Natural Phytochemicals, Luteolin and Isoginkgetin, Inhibit 3C Protease and Infection of FMDV, In Silico and In Vitro.

Foot-and-mouth-disease virus (FMDV) is a picornavirus that causes a highly contagious disease of cloven-hoofed animals resulting in economic losses worldwide. The 3C protease (3Cpro) is the main protease essential in the picornavirus life cycle, which is an attractive antiviral target. Here, we used computer-aided virtual screening to filter potential anti-FMDV agents from the natural phytochemical compound libraries. The top 23 filtered compounds were examined for anti-FMDV activities by a cell-based assay, two of which possessed antiviral effects. In the viral and post-viral entry experiments, luteolin and isoginkgetin could significantly block FMDV growth with low 50% effective concentrations (EC50). Moreover, these flavonoids could reduce the viral load as determined by RT-qPCR. However, their prophylactic activities were less effective. Both the cell-based and the fluorescence resonance energy transfer (FRET)-based protease assays confirmed that isoginkgetin was a potent FMDV 3Cpro inhibitor with a 50% inhibition concentration (IC50) of 39.03 ± 0.05 and 65.3 ± 1.7 µM, respectively, whereas luteolin was less effective. Analyses of the protein-ligand interactions revealed that both compounds fit in the substrate-binding pocket and reacted to the key enzymatic residues of the 3Cpro. Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production.

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

Validation of a binary ethylenimine (BEI) inactivation procedure for biosafety treatment of foot-and-mouth disease viruses (FMDV), vesicular stomatitis viruses (VSV), and swine vesicular disease virus (SVDV).

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV.

A randomized controlled field trial assessing foot and mouth disease vaccine effectiveness in Gondar Zuria district, Northwest Ethiopia.

Foot and mouth disease (FMD) is a highly transmissible viral disease of cloven-hoofed animals, which is endemic in many developing countries. Vaccination is the main tool for FMD control in resource limited endemic countries like Ethiopia. Vaccine quality, which is often questionable in developing countries, is a critical element for effective disease control. The present study was aimed at evaluating the field effectiveness of a trivalent FMD vaccine (containing serotypes O, A and SAT 2), produced and widely used in Ethiopia, in terms of preventing clinical infection and severe disease. A randomized controlled field trial design was employed in the study in which the attack rate of clinical FMD infection in vaccinated cattle was compared with the attack rate in unvaccinated controls in cattle population of 16 villages in Gondar Zuria district, Northwest Ethiopia. The vaccine was administered as a single dose course in the face of an impending FMD outbreak and the trial groups were monitored for clinical infection until the end of the outbreak. The attack rate of clinical FMD 20 days post vaccination in the vaccinated cattle (34 %) was significantly lower than the attack rate in the unvaccinated controls (49 %) (p < 0.001). However, the effectiveness of the vaccine was only 31 % (95 %CI: 20-40 %). This vaccine effectiveness increased to 52 % ((95 %CI: 33-66 %) 42 days post vaccination. The proportion of severely affected cattle in the vaccinated group (5.7 %) was significantly lower than in the unvaccinated group (9.4 %) (p < 0.001), resulting in 39 % (95 %CI: 18-55 %) vaccine effectiveness against severe disease. Generally, the observed level of vaccine effectiveness was lower than the internationally recommended 75 % plus expected percentage of protection for a standard potency 3PD50/dose FMD vaccine. Moreover, the level of effectiveness was insufficient to provide herd immunity to control the disease at the population level. Nevertheless, given the significant difference in the incidence of clinical disease and severity between vaccinated and unvaccinated cattle, it might still be worth using the current vaccine to reduce production losses associated with the disease provided it is cost effective and affordable for the farmers. Factors that cause low effectiveness of the vaccine need to be identified and addressed for effective control of the disease at population level.

Substrate Optimization in Baby Hamster Kidney Cell Culture for Foot and Mouth Disease Virus Vaccine Using the Taguchi Method.

