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Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. FMD poses an economic threat to the livestock industry in the United States. Due to the potential use of vaccines composed of partially purified structural proteins of the FMD virus (FMDV), it is important to test samples from infected and vaccinated animals with a competitive ELISA that detects antibodies against nonstructural proteins (NSPs) of FMDV. Our study extends the diagnostic validation of the Prionics ELISA (Thermo Fisher) and the VMRD ELISA. We used diverse serum sample sets from bovine, porcine, and other cloven-hoofed animals to evaluate the analytical specificity and sensitivity, diagnostic specificity and sensitivity, and differentiation of infected from vaccinated animals (DIVA) per validation guidelines outlined by the World Organisation for Animal Health (WOAH). The 2 tests were analytically 100% accurate. The VMRD test was diagnostically more sensitive than Prionics, but Prionics was diagnostically more specific than the VMRD test. Both tests could tell if animals were infected or vaccinated. Considering these data, both VMRD and Prionics ELISAs can be used for serodetection of FMDV antibodies at the Foreign Animal Disease Diagnostic Laboratory and within the National Animal Health Laboratory Network laboratories. [ABSTRACT FROM AUTHOR]

Simple Summary: Foot-and-mouth disease (FMD), caused by the foot-and-mouth disease virus (FMDV), is a prevalent infectious disease in swine. Timely detection and isolation of pigs infected with this virus are among the most effective strategies to prevent the disease's spread and mitigate potential economic damage. We have established a set of RT-qPCR detection methods for FMDV, which offer significant advantages. Notably, this detection method can effectively identify foot-and-mouth disease virus even in the presence of both exogenous and endogenous interfering substances. Furthermore, it exhibits high sensitivity, strong specificity, and good repeatability, enabling 100% detection of FMDV. The global livestock industry is facing a serious threat from a widespread foot-and-mouth disease virus (FMDV) epidemic. The timely detection of FMDV can significantly mitigate its harmful effects. This study aimed to establish and evaluate a TaqMan fluorescence quantitative PCR assay to assess its sensitivity, specificity, reproducibility, and stability. The standard curve equation range is 6.43 × 109–6.43 × 101 copies/µL, with an R2 value of 0.996 and a standard curve equation of y = −3.586x + 36.245. The method successfully detected 64.3 copies/µL of the target gene for FMDV and exhibited high specificity for FMDV. Repeatability tests demonstrated low coefficients of variation within and between groups (<2%), indicating good reproducibility. The clinical samples analyzed using this method showed results consistent with those of the SYBR Green I RT-qPCR assay, confirming the reliability of the method. Overall, the developed test method displayed high sensitivity, specificity, reproducibility, and stability, making it suitable for the rapid diagnosis of foot-and-mouth disease in clinical settings. [ABSTRACT FROM AUTHOR]

ELISA kits that detect antibodies to the non-structural protein (NSP) of the foot-and-mouth disease virus (FMDV), commonly referred to as NSP-ELISA, can distinguish between vaccinated and naturally infected animals. They can play an essential role in demonstrating 'proof-of-freedom' during the control of FMD. Although various NSP-ELISA kits are available in Thailand, information regarding their performance is lacking. To select the most appropriate NSP-ELISA kit for our specific purpose, we must compare their performance using carefully characterized sera. This will ensure that we maximize the benefits of our testing. In this study, six NSP-ELISA kits sold in Thailand―Biovet, ID Screen, VDPro, IDEXX, PrioCHECK, and KUcheck-F―were evaluated and compared. A total of 800 serum samples were examined, including samples from 357 cattle and 29 buffaloes in outbreak areas, as well as 14 swine serum samples from the Vaccine Quality Control Unit of the Bureau of Veterinary Biologics, Ministry of Agriculture and Cooperation, Thailand. Four hundred samples were confirmed to originate from animals infected with FMDV through ELISA typing (n = 11, tested as representative samples in each farm) and/or RT-PCR (n = 400, all samples), serving as positive control sera. Additionally, 400 negative control sera were obtained from Japan (97 cattle and 300 pigs) and Australia (3 goats), certified by the World Organisation for Animal Health as 'free of FMD'. The sensitivity and specificity of the six tests were determined based on the results obtained from two-by-two tables. Cohen's kappa statistics were calculated for the six tests to assess their concordance, and the diagnostic accuracy of the assays was also determined. For all six NSP-ELISA kits, the sensitivity ranged from 97.75 to 99.50%, and the specificity ranged from 97.25 to 100%. Cohen's kappa statistics ranged from 0.96 to 1.00, and diagnostic accuracy ranged from 98.13 to 99.75%. The study results indicated that the test kits have statistically similar sensitivity, specificity, concordance, and diagnostic accuracy, suggesting they can be used interchangeably. However, ID Screen demonstrated the highest sensitivity and specificity among all kits tested. Therefore, if a single kit were to be selected from the six evaluated, ID Screen would be the most appropriate choice. These findings can aid in selecting the most suitable test kit. Therefore, it is recommended to consider purchasing a diverse range of effective test kits. Furthermore, these findings can provide guidance for expanding the use of test kits, particularly with the growing availability of NSP-ELISA kits in the market. [ABSTRACT FROM AUTHOR]

The life cycle of foot-and-mouth disease virus (FMDV) is tightly regulated by host cell lipid metabolism. In previous studies, we reported downregulated expression of stearoyl coenzyme A desaturase-1 (SCD1), a key enzyme of fatty acid metabolism, in BHK-VEC cells (a virus-negative cell line derived from BKH-21 cells with persistent FMDV infection) on comparing transcriptomic data for BHK-VEC and BHK-21 cells (Y. Yuan et al., Front Cell Infect Microbiol 12:940906, 2022, https://doi.org/10.3389/fcimb.2022.940906; L. Han et al., Vet Microbiol 263:109247, 2021, https://doi.org/10.1016/j.vetmic.2021.109247). In the present study, we identify that SCD1 regulates FMDV replication. SCD1 overexpression or exogenous addition of oleic acid (OA), a product of the enzymatic activity of SCD1, increased FMDV replication in both BHK-21 cells and SCD1-knockdown cells. Overexpression of SCD1 or exogenous addition of OA restored FMDV infection and replication in BHK-VEC cells, and OA also promoted FMDV replication in BHK-21 cells with persistent FMDV infection. SCD1 recruited the nonstructural FMDV protein 2C to a detergent-resistant membrane located in the perinuclear region of cells to form replication complexes. Inhibiting SCD1 enzyme activity resulted in a significantly decreased number of FMDV replication complexes with abnormal morphology. Inhibition of SCD1 activity also effectively decreased the replication of other RNA viruses such as respiratory enteric orphan virus-3-176, poliovirus-1, enterovirus 71, and vesicular stomatitis virus. Our results demonstrate that SCD1, as a key host regulator of RNA virus replication, is a potential target for developing novel drugs against infections by RNA viruses. [ABSTRACT FROM AUTHOR]

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.

