Search Results (210)

Background/Objectives: Classical swine fever (CSF) and pseudorabies (PR), caused by classical swine fever virus (CSFV) and pseudorabies virus (PRV) respectively, are economically significant viral diseases worldwide that severely compromise swine health and constrain international trade. Previously, we generated a recombinant PRV (rPRVTJ-UL44-E2) expressing the CSFV E2 protein that elicited rapid E2-specific antibody responses in rabbits as early as 7 days post-immunization (dpi). However, its immunogenicity and protective efficacy in pigs remain uncharacterized. In this study, we evaluated the protective performance of rPRVTJ-UL44-E2 in pigs via needle-free immunization. Methods: Pigs (n = 5) received a prime immunization with 10⁷ median tissue culture infective doses (TCID₅₀) of rPRVTJ-UL44-E2 via needle-free immunization, followed by a boost immunization with the same dose at 21 dpi, and were then challenged with 10⁵ TCID₅₀ of the virulent CSFV Shimen strain (CSFV-SM) at 33 dpi. Results: Pigs immunized with rPRVTJ-UL44-E2 developed E2-specific antibodies (blocking rates > cutoff value (40%) at 25 dpi) and gB-specific antibodies that were detectable as early as 7 dpi, with anti-CSFV neutralizing antibody titers comparable to those induced by the C-strain vaccine and anti-PRV neutralizing antibody titers also reaching detectable levels at 28 dpi, whereas no specific antibodies were detected in the DMEM control group. All pigs immunized with rPRVTJ-UL44-E2 and C-strain survived the lethal CSFV challenge with no clinical signs or detectable viremia. In contrast all DMEM control pigs succumbed to infection within 9 days post-challenge (dpc). Conclusions: These findings demonstrate that needle-free delivery of rPRVTJ-UL44-E2 confers complete protection against lethal CSFV challenge in pigs. Full article

Porcine reproductive and respiratory syndrome (PRRS) represents a critical infectious disease caused by the PRRS virus (PRRSV), posing a substantial threat to the global swine industry. In China, there is currently an epidemic trend characterized by the coexistence of multiple evolving genotypes. Effective prevention and control measures are contingent upon the availability of rapid, precise, and sensitive pathogen detection technologies. Addressing the need for swift differentiation of the predominant circulating strains, including the classical strains (PRRSV-C), the highly pathogenic strains (PRRSV-HP), and NADC30-like strains (PRRSV-NA), this study focuses on the NSP2 region of each lineage. It establishes a triple TaqMan-qPCR method capable of simultaneously genotyping these three lineages. The method demonstrated no cross-reactivity with other viruses, including porcine parvovirus (PPV), porcine transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV), classical swine fever virus (CFSV), African swine fever virus (ASFV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (RV), and porcine circovirus (PCV2), thereby fully affirming its specificity. The sensitivity analysis demonstrated that the limit of detection (LOD) for the NSP2 gene in each lineage was 1 copy/μL based on the purified plasmids. Both inter-group and intra-group coefficients of variation (CV) were less than 4%, indicating high reproducibility. Comparative studies with commercial kits revealed that the developed TaqMan-qPCR method exhibited 100% relative sensitivity and a relative conformity rate exceeding 98%, suggesting its potential as a viable alternative to commercial kits. Furthermore, the analysis of 1049 clinical samples using the qPCR method indicated that the PRRSV-NADC30-like strains are currently the predominant circulating strain in clinical settings in Hebei Province. In conclusion, this study developed a triple TaqMan-qPCR method capable of simultaneously identifying PRRSV-C, PRRSV-HP and PRRSV-NA, enabling rapid and accurate identification of the PRRSV genotypes prevalent in pig populations. This provides a robust technical tool for the development of targeted immunization and prevention strategies. Full article