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.

Evaluation of the disinfectant concentration used on livestock facilities in Korea during dual outbreak of foot and mouth disease and high pathogenic avian influenza.

BACKGROUND: A nationwide outbreak of foot-and-mouth disease (FMD) in South Korea caused massive economic losses in 2010. Since then, the Animal and Plant Quarantine Agency (QIA) has enhanced disinfection systems regarding livestock to prevent horizontal transmission of FMD and Avian influenza (AI). Although the amount of disinfectant used continues to increase, cases of FMD and AI have been occurring annually in Korea, except 2012 and 2013. OBJECTIVES: This study measured the concentration of the disinfectant to determine why it failed to remove the horizontal transmission despite increased disinfectant use. METHODS: Surveys were conducted from February to May 2017, collecting 348 samples from disinfection systems. The samples were analyzed using the Standards of Animal Health Products analysis methods from QIA. RESULTS: Twenty-three facilities used inappropriate or non-approved disinfectants. Nearly all sampled livestock farms and facilities-93.9%-did not properly adjust the disinfectant concentration. The percentage using low concentrations, or where no effective substance was detected, was 46.9%. Furthermore, 13 samples from the official disinfection station did not use effective disinfectant, and-among 72 samples from the disinfection station-88.89% were considered inappropriate concentration, according to the foot-and-mouth disease virus guidelines; considering the AIV guideline, 73.61% were inappropriate concentrations. Inappropriate concentration samples on automatic (90.00%) and semi-automatic (90.90%) disinfection systems showed no significant difference from manual methods (88.24%). Despite this study being conducted during the crisis level, most disinfectants were used inappropriately. CONCLUSIONS: This may partially explain why horizontal transmission of FMD and AI cannot be effectively prevented despite extensive disinfectant use.

Recovery and chemical disinfection of foot-and-mouth disease and African swine fever viruses from porous concrete surfaces.

AIMS: Develop an effective laboratory method to consistently recover viral loads from porous concrete coupons sufficient for disinfectant efficacy testing. Investigate the role of concrete matrix pH on the recovery of foot-and-mouth disease virus (FMDV) and African Swine Fever virus (ASFV) from porous concrete. Compare parameters off FMDV and ASFV inactivation on porous and nonporous surfaces in quantitative carrier tests of a liquid chemical disinfectant. METHODS AND RESULTS: Concrete test coupons were fabricated from commercial and industrial sources and carbonated by exposure to 5% CO2 in a humidified incubator, lowering the matrix pH. Neither dried FMDV nor ASFV were recovered from high-pH concrete control coupons. Recovery of infectious virus from lower pH carbonated concrete was similar to stainless steel coupon controls. Exposure to the liquid disinfectant Virkon™ S inactivated FMDV and ASFV on porous concrete. CONCLUSIONS: Concrete matrix pH had a greater impact than surface porosity on the ability to recover viable virus from unsealed concrete. SIGNIFICANCE AND IMPACT OF THE STUDY: Concrete is commonly found in environments where virus decontamination is required. This study demonstrates a reproducible method to recover sufficient viral loads from porous concrete coupons to facilitate quantitative carrier testing. This method provides a basis for evidence-based validation testing of chemical disinfectants to inactivate pH-sensitive viruses on unsealed concrete.

Inactivation of foot-and-mouth disease virus A/IRN/8/2015 with commercially available lysis buffers.

Laboratories working with foot-and-mouth disease virus (FMDV) must maintain a high level of biocontainment. However, if infectious virus is reliably inactivated during sample processing, molecular and serological testing can be performed at a lower level of containment. In this study, three commercial lysis buffers (AL, AVL, and MagMAX CORE) were tested in two laboratories for their ability to inactivate FMDV A/IRN/8/2015 in different sample matrices (cell culture supernatant, epithelial tissue suspension and milk). Residual infectivity after the addition of lysis buffer was evaluated by inoculating susceptible cell cultures. No cytopathic effect was observed for all three lysis buffers, indicating that the buffers are capable of reducing viral infectivity (estimated range 3.1 to >5.1 Log10). These results highlight the capacity of lysis buffers to decrease FMDV infectivity; however, additional validation experiments should be conducted, particularly if different sample matrices and/or lysis buffers are used.

