Search Results (206)

Background/Objectives: An accurate quantification of the effective antigens from different serotypes is essential for the quality control of multivalent vaccines, but it remains challenging. Herein, we developed a simple and high-throughput method using differential scanning fluorimetry (DSF) for quantifying foot-and-mouth disease virus (FMDV) antigens in monovalent and bivalent vaccines. Methods: Purified serotypes A and O FMDV were used to establish and validate the method. The DSF parameters, including the dye concentration, thermal scanning velocity, and PCR tube material, were optimized at different FMDV concentrations. The established DSF method was validated for the quantification of monovalent and A/O bivalent FMDV, and was compared with the ultracentrifugation of 86 samples from different processing stages and serotypes. Results: The DSF showed that the melting temperature (T_m) of type A (56.2 °C) was significantly higher than that of type O FMDV (50.5 °C), indicating that their T_m can be distinguished in bivalent antigens. After optimizing the DSF parameters, a strong correlation (R² > 0.998) was observed between the 146S concentration and the maximum of the first derivative of the DSF fluorescence (d(RFU)/dT) for both serotypes A and O FMDV. The method demonstrated good reproducibility (RSD < 10%) and high sensitivity (limit of detection: 0.7 μg/mL). Using a multiple linear regression analysis, the simultaneous quantification of A and O FMDV in the bivalent mixtures achieved recovery rates of 82.4–105.5%, with an RSD < 10% for most of the samples. Additionally, the DSF results correlated well with the ultracentrifugation data (Pearson ρ = 0.9789), validating its accuracy and broad applicability. Conclusions: In summary, DSF represents a simple, rapid, and high-throughput tool for the quality control of monovalent and bivalent FMDV vaccines. Full article

Foot-and-mouth disease (FMD) significantly impacts global livestock industries, with raw milk transportation posing a recognized pathway for viral dissemination, particularly in endemic regions. This study aimed to evaluate the risk of FMD virus (FMDV) introduction and transmission to dairy farms via raw milk transportation in Ban Thi District, Thailand. A qualitative risk assessment methodology, adhering to WOAH guidelines, was employed. Data were collected through structured farmer surveys (n = 109), expert interviews (n = 12), and reviews of national disease surveillance data and scientific literature. The risk assessment, utilizing a scenario tree approach for domestic dairy cattle, revealed a moderate overall risk of FMDV transmission. This finding is primarily attributed to critical gaps in on-farm biosecurity practices, potential contamination at milk collection centers, and significant challenges in detecting subclinical carrier animals. While the qualitative approach presented inherent limitations and uncertainties, the study successfully highlighted key vulnerabilities. The results underscore the urgent necessity for implementing targeted biosecurity protocols, developing more robust surveillance strategies for FMDV carriers, and establishing standardized risk assessment frameworks to mitigate potential outbreaks and protect the regional dairy industry. Full article

PLA2G16 is a member of the phospholipase A2 family that catalyzes the generation of lysophosphatidic acids (LPAs) and free fatty acids (FFAs) from phosphatidic acid. Previously, PLA2G16 was found to be a host factor for picornaviruses. Here, we discovered that the Foot-and-Mouth Disease Virus (FMDV) infection led to an elevation in PLA2G16 transcription. We established PLA2G16 overexpression and knockdown cell lines in PK-15 cells to investigate the potential role of PLA2G16 in FMDV infection. Our findings revealed that during FMDV infection, PLA2G16-overexpressing cells had increased levels of phosphorylated STAT1 and the interferon-stimulating factors ISG15 and ISG56. In PLA2G16-overexpressing cells, p-STAT1 was observed at higher levels and earlier than in wild-type cells. Subsequent research demonstrated that PLA2G16 specifically promoted an antiviral innate immune response against FMDV. The host could detect the early release of FMDV viral nucleic acid in PLA2G16-overexpressing cells and trigger the interferon signaling pathway. Additionally, we discovered that the supernatants of PLA2G16-overexpressing cells stimulated the production of higher levels of ISG56 and phosphorylated STAT1. This suggests that PLA2G16-overexpressing cells can activate the innate immune pathway of uninfected cells after FMDV infection. Full article