Classical swine fever (CSF) is a highly contagious transboundary animal disease that causes major economic losses to the global swine industry and remains notifiable to the World Organisation for Animal Health (WOAH). Handling and transport of infectious classical swine fever virus (CSFV)-positive samples require biosafety level 3 (BSL-3) containment, limiting diagnostic capacity and interlaboratory exchange in many regions. In this study, we evaluated the efficacy of PrimeStore^® Molecular Transport Medium (PS-MTM) for eliminating detectable CSFV infectivity while preserving viral ribonucleic acid (RNA) detectability for downstream molecular detection. Validation assays were performed using the CSFV Alfort/187 reference strain and clinical samples collected from pigs experimentally infected with the Catalonia01 and Margarita CSFV strains. Residual infectivity was assessed by virus isolation, whereas viral RNA detection and stability were evaluated using the WOAH-recommended real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR) assay. Following PS-MTM treatment, no residual infectivity was detected in either cell culture-derived virus stocks or clinical samples, including extensive replicate testing and three subsequent blind passages. RT-qPCR analyses confirmed preservation and stability of detectable CSFV RNA for at least 60 days under the storage conditions evaluated, including refrigerated storage and room temperature storage for Catalonia01-derived samples. Overall, these findings demonstrate that PS-MTM eliminates detectable CSFV infectivity while preserving viral RNA for molecular diagnosis. The implementation of PS-MTM may facilitate safer handling, transport, and interlaboratory exchange of CSFV-positive samples, contributing to improved biosafety, diagnostic harmonization, and global surveillance capacity for this transboundary animal disease. Full article

Traditional injectable vaccine adjuvants are limited by local granulomatous reactions, chronic inflammation, and reduced compliance, creating an urgent need for safe, host-directed strategies to enhance vaccine immunogenicity. Proanthocyanidins (PACs)—natural polyphenols abundant in grapes, cocoa, and tea—are food-derived bioactive compounds with antioxidant, anti-inflammatory, and immunomodulatory activities, positioning them as attractive candidates for oral immunostimulants. Using classical swine fever virus (CSFV), a Pestivirus within the Flaviviridae that shares conserved genomic and immunological features with human pathogens such as hepatitis C and dengue virus, we systematically evaluated oral PACs (15, 30, and 60 mg/kg/d) combined with attenuated live or subunit CSFV vaccines in mice. PACs were well tolerated across the full dose range, with no impact on body weight or major organs, while selectively increasing the splenic index—suggesting spleen-targeted immune activation without systemic inflammation. Compared with vaccine alone, the moderate dose (30 mg/kg/d) produced the strongest immunostimulation, particularly with the subunit vaccine: peak antigen-specific IgG titer reached 1:409,600, a 4-fold increase versus 1:102,400 for subunit alone and exceeding the 3.4-fold gain observed with the attenuated vaccine. IgG2a and IgG2b rose by 199.4% and 269.1%, respectively, alongside coordinated elevations of Th1 (IL-2 +160.6%, IFN-γ +91.9%) and Th2 (IL-4, IL-10) cytokines, enhanced B- and T-lymphocyte proliferation, and expansion of CD4⁺ and CD8⁺ subsets, establishing a balanced and durable Th1/Th2 response. These findings provide preclinical evidence that food-derived PACs can serve as safe oral immunostimulants for low-immunogenicity subunit vaccines, supporting their further development in human and veterinary vaccinology. Full article