Three-percent sucrose acts as a thermostabilizer for cell-adapted foot-and-mouth disease virus without any negative effect on viral growth.

AIMS: As cell-adapted foot-and-mouth disease virus (FMDV) with H56R mutation in VP3 has reduced thermostability, this study aimed to investigate the effect of thermostabilizers on cell-adapted FMDV for vaccine production. METHODS AND RESULTS: We examined the effect of 3% sucrose, 10% (or 25%) glycerol or 10% FBS on cell-adapted FMDV O/SKR/JC/2014, containing H56R mutation in VP3, as vaccine seed virus at -80, 4, 25 or 37°C for 2, 4 or 7 days. The stabilizing effect of 3% sucrose on O/SKR/JC/2014 was observed at 25, 37°C, and after repeated freeze-thaw cycles. Additionally, we tested the effect of 3% sucrose on the growth of FMDV or cells and did not observe any decrease in either viral growth or cell viability. CONCLUSIONS: Our study showed the protective effect of 3% sucrose on FMDV infectivity at various temperatures; this virus stock in 3% sucrose could be used for infecting cells without the removal of sucrose. SIGNIFICANCE AND IMPACT OF THE STUDY: We suggest that 3% sucrose-containing medium could be beneficial for the stable storage and transport of cell-adapted FMDV.

Inactivation of foot-and-mouth disease virus in epithelium samples for safe transport and processing in low-containment laboratories.

During a foot-and-mouth disease (FMD) outbreak, transport and testing of potentially infectious samples, including epithelium from suspect lesions, presents a biosafety risk, particularly in FMD-free countries. Therefore, treatment to inactivate virus prior to transport is important. Tongue epithelium from cattle infected with FMD virus (FMDV) serotype O (O ALG/3/2014 - Lineage O/ME-SA/Ind-2001d) or A (A IRN/22/2015 - Lineage A/ASIA/G-VII) was incubated in RNAlater, RNA Shield or phosphate-buffered saline (pH 7.4) at room temperature for 2, 6, 24 or 48 h. After incubation, tissues were homogenised and tested by virus titration. Viral RNA in the homogenate was quantified by RT-qPCR, used for sequencing, and transfected into LFBKαVß6 cells to recover infectious virus. RNAlater reduced A IRN/22/2015 titres by 4 log10 after 24 h, and completely after 48 h incubation. While O ALG/3/2014 was detected by VI after 2, 6 and 24 h, titration yielded no infectious virus, likely as a result of freeze-thawing. RNA Shield was cytotoxic at high concentrations but was effective at inactivating both strains after 24 h. Regardless of reagent or inactivation period, RT-qPCR, VP1 sequencing, and transfection of RNA to recover infectious virus were possible. RNA Shield appears a better choice for FMDV inactivation in tissues, however 24 h incubation is recommended.

Antiviral effects of selected IMPDH and DHODH inhibitors against foot and mouth disease virus.

Foot-and-mouth disease virus (FMDV) is an important pathogen that affects livestock breeding and causes huge economic losses worldwide. Currently, the development of antiviral agents to combat FMDV infection at the early stages is being explored. As viral replication critically depends on the host for nucleoside supply, host enzymes involved in nucleotides biosynthesis may represent potential targets for the development of antiviral agents. In the present study, the effects of IMP dehydrogenase (AVN-944 and mycophenolate mofetil) and dihydroorotate dehydrogenase (teriflunomide) inhibitors were evaluated both in vitro and in vivo. The results revealed that these compounds were effective in suppressing FMDV (O/MY98/BY/2010 and A/GD/MM/2013) infection. With regard to the antiviral mechanism, time-of-addition experiments revealed that these compounds were effective when added at the early stages of viral lifecycle (0-8 h post infection). However, exogenous guanosine/uridine eliminated the antiviral activity of these compounds. Importantly, treatment AVN-944 and teriflunomide significantly improved the survival of mice that were subcutaneously treated with FMDV. Together, the results of the present study indicate the broad-spectrum activities of anti-FMDV agents targeting IMP dehydrogenase or dihydroorotate dehydrogenase, which could be useful in developing strategies to prevent FMD.

