addition, the expression levels of cytokines [interferon (IFN)α, IFNβ, IFNγ, interleukin (IL)-1β, IL-2, and IL-12p40] were evaluated at the early stages of vaccination to confirm the simultaneous induction of cellular and humoral immune responses induced by the test vaccine. Result:The groups that received vaccine containing furfurman showed a strong early, mid-term, and long-term immune response and host defense against viral infections compared to the control groups. The significant upregulation observed in cytokine levels in the furfurman group compared to those in the control groups strongly suggest that the test vaccine strengthens cellular immune response and effectively induces a humoral immune response. Conclusion:Our study demonstrated that furfurman, as an FMD vaccine adjuvant, achieves long-lasting immunity and host defense against viral infections by eliciting potent cellular and humoral immune responses. Therefore, our findings contribute to the design of next-generation FMD vaccines and highlight the potential application of furfurman as an adjuvant for other viral diseases.

Foot-and-mouth disease (FMD) vaccines are currently the most powerful protective and preventive measures used to control FMD. In this study, the chimeric vaccine strain containing antigenic epitopes from the FMD virus serotype A, which belongs to the ASIA topotype, was produced and evaluated. The chimeric vaccine strains contain sea-97/G1 (VP4, VP2, VP3) and A22 Iraq (VP1) or G-VII (VP1) for use in FMD vaccines in Asia. The 50% protective dose was determined in mice. Vaccinated mice were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) seven days post-vaccination (dpv), and mice that received the vaccine candidates were protected against the three viruses. The protective capability of one of the vaccine candidates was evaluated in pigs. Vaccinated pigs were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) at 28 dpv, and pigs that received the vaccine candidate were protected against the three viruses. The results showed that this vaccine candidate, which was designed to provide protection against FMD in Asia, efficiently protected pigs against virus challenge and thus has potential as a broad-spectrum vaccine for various epidemic FMD viruses.

Background/Objectives: Foot-and-mouth disease (FMD) remains a significant global threat to livestock farming. Current commercial FMD vaccines present several challenges, including the risk of infection and adverse injection site reactions due to oil-based adjuvants. The complex immune environment of the gut-associated lymphoid tissue has the potential to induce broad and diverse immune responses. Therefore, we aimed to explore the potential of zinc sulfate as an oral adjuvant to enhance intestinal mucosal immunity and complement the effects of intramuscular (IM) FMD vaccination. Methods: We conducted serological analyses on mice and pigs, measuring secretory IgA (sIgA) levels and evaluating the expression of mucosal immunity-related genes in pigs. These assessments were used to investigate the systemic and mucosal immune responses induced by oral zinc sulfate administration in combination with an IM FMD vaccine. Results: This combination strategy significantly increased structural protein antibody titers and virus neutralization titers in experimental animals (mice) and target animals (pigs) across early, mid-, and long-term periods. Additionally, this approach enhanced the expression of key cytokines associated with mucosal immunity and increased sIgA levels, which are critical markers of mucosal immunity. Conclusions: Oral zinc sulfate administration may synergize with inactivated FMD vaccines, leading to sustained and enhanced long-term immune responses. This novel strategy could reduce the frequency of required vaccinations or allow for a lower antigen dose in vaccines, effectively stimulating the mucosal immune system and boosting systemic immunity. This approach has the potential to improve the overall efficacy of commercial FMD vaccines.

Foot-and-mouth disease (FMD) is an economically important disease, and the FMD virus (FMDV) can spread rapidly in susceptible animals. FMD is usually controlled through vaccination. However, commercial FMD vaccines are only effective 4–7 days after vaccination. Furthermore, FMDV comprises seven serotypes and various topotypes, and these aspects should be considered when selecting a vaccine. Antiviral agents could provide rapid and broad protection against FMDV. Therefore, this study aimed to develop a fusion protein of consensus porcine interferon-α and Fc portion of porcine antibody IgG (poIFN-α-Fc) using a baculovirus expression system to develop a novel antiviral agent against FMDV. We measured the antiviral effects of the poIFN-α-Fc protein against FMDV and the enhanced duration in vitro and in vivo. The broad-spectrum antiviral effects were tested against seven FMDV serotypes, porcine reproductive and respiratory syndrome virus (PRRSV), and bovine enterovirus (BEV). Furthermore, the early protective effects and neutralizing antibody levels were tested by co-injecting poIFN-α-Fc and an FMD-inactivated vaccine into mice or pigs. Sustained antiviral effects in pig sera and mice were observed, and pigs injected with a combination of the poIFN-α-Fc and an inactivated FMD vaccine were protected against FMDV in a dose-dependent manner at 2- and 4-days post-vaccination. In addition, combined with the inactivated FMD vaccine, poIFN-α-Fc increased the neutralizing antibody levels in mice. Therefore, poIFN-α-Fc is a potential broad-spectrum antiviral and adjuvant candidate that can be used with inactivated FMD vaccines to protect pigs against FMDV.