Foot-and-mouth disease virus (FMDV) continues to pose a significant threat to livestock health and the global agricultural economy, particularly in endemic regions of Asia, Africa, and the Middle East. Current vaccines based on chemically inactivated FMDV present several challenges, including biosafety risks, high production costs, and limited effectiveness against emerging viral variants. To overcome these limitations, we developed virus-like particle (VLP) vaccines targeting FMDV serotypes O, A, and Asia1 using a recombinant Escherichia coli expression system. The resulting VLPs self-assembled into 25–30 nm particles with native-like morphology and antigenic properties, as confirmed by transmission electron microscopy, SDS-PAGE, and Western blot analysis. Immunogenicity was evaluated in mice and pigs using ELISA and virus neutralization tests (VNT), and protective efficacy was assessed through viral challenge studies. All VLPs induced strong serotype-specific antibody responses, with ELISA PI values exceeding 50% and significantly increased VNT titers after booster immunization. In mice, PD₅₀ values were 73.5 (A-type), 32.0 (O-type), and 55.7 (Asia1-type); in pigs, PD₅₀ values reached 10.6 (O-type) and 22.6 (Asia1-type). Notably, the vaccines induced robust immune responses even at lower antigen doses, suggesting the feasibility of dose-sparing formulations. These findings demonstrate that FMDV VLPs produced in E. coli are highly immunogenic and capable of eliciting protective immunity, highlighting their promise as safe, scalable, and cost-effective alternatives to conventional inactivated FMD vaccines. Full article

Background: The 2C protein of foot-and-mouth disease virus (FMDV), a member of helicase superfamily 3 (SF3), drives viral genome replication and serves as a critical target for antiviral drug development. Methods: A fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) platform was developed to identify 2C helicase inhibitors. Primary screening evaluated 4424 compounds for helicase inhibition. Molecular docking analyzed inhibitor interactions with the N207 residue within the catalytic core and helicase inhibition assays classified the inhibitor type (mixed, competitive, noncompetitive). Differential scanning fluorimetry (nanoDSF) quantified 2C thermal destabilization. Antiviral activity was assessed via indirect immunofluorescence, RT-qPCR, and plaque reduction assays. Results: Six compounds inhibited 2C helicase activity at >620 μM. Molecular docking revealed hydrogen bonding, hydrophobic interactions, and π-cation stabilization at the catalytic core. 2-MPO and MPPI were classified as mixed-type inhibitors, 5-TzS⁻ and 2-PyOH as competitive, and DCMQ/Spiro-BD-CHD-dione as noncompetitive. NanoDSF showed a ΔT_m ≥ 1.5 °C (2.5 mM compounds), with reduced destabilization in N207A mutants. Antiviral assays identified 2-MPO and MPPI as optimal inhibitors. MPPI achieved effective FMDV suppression at 160 μM, exhibiting two orders of magnitude higher potency than 2-MPO (400 μM). Conclusions: The established FRET-based HTS platform targeting 2C helicase facilitates anti-FMDV lead discovery, while 2C inhibitors may serve as an effective therapeutic strategy against other picornaviruses. Full article

Background/Objectives: Foot-and-mouth disease virus (FMDV) poses a continuous threat to livestock health and agricultural economies. Current vaccines require high biosafety standards and are costly to produce. While novel vaccine technologies have been explored, most fail to meet industrial scalability, cost-efficiency, or multiserotype flexibility required for effective FMD control. This study aimed to evaluate the feasibility of using a high-cell density transient gene expression (TGE) system in CHO cells for the production of FMDV virus-like particles (VLPs) as a recombinant vaccine platform. Methods: VLP expression was optimized by adjusting cDNA and polyethyleneimine (PEI) concentrations. Expression yields were compared at 24 and 48 h post-transfection to determine optimal harvest timing. We further tested the system’s capacity to express different serotypes and chimeric constructs, incorporating VP1 sequences from various FMDV strains. Immunogenicity was evaluated in swine using VLPs from the A2001 Argentina strain as a model. Results: Optimal VLP expression was achieved at 24 h post-transfection. Chimeric constructs incorporating heterologous VP1 regions were successfully expressed. Immunized pigs developed protective antibody titers as measured by a virus neutralization test (VNT, log₁₀ titer 1.43) and liquid-phase blocking ELISA (LPBE, titer 2.20) at 28 days post-vaccination (dpv). Titers remained above protective thresholds up to 60 dpv with a single dose. A booster at 28 dpv further elevated titers to levels comparable to those induced by the inactivated vaccine. Conclusions: Our results demonstrate the feasibility of using CHO cell-based TGE for producing immunogenic FMDV VLPs. This platform shows promise for scalable, cost-effective, and biosafe development of recombinant FMD vaccines. Full article