Hydrogen peroxide vapour is an effective replacement for Formaldehyde in a BSL4 Foot and mouth disease vaccine manufacturing facility.

An evaluation of the efficacy of 35% hydrogen peroxide vapour (HPV) against two strains of FMDV was conducted over a period of 6 months. FMDV biological indicators were produced on-site using strains obtained from a commercial FMDV vaccine manufacturing process. FMDV biological indicators were distributed within a BSL4 laboratory and exposed to short duration hydrogen peroxide cycles. Variations in titre, support matrix (soiling), temperature and humidity were evaluated in a series of 16 exposures using over 200 individual FMDV indicators. Additional verification testing was performed in an operational material transfer lock to replicate real-world use. HPV was found to be efficacious in inactivating FMDV strains; the inoculum titre influenced the level of reduction achieved with the specified cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: The classification of formaldehyde as a presumed human carcinogen has presented regulatory challenges for its continued use as a biocidal product. Institutions are actively seeking fumigants to replace formaldehyde and undertaking studies to validate biocidal efficacy, particularly in high-level biosafety facilities where the consequences of pathogen release can be extremely severe. This study builds on the already substantial scientific efficacy base of 35% hydrogen peroxide vapour and provides a comprehensive evaluation of the applicability of hydrogen peroxide vapour as a replacement for formaldehyde within a Foot & Mouth Disease (FMDV) vaccine manufacturing facility.

Antiviral activity of merimepodib against foot and mouth disease virus in vitro and in vivo.

Foot and mouth disease virus (FMDV), a member of family Picornaviridae, belongs to the genus Aphthovirus, which causes foot and mouth disease (FMD), a highly transmissible disease that affects cloven-hoof animals. In spite of the fact that efficient vaccines are available, effective antiviral molecules for FMD are needed to reduce viral infection during early stages of infection. In this study, merimepodib was found to efficiently inhibit FMDV replication in a dose-dependent manner. The 50% inhibitory concentration (IC50) of merimepodib antiviral activity against two distinct FMDV strains (O/MYA98/BY/2010 and A/GD/MM/CHA/2013) was estimated to be 7.859 and 2.876 µM, respectively, while the 50% cytotoxic concentration (CC50) of merimepodib was found to be 47.74 µM. Furthermore, treatment with 30 µg merimepodib efficiently prolonged the survival time of suckling mice infected with FMDV. Taken together, these results suggested that merimepodib has the potential to be a novel antiviral agent against FMDV.

Antiviral effect of amiloride on replication of foot and mouth disease virus in cell culture.

Recently, amiloride was shown to potently suppress Coxsackievirus B3 (CVB3) replication. In the current study, we investigated whether amiloride could also exhibit antiviral activity against foot-and-mouth disease virus (FMDV), which belongs to the same family (Picornaviridae) as CVB3. We found that amiloride exerted antiviral activity in a dose-dependent manner against two strains of FMDV in IBRS-2 cells, with slight cytotoxicity at 1000 µM. Besides, amiloride did not inhibit the attachment and entry of FMDV in IBRS-2 cells, but prevented early viral replication. These data implied that amiloride could be a promising candidate for further research as a potential antiviral drug against FMDV infection.

The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus genome replication.