Foot-and-mouth disease (FMD) is a fatal contagious viral disease that affects cloven-hoofed animals and causes severe economic damage at the national level. There are seven serotypes of the causative foot-and-mouth disease virus (FMDV), and type O is responsible for serious outbreaks and shows a high incidence. Recently, the Cathay, Southeast Asia (SEA), and ME-SA (Middle East-South Asia) topotypes of type O have been found to frequently occur in Asia. Thus, it is necessary to develop candidate vaccines that afford protection against these three different topotypes. In this study, an experimental FMD vaccine was produced using a recombinant virus (TWN-JC) with the JC epitope (VP1 140–160 sequence of the O/SKR/Jincheon/2014) between amino acid 152 and 153 of VP1 in TWN-R. Immunization with this novel vaccine candidate was found to effectively protect mice against challenge with the three different topotype viruses. Neutralizing antibody titers were considerably higher after a second vaccination. The serological differences between the topotype strains were identified in guinea pigs and swine. In conclusion, a significant serological difference was observed at 56 days post-vaccination between animals that received the TWN-JC vaccine candidate and those that received the positive control virus (TWN-R). The TWN-JC vaccine candidate induced IFNγ and IL-12B.

BackgroundMost commercial foot-and-mouth disease (FMD) vaccines have various disadvantages, such as low antibody titers, short-lived effects, compromised host defense, and questionable safety.ObjectivesTo address these shortcomings, we present a novel FMD vaccine containing Dectin-1 agonist, β-D-glucan, as an immunomodulatory adjuvant. The proposed vaccine was developed to effectively coordinate innate and adaptive immunity for potent host defense against viral infection.MethodsWe demonstrated β-D-glucan mediated innate and adaptive immune responses in mice and pigs in vitro and in vivo. The expressions of pattern recognition receptors, cytokines, transcription factors, and co-stimulatory molecules were promoted via FMD vaccine containing β-D-glucan.Resultsβ-D-glucan elicited a robust cellular immune response and early, mid-, and long-term immunity. Moreover, it exhibited potent host defense by modulating host’s innate and adaptive immunity.ConclusionOur study provides a promising approach to overcoming the limitations of conventional FMD vaccines. Based on the proposed vaccine’s safety and efficacy, it represents a breakthrough among next-generation FMD vaccines.

Foot-and-mouth disease (FMD) is a rapidly propagating infectious disease of cloven-hoofed animals, especially cattle and pigs, affecting the productivity and profitability of the livestock industry. Presently, FMD is controlled and prevented using vaccines; however, conventional FMD vaccines have several disadvantages, including short vaccine efficacy, low antibody titers, and safety issues in pigs, indicating the need for further studies. Here, we evaluated the efficacy of a novel bivalent vaccine containing zinc sulfate as an immunostimulant and FMD type O and A antigens (O PA2 and A YC, respectively) against FMD virus in mice and pigs. Zinc sulfate induced cellular immunity in murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs) by increasing IFNγ secretion. Additionally, FMD vaccine containing O PA2 and A YC antigens and zinc sulfate induced early, mid-, and long-term immune responses in mice and pigs, and enhanced cellular and humoral immunity by regulating the expression of pathogen recognition receptors (PRRs), transcription factors, co-stimulatory molecules, and cytokines in porcine PBMCs from vaccinated pigs. Overall, these results indicated that the novel immunostimulant zinc sulfate induced potent cellular and humoral immune responses by stimulating antigen-presenting cells (APCs) and T and B cells, and enhanced long-term immunity by promoting the expression of co-stimulatory molecules. These outcomes suggest that zinc sulfate could be used as a novel vaccine immunostimulant for difficult-to-control viral diseases, such as African swine fever (ASF) or COVID-19.