Foot-and-mouth disease (FMD) is a devastating infectious viral disease of cloven-hoofed animals. Differentiating FMD from other vesicular diseases is difficult based on only clinical symptoms, requiring an appropriate laboratory diagnostic test. The double-antibody sandwich (DAS)-ELISA is a reliable diagnostic technique for antigen detection and serotyping of FMDV. However, classical DAS-ELISAs use polyclonal antibodies (pAbs), which are inconsistent in yields and limited in large-scale applications compared to hybridoma cell-secreted laboratory-made monoclonal antibodies (mAbs). Therefore, this study aimed to develop simplified and sensitive FMD serotype-specific DAS-ELISAs using HRP-conjugated mAbs and a TMB substrate. Six FMDV serotype-specific mAb-DAS-ELISAs were developed. All assays were optimized using BEI-inactivated FMD antigens. Real-time reverse-transcriptase PCR (RRT-PCR) was also used to verify the detection efficiency of all assays. Known negative and positive 10% tissue suspensions of different animal origins were examined to calculate the diagnostic specificity (DSp) and sensitivity (DSe). Serotype-specific mAb-DAS-ELISAs demonstrated 100%, 97%, 97%, 99%, 99%, and 94% DSp and 100%, 95%, 90%, 95%, 100%, and 100% DSe for serotypes O, A, Asia-1, SAT-1, SAT-2, and SAT-3, respectively. The detection efficiency of mAb-DAS-ELISAs was better than that of classical DAS-ELISAs. Also, all assays demonstrated minimal cross-reactivity and optimal reproducibility. Therefore, the mAb-DAS-ELISAs developed in this study could be useful for detecting and serotyping FMDV and ultimately replacing the classical DAS-ELISA. Full article

Vaccination plays a pivotal role in the control and prevention of animal infectious diseases. However, no efficient and safe universal vaccines are currently registered for major pathogens such as influenza A virus, foot-and-mouth disease virus (FMDV), simian immunodeficiency virus (SIV), and small ruminant lentiviruses (SRLV). Here, we review the development of Sendai virus (SeV) vectors as a promising vaccine platform for animal diseases. Recombinant SeV vectors (rSeVv) possess several key features that make them highly suitable for developing vaccination strategies: (1) SeV has exclusively cytoplasmic replication cycle, therefore incapable of transforming host cells by integrating into the cellular genome, (2) rSeVv can accommodate large foreign gene/s inserts (~5 kb) with strong but adjustable transgene expression, (3) can be propagated to high titers in both embryonated chicken eggs and mammalian cell lines, (4) exhibits potent infectivity across a broad range of mammalian cells from different animals species, (5) undergo transient replication in the upper and lower respiratory tracts of non-natural hosts, (6) has not been associated with disease in pigs, non-humans primates, and small ruminants, ensuring a favorable safety profile, and (7) induce a robust innate and cellular immune responses. Preclinical and clinical studies using rSeVv-based vaccines against influenza A virus, FMDV, SIV, and SRLV have yielded promising results. Therefore, this review highlights the potential of rSeVv-based vaccine platforms as a valuable strategy for combating animal viruses. Full article

Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ. Full article

Background/Objectives: Intact (146S) foot-and-mouth disease virus (FMDV) particles easily dissociate into 12S particles with a concomitant decreased immunogenicity. Vaccine quality control with 146S-specific single-domain antibodies (VHHs) is hampered by the high strain specificity of most 146S-specific VHHs. This study aimed to isolate 146S-specific VHHs that recognize all serotype O strains. Methods: Biopanning was performed with the FMDV strain O/SKR/7/2010 146S, using a secondary library of mutagenized M170F VHH that did not recognize O/SKR/7/2010 or using phage-display libraries from llamas immunized with other serotype O strains. Novel VHHs were yeast-produced and their strain-, particle-, and antigenic-site specificities were determined by ELISA. Results: M170F mutagenesis did not improve the cross-reaction with O/SKR/7/2010. However, selection from immune libraries resulted in four VHHs that exhibited high 146S specificity for all five serotype O strains analyzed. These VHHs presumably recognize all serotype O strains since the five strains analyzed represent different phylogenetic clades. They bind the same antigenic site as M170F, which was previously shown to be a conserved site in serotypes A and O, and which has an altered 3D structure when 146S dissociates into 12S particles. M916F had the lowest limit of detection, which varied from 0.7 to 5.9 ng/mL 146S particles for three serotype O strains. Conclusions: We identified four VHHs (M907F, M910F, M912F, and M916F) that specifically bind 146S particles of probably all serotype O strains. They enable further improved FMDV vaccine quality control. Full article

Background/Objectives: CAR T cell therapy, as a rapidly advancing immuno-oncology modality, has achieved significant success in the treatment of leukaemia and lymphoma. However, its application in solid tumours remains limited. The challenges include the heterogeneity of tumours, local immunosuppression, poor trafficking and infiltration, life-threatening toxicity and the lack of precise representative immunocompetent research models. Considering its typically dense and immunosuppressive tumour microenvironment (TME) and early metastasis, pancreatic ductal adenocarcinoma (PDAC) was employed as a model to address the challenges that hinder CAR T cell therapies against solid tumours and to expand immunotherapeutic options for advanced disease. Methods: A novel murine A20FMDV2 (A20) CAR T cell targeting integrin αvβ6 (mA20CART) was developed, demonstrating efficient and specific on-target cytotoxicity. The mA20CART cell as a monotherapy for orthotopic pancreatic cancer in an immunocompetent model demonstrated modest efficacy. Therefore, a novel triple therapy regimen, combining mA20CART cells with oncolytic vaccinia virus encoding IL-21 and a TGF-β-blocking antibody was evaluated in vivo. Results: The triple therapy improved overall survival, improved the safety profile of the CAR T cell therapy, attenuated metastasis and enhanced T cell infiltration. Notably, the potency of mA20CART was dependent on IL-2 supplementation. Conclusions: This study presents an αvβ6-targeting murine CAR T cell, offering a novel approach to developing CAR T cell technologies for solid tumours and a potential adjuvant therapy for pancreatic cancer. Full article

Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription–recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks. Full article

Background/Objectives: Vaccination is important for controlling foot-and-mouth disease (FMD) in endemic regions and to lessen the effects of outbreaks in FMD-free countries. The adaptation of FMD virus to BHK cells is a necessary but time-consuming and costly step in vaccine production and can prove problematic for some isolates. Adaptation is, in part, driven by receptor availability and selects variants with altered receptor specificity that result from amino acid substitutions in the capsid proteins. Methods: To bypass the need for cell culture adaptation, we generated chimeric viruses with field-strain capsids and introduced amino acid substitutions associated with cell culture adaptation. We targeted two sites on the capsid: the canonical heparan sulphate binding site and the icosahedral 5-fold symmetry axes. Results: Our results show that some viruses with unmodified wild-type (wt) capsids grew well in BHK cells (suspension and adherent), whereas others showed poor growth. For viruses that showed good growth, the introduction of amino acid changes associated with cell culture adaptation improved the rate of growth but not virus titres or yields of 146S particles, whereas growth and 146S yields for viruses that grew poorly in BHK cells were greatly enhanced by some of the amino acid changes. For the latter viruses, the introduced changes did not appear to adversely affect virion stability or antigenicity. Conclusions: For FMD viruses that grow poorly in BHK cells, this approach could be a viable alternative to protracted adaptation by serial passage and could expedite the production of a new vaccine strain from a field virus. Full article

Background/Objectives: Foot-and-mouth disease (FMD) is a highly contagious class 1 animal disease that affects cloven-hoofed animals, such as cattle, pigs, and goats. Diagnosis and research on live FMD virus (FMDV) typically require biosafety level 3 facilities, which are challenging to maintain due to strict protocols and high costs. The development of NanoBiT-based assays has accelerated in response to the coronavirus disease pandemic, providing safer alternatives for viral research, and is now applicable for general laboratories. This study aimed to develop a NanoBiT-based virus-like particle (VLP) assay for the rapid and safe screening of neutralizing antibodies against FMDV Asia1 Shamir (AS). Methods: We developed an AS VLP with an inserted HiBiT tag that enabled the detection of entry into LgBiT cells through luminescence signals. Results: HiBiT-tagged AS VLPs mixed with anti-serum and introduced into LgBiT-expressing cells led to a reduction in luciferase activity. Therefore, we established a NanoBiT-based viral neutralizing antibody test (VNT) that demonstrated a high correlation (R² = 0.881) with the traditional gold standard VNT. Conclusions: The assay demonstrated high sensitivity and could be performed in BL-2 facilities, offering a safer and more efficient alternative to traditional assays while reducing the need to handle live viruses in high-containment facilities. This method provides a valuable tool for rapid screening of neutralizing antibodies and can be adapted for broader applications in FMDV research. Full article

Foot-and-mouth disease virus (FMDV) and Senecavirus A (SVA) have similar pathogenic characteristics, and both are important pathogens that harm the livestock industry. Studies have shown that lactoferrin peptides can inhibit the replication of various viruses and enhance the body’s immune functions. Based on this, in the present study, we aimed to investigate the effects of bovine lactoferricin-lactoferrampin (LFCA) on replicating FMDV and SVA and to analyze its role in the cellular antioxidant response caused by viral infection; in addition, we fed mice with constructed recombinant Lactobacillus reuteri expressing LFCA. Treatment with LFCA at different stages significantly inhibited the replication of both SVA and FMDV. Pretreatment before SVA infection achieved an inhibition rate of up to 94.9%, while treatment during the FMDV replication stage achieved an inhibition rate of 74.3%. After infection with either virus, intracellular ROS and MDA levels were significantly reduced, as was GSH-Px activity. However, SOD activity showed no significant difference, compared with the virus-exposed group, and remained at a high level, suggesting an increased cellular antioxidant capacity. LFCA treatment significantly increased the transcription levels of the Nrf2, Ho-1, and Nqo1 genes. In mouse experiments, the LFCA-treated group showed significantly lower viral loads in lung and intestinal tissues, compared with the SVA infection group, validating LFCA’s protective effect against SVA infection. These findings demonstrate the potential of LFCA as an antiviral drug. Full article