Timely detection of specific antibodies against foot-and-mouth disease virus (FMDV) is critical to effective prevention and control of the disease. In this study, we developed a competitive chemiluminescent enzyme immunoassay (cCLEIA) based on recombinant FMDV VP2 protein (FMDV-VP2 cCLEIA) for simultaneous detection of antibodies against FMDV serotypes O and A in serum samples. Using a cut-off of 35% percentage inhibition (PI), the FMDV-VP2 cCLEIA achieved a relative sensitivity of 101.5% and a relative specificity of 102% compared with commercial FMDV serotypes O and A liquid-phase blocking ELISA (LPBE) kits. The assay showed high specificity for detection of antibodies against FMDV serotypes A and O, with no cross-reactivity observed against positive sera for classical swine fever virus, porcine reproductive and respiratory syndrome virus, and Senecavirus A. Both intra-batch and inter-batch coefficients of variation were below 15%, confirming excellent assay reproducibility. The testing of 40 field serum samples yielded a positive detection rate of 95% using the FMDV-VP2 cCLEIA, compared with 92.5% using the FMDV serotypes O and A LPBE kits. In conclusion, the FMDV-VP2 cCLEIA developed is a rapid, sensitive assay for simultaneous detection of antibodies against FMDV serotypes O and A, which may offer a valuable tool for foot-and-mouth disease (FMD) surveillance and control programs. Full article

The genus Orthopestivirus, within the family Flaviviridae, includes important pathogens such as classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV-1 and BVDV-2), and border disease virus (BDV). Although Costa Rica is endemic for BVDV, it remains officially free of CSFV and BDV, highlighting the need for reliable molecular tools for pestivirus surveillance and differential diagnosis in swine populations. This study validated a conventional broad-range in-house RT-PCR assay targeting the 5′ untranslated region (5′UTR) of pestiviruses, a genomic region that is highly conserved among members of the genus Orthopestivirus. A wide variety of PCR-based assays have been developed for pestivirus detection; however, many are designed specifically for the diagnosis of bovine pestiviruses, such as bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2, and HoBi-like pestivirus (HoBiPeV) in cattle. In contrast, the assay described in this study was standardized and validated to detect multiple pestivirus species, thereby providing a useful tool for surveillance and diagnostic applications across different host species. A total of 66 reference samples were analyzed to determine diagnostic sensitivity and specificity, while analytical sensitivity, repeatability, reproducibility, and selectivity were also evaluated. Additionally, pestivirus surveillance was conducted from 2014 to 2025. The assay showed a limit of detection of 1.2 copies/µL for CSFV and 11.7 copies/µL for BVDV, with diagnostic sensitivity and specificity of 98% and 100%, respectively. A total of 1072 surveillance samples were tested, including 11 bovine and 1061 porcine samples. Two porcine samples collected in 2015 were positive for pestivirus and clustered within the BVDV-2a group, representing the first molecular detection of BVDV-2a in pigs in Costa Rica. Circulation of BVDV-1b in cattle was also confirmed. The assay demonstrated satisfactory performance as a broad-range pestivirus surveillance tool and may support differential diagnosis in CSFV-free countries when combined with sequencing confirmation for pestivirus species identification. Full article

Porcine circovirus type 2 (PCV2) is the primary causative agent of a spectrum of porcine circovirus-associated diseases (PCVDs) and remains a major threat to the global swine industry. In this study, ten monoclonal antibodies (mAbs) targeting the Cap protein of PCV2 were generated and characterized. One mAb, designated 4C4, which exhibited high reactivity, strong neutralizing activity, and superior blocking efficacy, was selected for horseradish peroxidase (HRP) labeling. After optimizing the reaction parameters, a blocking ELISA was developed for the detection of the anti-PCV2 antibody. Using receiver operating characteristic (ROC) curve analysis, a cutoff value of 40% was established to distinguish positive from negative serum samples. The sensitivity and specificity of this blocking ELISA method were 98.66% and 100%, respectively. No cross-reactivity was observed with serum antibodies against classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus (PRRSV), or pseudorabies virus (PRV). Intra-assay and inter-assay repeatability tests yielded coefficients of variation (CVs) all below 10%, confirming the assay’s excellent reproducibility. Simultaneous testing of 312 clinical porcine serum samples using the developed bELISA and a commercial indirect ELISA kit revealed an overall coincidence rate of 99.04%. In addition, the percentage inhibition (PI) in the bELISA was strongly correlated with serum anti-PCV2 neutralizing antibody titers. In conclusion, the blocking ELISA developed herein demonstrates high sensitivity, strong specificity, and good reproducibility, serving as a potentially effective tool for the detection of the anti-PCV2 antibody and epidemiological investigation. Full article