An inactivated whole-virus vaccine is currently used to prevent foot-and-mouth disease (FMD). Although this vaccine is effective, it offers short-term immunity that requires regular booster immunizations and has several side effects, including local reactions at the vaccination site. To address these limitations, herein, we evaluated the efficacy of bestatin as a novel small molecule adjuvant for inactivated FMD vaccines. Our findings showed that the FMD vaccine formulated with bestatin enhanced early, intermediate-, and particularly long-term immunity in experimental animals (mice) and target animals (pigs). Furthermore, cytokines (interferon (IFN)α, IFNβ, IFNγ, and interleukin (IL)-29), retinoic acid-inducible gene (RIG)-I, and T-cell and B-cell core receptors (cluster of differentiation (CD)28, CD19, CD21, and CD81) markedly increased in the group that received the FMD vaccine adjuvanted with bestatin in pigs compared with the control. These results indicate the significant potential of bestatin to improve the efficacy of inactivated FMD vaccines in terms of immunomodulatory function for the simultaneous induction of potent cellular and humoral immune response and a long-lasting memory response.

Foot-and-mouth disease (FMD) type O includes 11 genetic topotypes. The Southeast Asia (SEA), Middle East–South Asia (ME-SA), and Cathay topotypes belong to FMD type O and occur frequently in Asia. Therefore, it is necessary to develop a potent vaccine strain with a broad antigenic coverage in order to provide complete protection against these three topotypes. In this study, an experimental vaccine was produced using chimeric vaccine strains (JC-VP1 or PA2-VP1) that contained VP4, VP2, and VP3 of the ME-SA topotype (O Manisa) and VP1 of the SEA topotype (Mya98 lineage; O/SKR/Jincheon/2014) or ME-SA topotype (PanAsia2 lineage; O/PAK/44). Mice were immunized with the experimental vaccines, and they were fully protected against the three topotypes. The neutralizing antibody titers of PA2-VP1 were significantly higher than those of JC-VP1 in the early vaccination phase in pigs. Here, we confirmed complete protection in pigs vaccinated with JC-VP1 or PA2-VP1, when challenged against the SEA (O/SKR/Jincheon/2014), ME-SA (O/SKR/Boeun/2017) and Cathay (O/Taiwan/97) topotype viruses, with moderately higher protection provided by PA2-VP1 than by JC-VP1.

Foot-and-mouth disease (FMD) is an acute contagious infectious disease that affects cloven-hoofed animals. Although current emergency FMD vaccines only take effect 7 days after vaccination, antiviral agents, such as quercetin, which is a common flavonoid, could reduce the spread of FMD virus (FMDV) during outbreaks. We investigated the in vitro and in vivo antiviral effects of quercetin against FMDV. Analysis of viral copy numbers showed that quercetin had a dose-dependent inhibitory effect on FMDV at concentrations between 19.5 and 1,250 μM in porcine cells. In addition, we observed a quercetin-induced interferon (IFN)-α protein and interferon-stimulated gene (ISG) upregulation in swine cells. Enzyme-linked immunosorbent assay of sera revealed that quercetin induces the production of IFN-α, IFN-β, IFN-γ, interleukin (IL)-12, and IL-15 in mice. Inoculation of mice with quercetin or a combination of quercetin with an inactivated FMD vaccine enhanced both the survival rate and neutralizing antibody titer. Therefore, we suggest the use of quercetin as a novel and effective antiviral agent for controlling FMDV infection; however, further investigation of its application in livestock is required.

Human embryonic kidney (HEK) 293 cells are widely used in protein and viral vector production owing to their high transfection efficiency, rapid growth, and suspension growth capability. Given their antiviral, anticancer, and immune-enhancing effects, type I interferons (IFNs) have been used to prevent and treat human and animal diseases. However, the binding of type I IFNs to the IFN-α and-β receptor (IFNAR) stimulates the expression of IFN-stimulated genes (ISGs). This phenomenon induces an antiviral state and promotes apoptosis in cells, thereby impeding protein or viral vector production. In this study, we generated an IFNAR subtype 1 knockout (KO) HEK 293 suspension (IFNAR-KO) cell line by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology. Upon treatment with human IFN-α, the IFNAR-KO cells showed a constant expression of ISGs, including 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (Mx1), protein kinase RNA-activated (PKR), and IFN-induced transmembrane protein 1 (IFITM1), when compared with the wild-type HEK 293 (WT) cells, wherein the ISGs were significantly upregulated. As a result, the titer of recombinant adenovirus expressing porcine IFN-α was significantly higher in the IFNAR-KO cells than in the WT cells. Furthermore, the IFNAR-KO cells continuously produced higher amounts of IFN-α protein than the WT cells. Thus, the CRISPR-Cas9-mediated IFNAR1 KO cell line can improve the production efficiency of proteins or viral vectors related to IFNs. The novel cell line may be used for producing vaccines and elucidating the type I IFN signaling pathway in cells.