Abstract ELISA kits that detect antibodies to the non-structural protein (NSP) of the foot-and-mouth disease virus (FMDV), commonly referred to as NSP-ELISA, can distinguish between vaccinated and naturally infected animals. They can play an essential role in demonstrating ‘proof-of-freedom’ during the control of FMD. Although various NSP-ELISA kits are available in Thailand, information regarding their performance is lacking. To select the most appropriate NSP-ELISA kit for our specific purpose, we must compare their performance using carefully characterized sera. This will ensure that we maximize the benefits of our testing. In this study, six NSP-ELISA kits sold in Thailand―Biovet, ID Screen, VDPro, IDEXX, PrioCHECK, and KUcheck-F―were evaluated and compared. A total of 800 serum samples were examined, including samples from 357 cattle and 29 buffaloes in outbreak areas, as well as 14 swine serum samples from the Vaccine Quality Control Unit of the Bureau of Veterinary Biologics, Ministry of Agriculture and Cooperation, Thailand. Four hundred samples were confirmed to originate from animals infected with FMDV through ELISA typing (n = 11, tested as representative samples in each farm) and/or RT-PCR (n = 400, all samples), serving as positive control sera. Additionally, 400 negative control sera were obtained from Japan (97 cattle and 300 pigs) and Australia (3 goats), certified by the World Organisation for Animal Health as ‘free of FMD’. The sensitivity and specificity of the six tests were determined based on the results obtained from two-by-two tables. Cohen’s kappa statistics were calculated for the six tests to assess their concordance, and the diagnostic accuracy of the assays was also determined. For all six NSP-ELISA kits, the sensitivity ranged from 97.75 to 99.50%, and the specificity ranged from 97.25 to 100%. Cohen’s kappa statistics ranged from 0.96 to 1.00, and diagnostic accuracy ranged from 98.13 to 99.75%. The study results indicated that the test kits have statistically similar sensitivity, specificity, concordance, and diagnostic accuracy, suggesting they can be used interchangeably. However, ID Screen demonstrated the highest sensitivity and specificity among all kits tested. Therefore, if a single kit were to be selected from the six evaluated, ID Screen would be the most appropriate choice. These findings can aid in selecting the most suitable test kit. Therefore, it is recommended to consider purchasing a diverse range of effective test kits. Furthermore, these findings can provide guidance for expanding the use of test kits, particularly with the growing availability of NSP-ELISA kits in the market.

Background and Aim: Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-footed animals. It is a major threat to livestock production worldwide, causing significant economic losses. Inactivation of FMD virus (FMDV) is crucial for vaccine development and control of outbreaks. However, traditional inactivation methods can sometimes damage the viral protein, affecting vaccine efficacy. Therefore, finding new inactivating agents that effectively inactivate the virus while preserving the integrity of its proteins is an important research area. This study investigated the optimal materials (0.04% formaldehyde, 0.001 M binary ethylenimine [BEI], or a combination) for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. Materials and Methods: This study used serotype O FMDV isolated from several areas of East Java. The virus was inoculated into baby hamster kidney-21 cells, and the titer was calculated using the TCID50 Assay. The virus was inactivated using 0.04% formaldehyde, 0.001 M BEI, or a combination of 0.04% formaldehyde and 0.001 M BEI. Inactive viral proteins were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. Results: Serotype O FMDV can be inactivated using 0.04% formaldehyde while preserving specific FMDV proteins, specifically VP0 and VP3 with a molecular weight (MW) of 36 kDa and VP3 with a MW of 24 kDa. Serotype O FMDV can be inactivated by 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 with a MW of 35 kDa, VP3 with a MW of 28 kDa, and VP1 with a MW of 23 kDa. FMDV serotype O can be inactivated using a combination of 0.04% formaldehyde and 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 and VP3 with a MW of 36 kDa and VP3 with a MW of 24 kDa. Conclusion: This study found that 0.04% formaldehyde, alone or in combination with 0.001 M BEI, was effective for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. The limitation of this study was the inactivations of the virus have not yet been tested for their potency on experimental animals. Further research is warranted to investigate the inactivation kinetics of these materials, including their potency on experimental animals. Additionally, a comparison of the inactivation rates between 0.04% formaldehyde alone and the combination with BEI would help to determine the optimal inactivation agent for future applications.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67–78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115–126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures. [ABSTRACT FROM AUTHOR]

The inactivated vaccine is effective in controlling foot-and-mouth disease (FMD), but it has drawbacks such as the need for a biosafety level 3 laboratory facility to handle live foot-and-mouth disease virus (FMDV), high production costs, and biological safety risks. In response to these challenges, we developed a new recombinant protein vaccine (2BT-pIgG-Fc) containing porcine IgG-Fc to enhance protein stability in the body. This vaccine incorporates two-repeat B-cell and one-single T-cell epitope derived from O/Jincheon/SKR/2014. Our study confirmed that 2BTpIgG-Fc and a commercial FMDV vaccine induced FMDV-specific antibodies in guinea pigs at 28 days post-vaccination. The percentage inhibition (PI) value of 2BT-pIgG-Fc was 90.43%, and the commercial FMDV vaccine was 81.75%. The PI value of 2BT-pIgG-Fc was 8.68% higher than that of commercial FMDV vaccine. In pigs, the primary target animals for FMDV, all five individuals produced FMDV-specific antibodies 42 days after vaccination with 2BT-pIgG-Fc. Furthermore, serum from 2BTpIgG-Fc-vaccinated pigs exhibited neutralizing ability against FMDV infection. Intriguingly, the 2BTpIgG-Fc recombinant demonstrated FMDV-specific antibody production rates and neutralization efficiency similar to commercial inactivated vaccines. This study illustrates the potential to enhance vaccine efficacy by strategically combining well-known antigenic domains in the development of recombinant protein-based vaccines. [ABSTRACT FROM AUTHOR]

Introduction: Understanding multi-pathogen infections/exposures in livestock is critical to inform prevention and control measures against infectious diseases. We investigated the co-exposure of foot-and-mouth disease virus (FMDV), Brucella spp., Leptospira spp., and Coxiella burnetii in cattle in three zones stratified by land use change and with different wildlife-livestock interactions in Narok county, Kenya. We also assessed potential risk factors associated with the transmission of these pathogens in cattle. Methods: We identified five villages purposively, two each for areas with intensive (zone 1) and moderate wildlife-livestock interactions (zone 2) and one for locations with low wildlife-livestock interactions (zone 3). We sampled 1,170 cattle from 390 herds through a cross-sectional study and tested the serum samples for antibodies against the focal pathogens using enzymelinked immunosorbent assay (ELISA) kits. A questionnaire was administered to gather epidemiological data on the putative risk factors associated with cattle's exposure to the investigated pathogens. Data were analyzed using the Bayesian hierarchical models with herd number as a random effect to adjust for the within-herd clustering of the various co-exposures among cattle. Results: Overall, 88.0% (95% CI: 85.0-90.5) of the cattle tested positive for at least one of the targeted pathogens, while 41.7% (95% CI: 37.7-45.8) were seropositive to at least two pathogens. FMDV and Brucella spp. had the highest co-exposure at 33.7% (95% CI: 30.9-36.5), followed by FMDV and Leptospira spp. (21.8%, 95% CI: 19.5-24.4), Leptospira spp. and Brucella spp. (8.8%, 95% CI: 7.2-10.6), FMDV and C. burnetii (1.5%, 95% CI: 0.7-2.8), Brucella spp. and C. burnetii (1.0%, 95% CI: 0.3-2.2), and lowest for Leptospira spp. and C. burnetii (0.3%, 95% CI: 0.0-1.2). Cattle with FMDV and Brucella spp., and Brucella spp. and Leptospira spp. co-exposures and those simultaneously exposed to FMDV, Brucella spp. and Leptospira spp. were significantly higher in zone 1 than in zones 2 and 3. However, FMDV and Leptospira spp. co-exposure was higher in zones 1 and 2 than zone 3. Discussion/conclusion: We recommend the establishment of a One Health surveillance system in the study area to reduce the morbidity of the targeted zoonotic pathogens in cattle and the risks of transmission to humans. [ABSTRACT FROM AUTHOR]