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is an acute, febrile, and highly contagious disease that has led to significant economic losses in the global swine industry. Although the attenuated lapinized CSF vaccine (C-strain) has effectively controlled CSF outbreaks in China since the 1950s, it remains challenging to serologically differentiate infected from vaccinated animals (DIVA). Currently, the application of E2 subunit vaccines allows for DIVA by detecting antibodies against the E^rns protein. Therefore, this study aimed to develop a blocking ELISA for CSFV E^rns antibody detection using porcine monoclonal antibodies (mAbs) derived from single B cell technology. Peripheral blood mononuclear cells (PBMCs) were isolated from immunized pigs, and single CD21⁺IgM⁻E^rns-His tag⁺ B cells were sorted via flow cytometry. Using one-step PCR, full-length genes of porcine IgG heavy and light chains were amplified separately, yielding 11 porcine mAbs against the CSFV E^rns protein. Among these, three mAbs (E0S3, E0S5, and E0S10) exhibited broad reactivity, while two (E0S1, E0S4) showed no cross-reaction with bovine viral diarrhea virus (BVDV). Using mAb E0S4 as the blocking antibody, a blocking ELISA was established and optimized. The assay demonstrated a detection limit of 1:128, no cross-reactivity with other swine viruses or BVDV, and intra- and inter-assay coefficients of variation below 10%. ROC curve analysis determined an optimal cut-off value of 48.4%, with high sensitivity and specificity. In conclusion, the developed blocking ELISA provides a reliable tool for high-throughput serological surveillance, facilitating the DIVA strategy and contributing to CSF eradication programs. Full article

The Getah virus (GETV) is a mosquito-borne pathogen that infects diverse hosts, including pigs, horses, and humans, which can cause swine reproductive disorders such as abortion and stillbirth, posing a potential threat to animal and public health. Therefore, there is an urgent need for efficient and accurate serological diagnostic methods for surveillance and control of GETV. However, commercial diagnostic kits for swine GETV infection remain unavailable. In this study, we developed a novel enzyme-linked immunosorbent assay (ELISA) based on a GETV-specific epitope peptide (E2EP3) for serological detection. The N-terminally biotinylated E2EP3 peptide was synthesized, and the reaction conditions were systematically optimized, resulting in a cut-off value of 0.363. The assay exhibited no cross-reactivity with Japanese encephalitis virus (JEV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), pseudorabies virus (PRV), or classical swine fever virus (CSFV). It demonstrated good reproducibility and high sensitivity, detecting GETV-positive serum diluted up to 1:640. The overall agreement rate reached 95%, consistent with a conventional recombinant GETV E2 protein-based ELISA. Benefiting from the biotin–streptavidin system, this assay achieved strong signal amplification and low background. Moreover, the procedure is simple, cost-effective, and stable, making it suitable for GETV large-scale serological surveillance and vaccine evaluation. Full article

This study aimed to evaluate effect of Gordonia alkanivorans on phagocytic activity of porcine alveolar macrophages (PAMs) and immune function in piglets. Quantitative PCR and fluorescence tracing were used to measure phagocytic efficiency of G. alkanivorans-intervened PAMs against PRRSV and E. coli. Sixty-four 45-day-old cross-bred piglets with equal sex were randomly divided into four groups (n = 16/group). Growth performance, immune function, and intestinal flora were analyzed. G. alkanivorans extract exhibited half cytotoxic concentration of 36.43 mg/mL, half effective concentration of 0.1009 mg/mL, and half inhibitory concentration of 0.0043 mg/mL in PAMs, significantly increasing their phagocytic efficiency by 98.5% against PRRSV and 2.31- to 13.46-fold against E. coli. Dietary supplementation with G. alkanivorans elevated antibody-positive rates against classical swine fever virus (47.92%) and pseudorabies virus (14.58%), modified serum cytokine: Interleukin (IL)-1β, IL-2, Tumor Necrosis Factor -α, Interferon (IFN)-α, IFN-γ, IL-4, and IL-10 (−144.51% to +191.72%). It increased intestinal operational taxonomic units by 152%, the Shannon index by 14.62%, and the Chao index by 11.37%, while reducing the Firmicutes/Bacteroidetes ratio by 713.90%. In conclusion, G. alkanivorans enhances immunity and antiviral activity in piglets by gut and immune regulation. Full article