Arbidol (ARB, also known as umifenovir) is used clinically in several countries as an anti-influenza virus drug. ARB inhibits multiple enveloped viruses in vitro and the primary mode of action is inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. ARB is also an effective inhibitor of non-enveloped poliovirus types 1 and 3. In the current report, we evaluate the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen. ARB inhibits the replication of FMDV RNA sub-genomic replicons. ARB inhibition of FMDV RNA replication is not a result of generalized inhibition of cellular uptake of cargo, such as transfected DNA, and ARB can be added to cells up to 3 h post-transfection of FMDV RNA replicons and still inhibit FMDV replication. ARB prevents the recovery of FMDV replication upon withdrawal of the replication inhibitor guanidine hydrochloride (GuHCl). Although restoration of FMDV replication is known to require de novo protein synthesis upon GuHCl removal, ARB does not suppress cellular translation or FMDV internal ribosome entry site (IRES)-driven translation. ARB also inhibits infection with the related Aphthovirus, equine rhinitis A virus (ERAV). Collectively, the data demonstrate that ARB can inhibit some non-enveloped picornaviruses. The data are consistent with inhibition of picornavirus genome replication, possibly via the disruption of intracellular membranes on which replication complexes are located.

In Vitro and in Vivo Antiviral Activity of Mizoribine Against Foot-And-Mouth Disease Virus.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which has significant economic consequences in affected countries. As the currently available vaccines against FMD provide no protection until 4-7 days post-vaccination, the only alternative method to control the spread of FMD virus (FMDV) during outbreaks is the application of antiviral agents. Hence, it is important to identify effective antiviral agents against FMDV infection. In this study, we found that mizoribine has potent antiviral activity against FMDV replication in IBRS-2 cells. A time-of-drug-addition assay demonstrated that mizoribine functions at the early stage of replication. Moreover, mizoribine also showed antiviral effect on FMDV in vivo. In summary, these results revealed that mizoribine could be a potential antiviral drug against FMDV.

Antiviral activity of brequinar against foot-and-mouth disease virus infection in vitro and in vivo.

Foot-and-mouth disease (FMD) is one of the most highly contagious animal disease that affects cloven-hoofed animals. However, the FMD vaccine does not provide effective protection until adaptive immune protection elicited by the vaccination occurs. Therefore, an alternative application of antiviral agents for inhibition of the FMD virus (FMDV) is needed. Here, we demonstrated that brequinar could exhibit antiviral activity in swine kidney cells (IBRS-2 cells) infected with two different FMDV serotypes. Subsequently, in vivo activity of brequinar was confirmed in a mouse model of infection. Specifically, brequinar at a concentration of 50 µg, provided 25% protection for 5 days following FMDV challenge. These results suggested that brequinar could be used as effective antiviral agent against FMD.

Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway.

DnaJ heat shock protein family (Hsp40) member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by coimmunoprecipitation and colocalization in FMDV-infected cells. The J domain of DNAJA3 (amino acids 1 to 168) and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss of function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the beta interferon (IFN-ß) signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on the IFN-ß signaling pathway. Poly(I⋅C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared to that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the host's antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-ß signaling pathway.IMPORTANCE This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study is also the first to clarify the mechanism by which VP1 suppressed IFN-ß signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-ß signaling pathway. These data suggested that DNAJA3 plays an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-ß signaling pathway.

Inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects cloven-hoof animals including cattle, swine, sheep, goats, and lots of wild species. Effectively control measures are urged needed. Here, we showed that homoharringtonine treatment exhibited a strong inhibitory effect against two different strains of FMDVs (O/MYA98/BY/2010 and A/GD/MM/2013) in swine kidney (IBRS-2) cells. Further experiments demonstrated that homoharringtonine did not affect virus attachment or entry. Using time-of-addition assays, we found that the antiviral activity of homoharringtonine occurred primarily during the early stage of infection. These results demonstrated that homoharringtonine might be an effective anti-FMDV drug. Further studies are required to explore the antiviral activity of homoharringtonine against FMDV replication in vivo.

Advances in novel vaccines for foot and mouth disease: focus on recombinant empty capsids.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.