Background and Aim: Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-footed animals. It is a major threat to livestock production worldwide, causing significant economic losses. Inactivation of FMD virus (FMDV) is crucial for vaccine development and control of outbreaks. However, traditional inactivation methods can sometimes damage the viral protein, affecting vaccine efficacy. Therefore, finding new inactivating agents that effectively inactivate the virus while preserving the integrity of its proteins is an important research area. This study investigated the optimal materials (0.04% formaldehyde, 0.001 M binary ethylenimine [BEI], or a combination) for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. Materials and Methods: This study used serotype O FMDV isolated from several areas of East Java. The virus was inoculated into baby hamster kidney-21 cells, and the titer was calculated using the TCID50 Assay. The virus was inactivated using 0.04% formaldehyde, 0.001 M BEI, or a combination of 0.04% formaldehyde and 0.001 M BEI. Inactive viral proteins were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. Results: Serotype O FMDV can be inactivated using 0.04% formaldehyde while preserving specific FMDV proteins, specifically VP0 and VP3 with a molecular weight (MW) of 36 kDa and VP3 with a MW of 24 kDa. Serotype O FMDV can be inactivated by 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 with a MW of 35 kDa, VP3 with a MW of 28 kDa, and VP1 with a MW of 23 kDa. FMDV serotype O can be inactivated using a combination of 0.04% formaldehyde and 0.001 M BEI while preserving specific FMDV proteins, specifically VP0 and VP3 with a MW of 36 kDa and VP3 with a MW of 24 kDa. Conclusion: This study found that 0.04% formaldehyde, alone or in combination with 0.001 M BEI, was effective for inactivating and preserving the specific molecular weight of Serotype O FMDV protein. The limitation of this study was the inactivations of the virus have not yet been tested for their potency on experimental animals. Further research is warranted to investigate the inactivation kinetics of these materials, including their potency on experimental animals. Additionally, a comparison of the inactivation rates between 0.04% formaldehyde alone and the combination with BEI would help to determine the optimal inactivation agent for future applications. [ABSTRACT FROM AUTHOR]

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The foot-and-mouth disease virus (FMDV) Leader proteinase Lpro inhibits host mRNA translation and blocks the interferon response which promotes viral survival. Lpro is not required for viral replication in vitro but serotype A FMDV lacking Lpro has been shown to be attenuated in cattle and pigs. However, it is not known, whether leaderless viruses can cause persistent infection in vivo after simulated natural infection and whether the attenuated phenotype is the same in other serotypes. We have generated an FMDV O/FRA/1/2001 variant lacking most of the Lpro coding region (ΔLb). Cattle were inoculated intranasopharyngeally and observed for 35 days to determine if O FRA/1/2001 ΔLb is attenuated during the acute phase of infection and whether it can maintain a persistent infection in the upper respiratory tract. We found that although this leaderless virus can replicate in vitro in different cell lines, it is unable to establish an acute infection with vesicular lesions and viral shedding nor is it able to persistently infect bovine pharyngeal tissues.

Background and Aim: Foot-and-mouth disease (FMD) is a highly contagious viral disease that endangers livestock and the environment with significant economic consequences. This study aimed to validate the inactivation of the Indonesian isolate of foot-and-mouth disease virus (FMDV) with various formaldehyde concentration. Materials and Methods: The experiment started with FMDV being adapted on BHK-21 cells until cytopathic effects (CPE) appeared. The biological titer of the virus was determined using the 50% tissue culture infectious dose (TCID50) assay. The virus was inactivated by exposing the isolate to different formaldehyde (FA) concentrations (0.025%, 0.05%, 0.1%, and 0.2%) at 37°C for 24 h, and residual infectivity was assessed using CPE scoring of reinoculated BHK-21 cells. Results: 72 h post-inoculation, the virulence of the FMDV isolate was indicated by complete CPE on BHK-21 monolayer cells, with a TCID50 value of 109/mL; CPE scoring did not signify significant differences (p < 0.05) among 0.025%, 0.05%, 0.1%, 0.2% FA, and the negative control. All treatment groups showed significant differences (p < 0.05) from the positive control (C+). FA concentrations inactivated the FMDV isolate under the given conditions. 0.025% and 0.05% FA continued to display CPE through the third passage, while 0.2% FA did not significantly differ from 0.1% FA (p > 0.05). 0.1% FA is the optimal concentration for safely and effectively inactivating FMDV. Conclusion: All of the formaldehyde concentrations can completely inactivate the FMDV isolate, with the most optimal and safe concentration being 0.1%.

Background and Aim: Foot-and-mouth disease (FMD) is a highly contagious viral disease that endangers livestock and the environment with significant economic consequences. This study aimed to validate the inactivation of the Indonesian isolate of foot-and-mouth disease virus (FMDV) with various formaldehyde concentration. Materials and Methods: The experiment started with FMDV being adapted on BHK-21 cells until cytopathic effects (CPE) appeared. The biological titer of the virus was determined using the 50% tissue culture infectious dose (TCID50) assay. The virus was inactivated by exposing the isolate to different formaldehyde (FA) concentrations (0.025%, 0.05%, 0.1%, and 0.2%) at 37 °C for 24 h, and residual infectivity was assessed using CPE scoring of reinoculated BHK-21 cells. Results: 72 h post-inoculation, the virulence of the FMDV isolate was indicated by complete CPE on BHK-21 monolayer cells, with a TCID50 value of 109/mL; CPE scoring did not signify significant differences (p < 0.05) among 0.025%, 0.05%, 0.1%, 0.2% FA, and the negative control. All treatment groups showed significant differences (p < 0.05) from the positive control (C+). FA concentrations inactivated the FMDV isolate under the given conditions. 0.025% and 0.05% FA continued to display CPE through the third passage, while 0.2% FA did not significantly differ from 0.1% FA (p > 0.05). 0.1% FA is the optimal concentration for safely and effectively inactivating FMDV. Conclusion: All of the formaldehyde concentrations can completely inactivate the FMDV isolate, with the most optimal and safe concentration being 0.1%. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease is a highly contagious disease affecting cloven-hoofed animals, resulting in considerable economic losses. Its causal agent is foot-and-mouth disease virus (FMDV), a picornavirus. Due to its error-prone replication and rapid evolution, the transmission and evolutionary dynamics of FMDV can be studied using genomic epidemiological approaches. To analyze FMDV evolution and identify possible transmission routes in an Argentinean region, field samples that tested positive for FMDV by PCR were obtained from 21 farms located in the Mar Chiquita district. Whole FMDV genome sequences were obtained by PCR amplification in seven fragments and sequencing using the Sanger technique. The genome sequences obtained from these samples were then analyzed using phylogenetic, phylogeographic, and evolutionary approaches. Three local transmission clusters were detected among the sampled viruses. The dataset was analyzed using Bayesian phylodynamic methods with appropriate coalescent and relaxed molecular clock models. The estimated mean viral evolutionary rate was 1.17 × 10− 2 substitutions/site/year. No significant differences in the rate of viral evolution were observed between farms with vaccinated animals and those with unvaccinated animals. The most recent common ancestor of the sampled sequences was dated to approximately one month before the first reported case in the outbreak. Virus transmission started in the south of the district and later dispersed to the west, and finally arrived in the east. Different transmission routes among the studied herds, such as non-replicating vectors and close contact contagion (i.e., aerosols), may be responsible for viral spread. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is considered as one of the most important contagious viral diseases affecting cloven-footed animals. For effective control of FMD, immunization along with herd immunity is essential in the field conditions. To assure and track the coverage and effectiveness of the vaccination program, the serological studies are very much required after the vaccination program. The present study was aimed to investigate the prevalence of antibodies against structural proteins of FMD virus (FMDV) serotypes of O, A and Asia-1 in seven districts of western Uttar Pradesh, India, and assure the efficacy of vaccination under National Animal Disease Control Program. A total of 308 sera samples were collected from apparent healthy vaccinated cattle and buffaloes from seven districts including Amroha, Baghpat, Bareilly, Bulandsahar, Gautam Budh Nagar, Meerut and Muzaffarnagar of western Uttar Pradesh, India. Determination of antibodies against structural proteins of FMDV was carried out using solid-phase blocking enzyme-linked immunosorbent assay. The protective level of the FMDV serotypes O, A and Asia-1 included in the inactivated trivalent vaccine was 66.55, 48.05 and 47.08% in bovines, respectively. To provide the higher level of protection against the circulating FMDV, the present study recommended the thorough investigation of the immunogenic interaction between the vaccine strains and the field strains. Further investigations should also be conducted with larger sample size and across diverse geographical regions to gain a more comprehensive understanding of herd immunity. [ABSTRACT FROM AUTHOR]