Classical swine fever virus (CSFV) remains a significant threat to the global swine industry, causing a highly contagious and often fatal disease in pigs. This review comprehensively examines the molecular biology of CSFV and the intricate mechanisms it employs to establish infection. We detail the structure and functions of viral proteins, highlighting their roles in virus entry, replication, and immune evasion. A major focus is placed on the virus–host interaction, specifically how CSFV subverts host innate immune responses and hijacks critical cellular processes, including metabolism and cell death pathways. The virus strategically manipulates host cell death programs (apoptosis, mitophagy, necroptosis) and exploits intracellular transport systems to promote its propagation. Furthermore, we summarize recent advances in understanding the cellular receptors involved in CSFV entry and the role of exosomes in viral spread. This synthesis of current knowledge aims to provide a deeper insight into the pathogenesis of CSFV and identify potential vulnerabilities that could be targeted for the development of novel antiviral strategies. Full article

Pestiviruses, such as bovine viral diarrhea virus (BVDV) or classical swine fever virus (CSFV), are members of the family Flaviviridae and infect a broad range of species, causing significant economic losses in livestock. A unique feature of pestiviruses is the E^rns protein, which is part of the glycoprotein complex at the surface of the virion, but it is also secreted as an RNase that functions as an interferon (IFN) antagonist. This dual nature makes E^rns a particularly complex and multifunctional protein, highlighting its importance for understanding pestivirus biology. Bungowannah pestivirus (BuPV) was reported to exhibit high genetic plasticity, making it suitable for engineering recombinant tools. In this study, we generated a recombinant BuPV expressing green fluorescent protein (GFP) fused to the N-terminus of the E^rns protein from BVDV. The GFP-E^rns fusion was detected by fluorescence microscopy and remained stable across five serial passages. The recombinant virus infected all tested mammalian cell lines but replicated more slowly than the parental BuPV stock. RNase activity assays confirmed retention of enzymatic function. These results demonstrate stable expression, broad infectivity, and preserved activity of GFP-E^rns in the recombinant BuPV, indicating that this might be a useful tool for further investigations on pestivirus pathogenesis. Full article

Background: Highly potent vaccines are essential for the effective control of classical swine fever (CSF). Since CSF re-emerged in 2018 in Japan, the live CSF virus (CSFV) vaccine—a guinea pig exaltation of Newcastle disease virus-negative strain vaccine (GPE⁻, genotype 1.1)—has been applied to domestic pigs, contributing to a reduction in outbreaks. Meanwhile, the persistence and continued expansion of CSFV in wild boar populations have raised concerns regarding potential antigenic divergence. Methods: We systematically evaluated the neutralizing reactivity of sera from GPE⁻-vaccinated pigs against CSFV strains (genotype 2.1) recently circulating in Japan against identified a representative strain that showed markedly reduced neutralization. We directly assessed the protective efficacy of the GPE⁻ vaccine against this strain in a controlled challenge experiment. At 4 weeks post-vaccination, both vaccinated and unvaccinated pigs were orally challenged with the representative Japanese strain and monitored for 3 weeks thereafter. Results: Among the Japanese CSFV strains, the JPN/SM/WB/2022 isolate exhibited markedly reduced neutralizing reactivity—over 32-fold lower than that against the vaccine strain—when tested with GPE⁻ vaccine-induced antisera. In the experimental infection in pigs, unvaccinated pigs exhibited typical clinical signs of CSF and viremia, and two pigs reached the humane endpoint. In contrast, none of the vaccinated pigs showed any clinical signs of infection. Robust humoral and cellular immune responses were induced in vaccinated pigs, which may correlate with the observed complete protection. Conclusions: The GPE^– live vaccine provides protective immunity against an antigenically distinct strain, prevents disease, and limits viral spread in domestic pigs. Full article