Background:Foot-and-mouth disease (FMD) is an extremely contagious viral disease that is fatal to young animals and is a major threat to the agricultural economy by reducing production and limiting the movement of livestock. The currently commercially-available FMD vaccine is prepared using an inactivated viral antigen in an oil emulsion, with aluminum hydroxide [Al(OH)3] as an adjuvant. However, oil emulsion-based options possess limitations including slow increases in antibody titers (up to levels adequate for defense against viral infection) and risks of local reactions at the vaccination site. Further, Al(OH)3 only induces a T helper 2 (Th2) cell response. Therefore, novel adjuvants that can address these limitations are urgently needed. Glycyrrhizic acid (extracted from licorice roots) is a triterpenoid saponin and has great advantages in terms of price and availability.Methods:To address the limitations of the currently used commercial FMD vaccine, we added glycyrrhizic acid as an adjuvant (immunostimulant) to the FMD bivalent (O PA2 + A YC) vaccine. We then evaluated its efficacy in promoting both innate and adaptive (cellular and humoral) immune reactions in vitro [using murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs)] and in vivo (using mice and pigs).Results:Glycyrrhizic acid has been revealed to induce an innate immune response and enhance early, mid-, and long-term immunity. The studied bivalent vaccine with glycyrrhizic acid increased the expression of immunoregulatory genes such as pattern-recognition receptors (PRRs), cytokines, transcription factors, and co-stimulatory molecules.Conclusion:Collectively, glycyrrhizic acid could have utility as a novel vaccine adjuvant that can address the limitations of commercialized FMD vaccines by inducing potent innate and adaptive immune responses.

Foot-and-mouth disease (FMD) is an acute contagious disease of cloven-hoofed animals such as cows, pigs, sheep, and deer. The current emergency FMD vaccines, to induce early protection, have limited use, as their protective effect in pigs does not begin until 7 days after vaccination. Therefore, the use of antiviral agents would be required for reducing the spread of foot-and-mouth disease virus (FMDV) during outbreaks. Vesatolimod (GS-9620), a toll-like receptor 7 agonist, is an antiviral agent against various human disease-causing viruses. However, its antiviral effect against FMDV has not been reported yet. The aim of this study was to investigate the antiviral effects of GS-9620 against FMDV both in vitro and in vivo. The inhibitory effect of GS-9620 on FMDV in swine cells involved the induction of porcine interferon (IFN)-α and upregulation of interferon-simulated genes. Protective effect in mice injected with GS-9620 against FMDV was maintained for 5 days after injection, and cytokines such as IFN-γ, interleukin (IL)-12, IL-6, and IFN-γ inducible protein-10 could be detected following the treatment with GS-9620. Furthermore, the combination of GS-9620 with an FMD-inactivated vaccine was found to be highly effective for early protection in mice. Overall, we suggest GS-9620 as a novel and effective antiviral agent for controlling FMDV infection.

Vaccination prevents and controls foot-and-mouth disease (FMD). However, the current FMD vaccine remains disadvantageous since it cannot overcome maternally-derived antibody (MDA) interference in weeks-old animals, which suppress active immunity via vaccination. To address this, we developed the immune-enhancing O PA2-C3d and A22-C3d FMD vaccine strains that can stimulate receptors on the surface of B cells by inserting C3d (a B cell epitope) into the VP1 region of O PA2 (FMDV type O) and A22 (FMDV type A). We purified inactivated viral antigens from these vaccine strains and evaluated their immunogenicity and host defense against FMDV infection in mice. We also verified its efficacy in inducing an adaptive immune response and overcome MDA interference in MDA-positive (MDA(+), FMD-seropositive) and -negative (MDA(-), FMD-seronegative) pigs. These results suggest a key strategy for establishing novel FMD vaccine platform to overcome MDA interference and induce a robust adaptive immune response.