The O/ME-SA/Ind-2001e foot-and-mouth disease virus (FMDV) lineage is a pandemic strain that has recently become dominant within East and Southeast Asia. During May 2023, this viral lineage spread to the Republic of Korea, where 11 outbreaks were detected on cattle and goat farms located in Cheongju and Jeungpyeong. Infected animals displayed typical FMD signs including vesicular lesions with drooling and anorexia. Molecular diagnostic testing and genetic analysis (VP1 sequencing) showed that the causative FMDVs belonged to the O/ME-SA/Ind-2001e lineage and shared the closest nucleotide identity (97.95– 99.21%) to viruses that have been collected from Mongolia and South-East Asian countries. Phylogenetic analyses showed that these sequences were distinct to those collected from the previous Korean O/ME-SA/Ind-2001e lineage outbreaks in 2019, demonstrating that these cases are due to a new incursion of the virus into the country. Prompt implementation of emergency vaccination using antigenically matched serotype O vaccines (r1 value: 0.74–0.93), together with intensive active surveillance on farms surrounding the infected premises has successfully prevented further spread of FMD. These recent FMD outbreaks reinforce the importance of research to understand the risks associated with transboundary pathways in the region, in order to reduce the possibility of a further reintroduction of FMD into the Republic of Korea. [ABSTRACT FROM AUTHOR]

The foot-and-mouth disease virus is a highly contagious and economically devastating virus of cloven-hooved animals, including cattle, buffalo, sheep, and goats, causing reduced animal productivity and posing international trade restrictions. For decades, chemically inactivated vaccines have been serving as the most effective strategy for the management of foot-and-mouth disease. Inactivated vaccines are commercially produced in cell culture systems, which require successful propagation and adaptation of field isolates, demanding a high cost and laborious time. Cell culture adaptation is chiefly indebted to amino acid substitutions in surface-exposed capsid proteins, altering the necessity of RGD-dependent receptors to heparan sulfate macromolecules for virus binding. Several amino acid substations in VP1, VP2, and VP3 capsid proteins of FMDV, both at structural and functional levels, have been characterized previously. This literature review combines frequently reported amino acid substitutions in virus capsid proteins, their critical roles in virus adaptation, and functional characterization of the substitutions. Furthermore, this data can facilitate molecular virologists to develop new vaccine strains against the foot-and-mouth disease virus, revolutionizing vaccinology via reverse genetic engineering and synthetic biology. [ABSTRACT FROM AUTHOR]

The thermal stability of the in-house-developed foot-and-mouth disease (FMD) type O and A viruses was evaluated, and the O Jincheon virus was found to exhibit the lowest thermal stability. To overcome this instability, we proposed a novel stabilizer, calcium chloride. The thermal stability of FMDVs increased up to a CaCl2 concentration of 10 mM, and it had a decreasing trend at >30 mM. The O Jincheon virus showed a significant decrease in the amount of antigen over time at 4 °C. In contrast, the samples treated with CaCl2 showed stable preservation of the virus without significant antigen loss. After the CaCl2-formulated vaccine was administered twice to pigs, the virus neutralization titer reached approximately 1:1000, suggesting that the vaccine could protect pigs against the FMDV challenge. In summary, the O Jincheon virus is difficult to utilize as a vaccine given its low stability during storage after antigen production. However, following its treatment with CaCl2, it can be easily utilized as a vaccine. This study evaluated CaCl2 as a novel stabilizer in FMD vaccines and may contribute to the development of stable vaccine formulations, especially for inherently unstable FMDV strains. [ABSTRACT FROM AUTHOR]

IntroductionUnderstanding multi-pathogen infections/exposures in livestock is critical to inform prevention and control measures against infectious diseases. We investigated the co-exposure of foot-and-mouth disease virus (FMDV), Brucella spp., Leptospira spp., and Coxiella burnetii in cattle in three zones stratified by land use change and with different wildlife-livestock interactions in Narok county, Kenya. We also assessed potential risk factors associated with the transmission of these pathogens in cattle.MethodsWe identified five villages purposively, two each for areas with intensive (zone 1) and moderate wildlife-livestock interactions (zone 2) and one for locations with low wildlife-livestock interactions (zone 3). We sampled 1,170 cattle from 390 herds through a cross-sectional study and tested the serum samples for antibodies against the focal pathogens using enzyme-linked immunosorbent assay (ELISA) kits. A questionnaire was administered to gather epidemiological data on the putative risk factors associated with cattle’s exposure to the investigated pathogens. Data were analyzed using the Bayesian hierarchical models with herd number as a random effect to adjust for the within-herd clustering of the various co-exposures among cattle.ResultsOverall, 88.0% (95% CI: 85.0–90.5) of the cattle tested positive for at least one of the targeted pathogens, while 41.7% (95% CI: 37.7–45.8) were seropositive to at least two pathogens. FMDV and Brucella spp. had the highest co-exposure at 33.7% (95% CI: 30.9–36.5), followed by FMDV and Leptospira spp. (21.8%, 95% CI: 19.5–24.4), Leptospira spp. and Brucella spp. (8.8%, 95% CI: 7.2–10.6), FMDV and C. burnetii (1.5%, 95% CI: 0.7–2.8), Brucella spp. and C. burnetii (1.0%, 95% CI: 0.3–2.2), and lowest for Leptospira spp. and C. burnetii (0.3%, 95% CI: 0.0–1.2). Cattle with FMDV and Brucella spp., and Brucella spp. and Leptospira spp. co-exposures and those simultaneously exposed to FMDV, Brucella spp. and Leptospira spp. were significantly higher in zone 1 than in zones 2 and 3. However, FMDV and Leptospira spp. co-exposure was higher in zones 1 and 2 than zone 3.Discussion/conclusionWe recommend the establishment of a One Health surveillance system in the study area to reduce the morbidity of the targeted zoonotic pathogens in cattle and the risks of transmission to humans.