The global swine industry suffers persistent economic losses and health challenges due to major viral pathogens such as African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine circovirus (PCV). Traditional diagnostic methods, including virus isolation, serology, and quantitative PCR (qPCR), are limited by time, equipment requirements, and field applicability. Recent advances in CRISPR-based diagnostics, particularly those leveraging the collateral cleavage activity of Cas12a and Cas13a, have enabled rapid, sensitive, and field-deployable nucleic acid detection. This review outlines the principles of CRISPR-Cas12a/13a systems, their integration with isothermal amplification techniques, and their application in detecting major swine viruses. Cas12a-based platforms (e.g., DETECTR) and Cas13a-based systems (e.g., SHERLOCK) achieve detection limits as low as single-copy/μL within 25–60 min at 37 °C, offering high specificity and compatibility with visual readouts. Applications include ASFV, PRRSV, CSFV, PCV, foot-and-mouth disease virus (FMDV), porcine rotavirus (PoRV), and porcine parvovirus 7 (PPV7). Despite significant advances, challenges remain, notably the reliance on nucleic acid extraction and the need for fully integrated “sample-in, result-out” systems. Ongoing innovations in extraction-free methods, lyophilized reagents, and multiplex detection will strengthen the role of CRISPR diagnostics in swine disease surveillance and control. From an application standpoint, the technology offers a low-capital, field-adaptable alternative to qPCR, with its value proposition rooted in early outbreak containment and loss prevention. Its adoption pathway is expected to vary across production systems—serving as a sentinel tool in intensive settings, a leapfrogging solution in rapidly intensifying regions, and through shared-service models in resource-limited contexts. However, translation to routine use still requires overcoming standardization hurdles, regulatory validation, and workflow integration. Full article

Curcumin, the major polyphenolic constituent of Curcuma longa, has been widely investigated as a hepatoprotective adjunct due to its antioxidant and immunomodulatory properties. This review evaluates the relevance of curcumin for the prevention and management of liver dysfunction and hepatitis in pigs by synthesizing available porcine evidence and integrating mechanistic insights from translational liver injury models where pig-specific data remain limited. Across experimental hepatic injury contexts, curcumin administration is most consistently associated with reduced biochemical and structural indicators of hepatocellular damage, including decreased aminotransferase activity, attenuation of lipid peroxidation, and enhancement of endogenous antioxidant defenses. These effects are mechanistically linked to suppression of pro-inflammatory signaling pathways, particularly NF-κB-related transcriptional activity and inflammasome-associated responses, together with reduced expression of key cytokines such as TNF-α, IL-1β, and IL-6. Concurrent activation of Nrf2-centered cytoprotective pathways and induction of phase II antioxidant enzymes (including HO-1, GST, and NQO1) appear to constitute a conserved axis supporting hepatic oxidative stress resilience. In swine-relevant infectious settings, available data further support antiviral activity against selected porcine pathogens, including classical swine fever virus and porcine reproductive and respiratory syndrome virus, potentially mediated through interference with lipid-dependent stages of viral replication and modulation of Kupffer cell activation. Although combination strategies with established hepatoprotective approaches are conceptually attractive, current synergy evidence remains heterogeneous and largely extrapolated. Overall, curcumin represents a plausible adjunct candidate for supporting porcine liver health; however, translation into practice will depend on resolving formulation-dependent bioavailability constraints and strengthening the pig-specific evidence base. Full article