Molecular epidemiological studies are an important tool for regional and global surveillance of foot-and-mouth disease (FMD). These tests are based on constantly progressing technologies of nucleic acid sequencing and phylogenetic analysis. The use of these technologies made it possible to assess the genetic diversity of the FMD virus, to analyze the evolution of the virus in the enzootic regions, and track the pathways of FMD epizootic and panzootic spread. Molecular epidemiological studies have shown that within the long-known seven serotypes of the FMD virus, there are numerous topotypes (geographical types), genetic lineages and sublineages. Usually, the foot-and-mouth disease virus of a certain topotype and genetic lineage evolves within a certain area, periodically causing regional epizootics. However, over the past 30 years, two FMD panzootics have occurred, invloving several continents. The first panzootic occurred in the late 1990s – early 2000s and was caused by O/ME-SA/PanAsia FMDV, and the second, caused by O/ME-SA/Ind-2001 virus, began in 2013 and continues to the present. The emergence of FMD panzootics is probably a consequence of the economic globalization. FMD is not enzootic in Russia, but sporadic outbreaks of this disease are periodically reported. Molecular epidemiological studies have shown that these outbreaks are caused by the infection introduction from neighboring Asian countries, mainly from China. The FMD virus, which has come to the Russian Federation from other countries, is characterized by great genetic diversity and belongs to three serotypes, five topotypes and eight genetic lineages: O/Cathay, O/ME-SA/PanAsia, O/SEA/Mya-98, O/ME-SA/Ind-2001, O/ME-SA/unnamed, A/Asia/Iran-05, A/Asia/Sea-97, Asia1/V. The results of molecular epidemiological studies are taken into account when vaccine strains are to be selected for preventive vaccination of livestock in FMD high-risk areas. The review is based on the analysis of 68 literature sources.

The global livestock industry is facing a serious threat from a widespread foot-and-mouth disease virus (FMDV) epidemic. The timely detection of FMDV can significantly mitigate its harmful effects. This study aimed to establish and evaluate a TaqMan fluorescence quantitative PCR assay to assess its sensitivity, specificity, reproducibility, and stability. The standard curve equation range is 6.43 × 109–6.43 × 101 copies/µL, with an R2 value of 0.996 and a standard curve equation of y = −3.586x + 36.245. The method successfully detected 64.3 copies/µL of the target gene for FMDV and exhibited high specificity for FMDV. Repeatability tests demonstrated low coefficients of variation within and between groups (

Foot-and-mouth disease (FMD) is a highly contagious viral infection causing acute and severe vesicular lesions in cattle and pigs, which has prompted global vaccination policies. This study presents a technique for enhancing antigen yield in SAT1 BOT and SAT3 ZIM by treatment with calcium chloride (CaCl2). We tested changes in cell viability in BHK-21 suspension cells treated with varying concentrations of CaCl2. The optimal CaCl2 concentration was determined based on antigen yield. The timing of CaCl2 supplementation relative to FMD virus inoculation was tested. Finally, the optimal medium for antigen production was identified. We observed a concentration-dependent decrease in BHK-21 cell viability at >7.5 mM CaCl2. A CaCl2 concentration of 3 mM yielded the most antigens. CaCl2 supplementation relative to FMD virus infection was optimal 2 h before or with viral inoculation. CD-BHK 21 medium supplemented with CaCl2 was the most productive medium. Specifically, SAT1 BOT and SAT3 ZIM showed improved antigen production in CD-BHK 21 medium with 3 mM CaCl2, while Provero-1 and Cellvento BHK-200 media showed no significant enhancement. Overall, CaCl2 supplementation enhanced FMD antigen productivity. This study provides a useful framework for enhancing antigen production efficiently in the FMD vaccine industry. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) remains a challenge for clovenhooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines. IMPORTANCE Although the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines. [ABSTRACT FROM AUTHOR]

To develop a novel water-in-oil-in-water (W/O/W) adjuvant and evaluate the effect on foot-and-mouth disease (FMD) inactivated vaccine, in this study, we prepared the novel nano-emulsion adjuvant based on QS-21 (BEA) which is composed of the mixture of mineral oil Marcol52, surfactant Tween80, oleate polyoxyethylene ether ester, polyoxyethylene palmitic acid ester and span80, cosurfactant polyethylene glycol and QS-21. The two-step emulsification method formed the W/O/W nano-emulsion with two films and three-phase structures. The effective particle diameter of the BEA was about 184 nm, and it has good thermal stability. Then, BEA was emulsified as an adjuvant to prepare for the inactivated FMDV vaccine, and BALB/c mice and pigs were immunized to evaluate its safety and immunization effect. The results showed that the inactivated BEA-FMDV vaccine significantly increased BALB/c mice and pigs' antibodies and cytokine IFN-γ in serum. Meanwhile, the pig-neutralizing antibodies were higher than control group. Safety tests found no symptoms of FMD or significant toxic reactions. After 28 days of immunization, the protection rate can reach 93.3%. The BEA vaccine had good stability at 4 °C, no stratification after 180 days, and the content of 146S in the vaccine did not decrease. In conclusion, the BEA prepared in this study is suitable for FMDV inactivated vaccine and is an effective adjuvant. [ABSTRACT FROM AUTHOR]

Simple Summary: Foot-and-mouth disease (FMD) is endemic in the United Arab Emirates (UAE); however, FMD virus (FMDV) genotyping in small ruminants or cattle has never been reported. This study focused on FMD outbreaks that occurred during 2021 in two different regions in the Emirate of Abu Dhabi, where cases affected sheep, goats, cattle, and Arabian oryx. VP1 sequences from different isolates were characterised, and phylogenetic analysis revealed epidemiological connections between FMDV sequences collected in the UAE and neighbouring countries, which highlights the importance of implementing measures such as vaccination, animal movement controls, and biosecurity to limit the spread of the disease. Foot-and-mouth disease (FMD) is an endemic disease in the United Arab Emirates (UAE) in both wild and domestic animals. Despite this, no systematic FMD outbreak investigation accompanied by molecular characterisation of FMD viruses (FMDVs) in small ruminants or cattle has been performed, and only a single report that describes sequences for FMDVs in wildlife from the Emirate has been published. In this study, FMD outbreaks that occurred in 2021 in five animal farms and one animal market in the Emirate of Abu Dhabi were investigated. Cases involved sheep, goats, and cattle, as well as Arabian oryx (Oryx leucoryx). Twelve samples were positive for FMDV via RT-qPCR, and four samples (Arabian oryx n = 1, goat n = 2, and sheep n = 1) were successfully genotyped using VP1 nucleotide sequencing. These sequences shared 88~98% identity and were classified within the serotype O, Middle East–South Asia topotype (O/ME-SA). Phylogenetic analysis revealed that the Arabian oryx isolate (UAE/2/2021) belonged to the PanAsia-2 lineage, the ANT-10 sublineage, and was closely related to the FMDVs recently detected in neighbouring countries. The FMDV isolates from goats (UAE/10/2021 and UAE/11/2021) and from sheep (UAE/14/2021) formed a monophyletic cluster within the SA-2018 lineage that contained viruses from Bangladesh, India, and Sri Lanka. This is the first study describing the circulation of the FMDV O/ME-SA/SA-2018 sublineage in the UAE. These data shed light on the epidemiology of FMD in the UAE and motivate further systematic epidemiological studies and genomic sequencing to enhance the ongoing national animal health FMD control plan. [ABSTRACT FROM AUTHOR]

According to the World Organisation for Animal Health, foot-and-mouth disease (FMD) is regularly reported in domestic and wild cloven-hoofed animals in Africa. G-I, G-IV, G-VI, G-VII, ASIA/Iran-05 genetic lineages of serotype А FMD virus are considered to be the most widespread on the African continent. Given the close economic and trade relations maintained by the Russian Federation with the countries of North Africa, of particular interest for us is studying the FMD virus of serotype A G-IV genetic lineage, which has been responsible for the infection outbreaks in the naturally susceptible animal population of the said region every year since 2012, and there is a risk of introduction of this virus genotype in to the Russian Federation. Therefore, the issues of FMD introduction risk assessment and timely diagnosis are relevant for the Veterinary Service of Russia. FMD virus A 2205/G-IV strain tested for its biological and antigenic properties in cell cultures and naturally susceptible animals (cattle and pigs) was adapted for its reproduction in initially trypsinized porcine kidney (PK) cell culture, continuous monolayer cell cultures (IB-RS-2, PSGK-30, YaDK-04, ВНК-21) by five serial passages. The virus was considered to be adapted when 90–95% cytopathic effect developed within 14–19 hours after the cell culture infection. The virus adapted to the cell cultures was tested for its infectivity with microtitration in IB-RS-2 cell culture. The virus strain tested for vaccine matching with microneutralization test (MNT) demonstrated significant difference from production A/Turkey/06, А22 No. 550/Azerbaijan/64, A22/Iraq/64, A/Iran/97, A No. 2155/Zabaikalsky/2013, A No. 2166/Krasnodarsky/2013, A No. 2269/ARRIAH/2015 strains of FMD virus.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67–78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115–126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures.

Foot-and-mouth disease (FMD) isa highlycontagiousviraldiseaseof cloven-hoovedanimals that can causeepidemicsandgreateconomic losses. Thedisease remains a huge problem in developing countries and poses a constant threat to developed countries. Detection of antibodies to FMD virus non-structural proteins in thebloodof susceptibleanimals isan important tool for thediseasecontrol. This is theonly way for reliabledifferentiation of vaccinatedanimals fromconvalescentandvirus-carrieranimals. Variousmodificationsof solid-phaseenzymeimmunosorbentassay (ELISA) havebeen developedfordetection of antibodies toFMD virus nonstructuralproteins. RecombinantFMD virus non-structuralproteins, mostly 3АВС or 3АВ, areusedasan antigen for such assay. In ashort time, recombinant FMD virusantigen-basedELISA hasevolvedfrom an in-houselaboratory methodtocommonlyavailablecommercial test systems, mostof which have high diagnostic specificityandsensitivity. Thesaid methodis widelyusedforFMD surveillance. In thecountriesandzones freefrom FMD withoutvaccination theELISA fordetection ofantibodiesagainstFMD virus non-structuralproteinsisusedasaprimarymethodforFMD serologicalmonitoring andretrospectivediagnosis. In thecountriesandzones freefromFMD with vaccination thisELISA isusedforconfirmation of thevirus infection absencein vaccinated herds. In South America, ELISA fordetection ofantibodiesagainstFMD virus non-structuralproteins wasusedfordetection of infectedanimalsduring thediseaseeradication. Currently, it isusedfor monitoringfor thevirus circulation in stillFMD infected Asian and African countries implementingprogressive FMD controlprogramme. In Russia havingzones where anti-FMD vaccination is carriedout this methodis a mandatory tool of thedisease surveillance. The review isbasedon theanalysisof 65 publications.

Background and Aim: Various methods can detect foot-and-mouth disease (FMD) in cows, but they necessitate resources, time, costs, laboratory facilities, and specific clinical specimen submission, often leading to FMD virus (FMDV) diagnosis delays. The 2022 FMD outbreak in East Java, Indonesia, highlighted the need for an easy, inexpensive, rapid, and accurate detection approach. This study aims to devise a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technique and phylogenetic analysis to detect the serotype O FMDV outbreak in East Java. Materials and Methods: Swab samples were collected from the foot vesicles, nasal secretions, and saliva of five suspected FMDV-infected cows in East Java between June and July 2022. The RT-LAMP design used hydroxy naphthol blue dye or SYBR Green I dye, with confirmatory analysis through reverse transcriptase polymerase chain reaction (RT-PCR) targeting 249 base pairs. PCR products underwent purification, sequencing, and nucleotide alignment, followed by phylogenetic analysis. Results: The RT-LAMP method using hydroxy naphthol blue dye displayed a positive reaction through a color shift from purple to blue in the tube. Naked-eye observation in standard light or ultraviolet (UV) light at 365 nm, with SYBR Green I stain, also revealed color change. Specifically, using SYBR Green I dye, UV light at 365 nm revealed a color shift from yellow to green, signifying a positive reaction. Nucleotide alignment revealed mutations and deletion at the 15th sequence in the JT-INDO-K3 isolate from the East Java FMDV outbreak. Despite differing branches, the phylogenetic tree placed it in the same cluster as serotype O FMDV from Malaysia and Mongolia. Conclusion: JT-INDO-K3 exhibited distinctions from Indonesian serotype O FMDV isolates and those documented in GenBank. Then, the RT-LAMP method used in this study has a detection limit 10 times higher latter than the conventional RT-PCR limit, without any cross-reactivity among strains.

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. Thismolecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O. [ABSTRACT FROM AUTHOR]

• Efficacious quadrivalent foot-and-mouth disease vaccine for East Africa. • Recombinant chimeric foot-and-mouth disease virus vaccine. • Foot-and-mouth disease vaccine gives cross-protection against East African strains. Foot-and-mouth disease (FMD) is a highly contagious viral disease of livestock which is prevalent across Africa, the Middle East, Asia, and South America where it has a severe economic impact on the agriculture industry. Vaccination with inactivated viral vaccines is used as the main control measure in these endemic regions of the world, however the presence of multiple serotypes, subtypes, and the continual emergence of new, antigenically divergent strains limits its effectiveness. East Africa (EA) has been identified as a region that would particularly benefit from updated FMD vaccines, since those currently in use contain older strains which do not provide good protection against contemporary strains. Four serotypes are currently circulating in EA, necessitating the development of a quadrivalent vaccine containing representative strains of each serotype. A key consideration in the selection of vaccine strains is the stability of the virus particle, since the capsids readily dissociate on exposure to elevated temperatures, but only intact capsids induce protective immunity to FMD. Therefore, with a view to producing a more stable, updated quadrivalent vaccine for EA, we recently screened a panel of foot-and-mouth disease virus (FMDV) isolates from the region to select strains with naturally higher thermostabilities and confirmed their immunogenicity in cattle. Herein we describe the formulation and serological assessment of wild-type and recombinant quadrivalent vaccine candidates comprising these stable strains, and demonstrate that both vaccines generate high neutralising antibody titres against the homologous strains and also to heterologous strains from EA. Importantly, the vaccine passed the criteria set by the AgResults vaccine challenge project and offers good cross-protection against a panel of regional FMDV strains. [ABSTRACT FROM AUTHOR]

Plant-derived antibodies fused to horseradish peroxidase (HRP) have been developed as efficient diagnostic reagents for detecting foot-and-mouth disease virus (FMDV). The antibodies were produced in Nicotiana benthamiana plants and targeted to the apoplast, resulting in high protein abundance and solubility. The plant-produced HRP-fused antibodies showed superior detection abilities compared to antibodies produced in mammalian CHO cells. They also exhibited high HRP activity and heme content, which are critical for HRP function. The plant-derived HRP-fused antibodies performed well in serodiagnosis assays, demonstrating high sensitivity in detecting FMDV antibodies in serum samples. This platform has potential for the development of reagents for disease detection in clinical settings. [Extracted from the article]

Foot-and-mouth disease (FMD) is endemic in many Asian countries, with outbreaks occurring regularly due to viruses from serotypes O, A, and Asia1 that co-circulate in the region. The ability to rapidly characterize new virus occurrences provides critical information to understand the epidemiology and risks associated with field outbreaks, and helps in the selection of appropriate vaccines to control the disease. FMD lineage-specific characterization is usually determined through sequencing; however, this capacity is not always readily available. In this study, we provide a panel of real-time RT-PCR (rRT-PCR) assays to allow differentiation of the FMD virus (FMDV) lineages known to have been co-circulating in Asia during 2020. This panel included five new rRT-PCR assays designed to detect lineages O/ME-SA/PanAsia-PanAsia-2, O/ME-SA/Ind-2001, O/SEA/Mya-98, O/CATHAY, and A/ASIA/Sea-97, along with three published rRT-PCR assays for A/ASIA/Iran-05, A/ASIA/G-VII, and Asia1 serotypes. Samples of known FMD lineage (n = 85) were tested in parallel with all eight lineage-specific assays and an established 3D pan-FMD rRT-PCR assay, and comparative limit of detection (LOD) experiments were conducted for the five newly developed assays. All samples (85/85) were assigned to the correct serotype, and the correct lineage was assigned for 70 out of 85 samples where amplification only occurred with the homologous assay. For 13 out of 85 of the samples, there was amplification in two assays; however, the correct lineage could be designated based on the strongest Ct values for 12 out of 13 samples. An incorrect lineage was assigned for 3 out of 85 samples. The amplification efficiencies for the five new rRT-PCR assays ranged between 79.7 and 100.5%, with nucleic acid dilution experiments demonstrating broadly equivalent limits of detection when compared to the 3D pan-FMD rRT-PCR assay. These new tests, together with other published lineage-specific rRT-PCR assays, constitute a panel of assays (or molecular toolbox) that can be selected for use in FMD endemic countries (individually or a subset of the assays depending on region/lineages known to be circulating) for rapid characterization of the FMDV lineages circulating in Asia at a relatively low cost. This molecular toolbox will enhance the ability of national laboratories in endemic settings to accurately characterize circulating FMDV strains and facilitate prompt implementation of control strategies, and may be particularly useful in settings where it is difficult to access sequencing capability.

FMD remains endemic in many Asian and African countries where multiple variants of serotypes O and A, among others, currently circulate. Due to lack of cross-protection between serotypes and incomplete protection between some strains even within a serotype, an important challenge for the application of effective vaccination programs is to select highly immunogenic and widely cross-reactive vaccine strains. Adaptation of a candidate field virus for use as a vaccine can be quite complex, so that whenever possible, the use of well-established vaccine viruses could have enormous advantages. FMD vaccine strains harmonized for use in South America have shown excellent results in FMD control, not only in the region, where it is still used systematically as a preventive measure, but also more recently in some Asian countries. To gain further insight into the immunogenic spectrum of these strains, VN tests (VNT) were performed with sera from cattle and/or pigs vaccinated with monovalent (type O) or trivalent (types O and A) formulations against 122 type O and 32 type A field viruses isolated from 35 countries in Asia and Africa, belonging to different lineages. Almost all VNT titers obtained were within the expected protective level, indicating the wide immunogenic spectrum of high potency FMD vaccines formulated with O 1 Campos, A24 Cruzeiro and A Argentina 2001 South American vaccine strains belonging to EURO-SA topotypes against currently active viruses from other topotypes. These in vitro results are in line with previously reported in vivo challenge tests in pigs against three A/ASIA/Sea-97 isolates and two isolates belonging to type O lineages O/SEA/Mya-98 and O/ME-SA/Ind-2001e. [ABSTRACT FROM AUTHOR]

Background and Aim: Various methods can detect foot-and-mouth disease (FMD) in cows, but they necessitate resources, time, costs, laboratory facilities, and specific clinical specimen submission, often leading to FMD virus (FMDV) diagnosis delays. The 2022 FMD outbreak in East Java, Indonesia, highlighted the need for an easy, inexpensive, rapid, and accurate detection approach. This study aims to devise a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technique and phylogenetic analysis to detect the serotype O FMDV outbreak in East Java. Materials and Methods: Swab samples were collected from the foot vesicles, nasal secretions, and saliva of five suspected FMDV-infected cows in East Java between June and July 2022. The RT-LAMP design used hydroxy naphthol blue dye or SYBR Green I dye, with confirmatory analysis through reverse transcriptase polymerase chain reaction (RT-PCR) targeting 249 base pairs. PCR products underwent purification, sequencing, and nucleotide alignment, followed by phylogenetic analysis. Results: The RT-LAMP method using hydroxy naphthol blue dye displayed a positive reaction through a color shift from purple to blue in the tube. Naked-eye observation in standard light or ultraviolet (UV) light at 365 nm, with SYBR Green I stain, also revealed color change. Specifically, using SYBR Green I dye, UV light at 365 nm revealed a color shift from yellow to green, signifying a positive reaction. Nucleotide alignment revealed mutations and deletion at the 15th sequence in the JT-INDO-K3 isolate from the East Java FMDV outbreak. Despite differing branches, the phylogenetic tree placed it in the same cluster as serotype O FMDV from Malaysia and Mongolia. Conclusion: JT-INDO-K3 exhibited distinctions from Indonesian serotype O FMDV isolates and those documented in GenBank. Then, the RT-LAMP method used in this study has a detection limit 10 times higher latter than the conventional RT-PCR limit, without any cross-reactivity among strains. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is an acute, highly contagious disease of cloven-hoofed animals caused by FMD virus (FMDV). Currently, the molecular pathogenesis of FMDV infection remains poorly understood. Here, we demonstrated that FMDV infection induced gasdermin E (GSDME)-mediated pyroptosis independent of caspase-3 activity. Further studies showed that FMDV 3Cpro cleaved porcine GSDME (pGSDME) at the Q271-G272 junction adjacent to the cleavage site (D268-A269) of porcine caspase-3 (pCASP3). The inhibition of enzyme activity of 3Cpro failed to cleave pGSDME and induce pyroptosis. Furthermore, overexpression of pCASP3 or 3Cpro-mediated cleavage fragment pGSDME-NT was sufficient to induce pyroptosis. Moreover, the knockdown of GSDME attenuated the pyroptosis caused by FMDV infection. Our study reveals a novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the pathogenesis of FMDV and the design of antiviral drugs. [ABSTRACT FROM AUTHOR]

Abstract Background Peste des petits ruminants (PPR), foot-and-mouth disease (FMD) and sheep pox and goat pox are three important infectious diseases that infect goats, sheep and other small ruminants. It is well-known that the prevention of three diseases rely mainly on their individual vaccines. However, the vaccines have a variety of different disadvantages, such as short duration of immunity, increasing the number of vaccinations, and poor thermal stability. The purpose of this study is to construct a recombinant goat pox virus (rGPV) capable of expressing the F gene of PPRV and the P12A3C gene of FMDV as a live vector vaccine. Results The IRES, FMDV P12A3C and PPRV F genes into the multi-cloning site of the universal transfer plasmid pTKfpgigp to construct a recombinant transfer plasmid pTKfpgigpFiP12A3C, and transfected GPV-infected lamb testis (LT) cells with liposomes and produced by homologous recombination Recombinant GPV (rGPV/PPRVF-FMDVP12A3C, rGPV). The rGPV was screened and purified by green florescence protein (GFP) and xanthine-guanine-phosphoribosyltransferase gene (gpt) of Escherichia coli as selective markers, and the expression of rGPV in LT cells was detected by RT-PCR and immunofluorescence techniques. The results showed that the virus strain rGPV/PPRVF-FMDVP12A3C containing FMDV P12A3C and PPRV F genes was obtained. The exogenous genes FMDV P12A3C and PPRV F contained in rGPV were normally transcribed and translated in LT cells, and the expression products could specifically react with PPRV and FMDV antiserum. Then, the rGPV was intradermally inoculated with goats, the animal experiments showed that rGPV/PPRVF-FMDVP12A3C could induce high levels of specific antibodies against GPV, PPRV and FMDV. Conclusions The constructed rGPV induced high levels of specific antibodies against GPV, PPRV and FMDV. The study provides a reference for “ one vaccine with multiple uses “ of GPV live vector vaccine.

It has been reported that a commercial solid-phase competition ELISA (SPCE) commonly used in year-round serum surveillance in South Korea may not be sufficient for use in the sero-monitoring of protective antibody responses following vaccination. Here, we established a liquid-phase blocking ELISA (LPBE) method based on the purified 146S antigen of the foot-and-mouth disease virus (FMDV) A/Yeoncheon/SKR/2017 strain, and its analytical performance was assessed using a wide range of pig sera. The established LPBE performed well regardless of whether antisera were elicited by a homologous (A/Yeoncheon/SKR/2017) or heterologous virus (A/Pocheon/SKR/2010) classified in the A/ASIA/Sea-97 lineage. The LPBE performance was also assessed using sera obtained from pigs in the field that underwent routine vaccination, demonstrating that the assay exhibited higher consistency with the virus neutralization test (standard reference test) than the commercial SPCE. This suggested that the LPBE performed well for antisera induced by the FMDV serotype A vaccine strains used in South Korea, namely, A22/IRQ/24/64, A/Argentina/2001 + A24/Cruzeiro and A/Zabaikalsky/2013. Overall, the established LPBE is considered to be a convenient serological assay for use in postvaccination sero-monitoring.

The construction of a full-length infectious clone, essential for molecular virological study and vaccine development, is quite a challenge for viruses with long genomes or possessing complex nucleotide sequence structures. Herein, we have constructed infectious clones of foot-and-mouth disease virus (FMDV) types O and A by joining each viral coding region with our pKLS3 vector in a single isothermal reaction using Gibson Assembly (GA). pKLS3 is a 4.3-kb FMDV minigenome. To achieve optimal conditions for the DNA joining, each FMDV coding sequence was divided into two overlapping fragments of approximately 3.8 and 3.2 kb, respectively. Both DNA fragments contain the introduced linker sequences for assembly with the linearized pKLS3 vector. FMDV infectious clones were produced upon directly transfecting the GA reaction into baby hamster kidney-21 (BHK-21) cells. After passing in BHK-21 cells, both rescued FMDVs (rO189 and rNP05) demonstrated growth kinetics and antigenicity similar to their parental viruses. Thus far, this is the first report on GA-derived, full-length infectious FMDV cDNA clones. This simple DNA assembly method and the FMDV minigenome would facilitate the construction of FMDV infectious clones and enable genetic manipulation for FMDV research and custom-made FMDV vaccine production. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is one of the most contagious livestock diseases in the world, posing a constant global threat to the animal trade and national economies. The chemokine C-X-C motif chemokine ligand 13 (CXCL13), a biomarker for predicting disease progression in some diseases, was recently found to be increased in sera from mice infected with FMD virus (FMDV) and to be associated with the progression and severity of the disease. However, it has not yet been determined which cells are involved in producing CXCL13 and the signaling pathways controlling CXCL13 expression in these cells. In this study, the expression of CXCL13 was found in macrophages and T cells from mice infected with FMDV, and CXCL13 was produced in bone-marrow-derived macrophages (BMDMs) by activating the nuclear factor-kappaB (NF-κB) and JAK/STAT pathways following FMDV infection. Interestingly, CXCL13 concentration was decreased in sera from interleukin-10 knock out (IL-10-/-) mice or mice blocked IL-10/IL-10R signaling in vivo after FMDV infection. Furthermore, CXCL13 was also decreased in IL-10-/- BMDMs and BMDMs treated with anti-IL-10R antibody following FMDV infection in vitro. Lastly, it was demonstrated that IL-10 regulated CXCL13 expression via JAK/STAT rather than the NF-κB pathway. In conclusion, the study demonstrated for the first time that macrophages and T cells were the cellular sources of CXCL13 in mice infected with FMDV; CXCL13 was produced in BMDMs via NF-κB and JAK/STAT pathways; and IL-10 promoted CXCL13 expression in BMDMs via the JAK/STAT pathway. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV), classical swine fever virus (CSFV), and foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases (TADs) that have a significant economic impact. The rapid and unequivocal identification of these pathogens and distinction from other animal diseases based on clinical symptoms in the field is difficult. Nevertheless, early pathogen detection is critical in limiting their spread and impact as is the availability of a reliable, rapid, and cost-effective diagnostic test. The purpose of this study was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples using next generation sequencing of short PCR products as a point-of-care diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products were then sequenced in Mongolia using the MinION nanopore portable sequencer. The resulting sequencing reads successfully identified the respective pathogens that exhibited 91–100% nucleic acid similarity to the reference strains. Phylogenetic analyses suggest that the Mongolian virus isolates are closely related to other isolates circulating in the same geographic region. Based on our results, sequencing short fragments derived by conventional (RT-) PCR is a reliable approach for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource countries. [ABSTRACT FROM AUTHOR]

The thermal stability of the in-house-developed foot-and-mouth disease (FMD) type O and A viruses was evaluated, and the O Jincheon virus was found to exhibit the lowest thermal stability. To overcome this instability, we proposed a novel stabilizer, calcium chloride. The thermal stability of FMDVs increased up to a CaCl2 concentration of 10 mM, and it had a decreasing trend at >30 mM. The O Jincheon virus showed a significant decrease in the amount of antigen over time at 4 °C. In contrast, the samples treated with CaCl2 showed stable preservation of the virus without significant antigen loss. After the CaCl2-formulated vaccine was administered twice to pigs, the virus neutralization titer reached approximately 1:1000, suggesting that the vaccine could protect pigs against the FMDV challenge. In summary, the O Jincheon virus is difficult to utilize as a vaccine given its low stability during storage after antigen production. However, following its treatment with CaCl2, it can be easily utilized as a vaccine. This study evaluated CaCl2 as a novel stabilizer in FMD vaccines and may contribute to the development of stable vaccine formulations, especially for inherently unstable FMDV strains.