Search Results (126)

African swine fever virus (ASFV) is an important transboundary animal pathogen with significant impacts on the global swine industry. Overwhelming proinflammatory responses are a major virulence mechanism for ASFV, but the dynamics of these changes during clinical disease are not completely understood. We constructed a detailed portrait of the innate immune responses during acute African swine fever (ASF) at the cellular, transcriptomic, and cytokine levels. Samples serially obtained from infected piglets show that progression of acute ASF is characterized by rapid increases in plasma type I interferons, TNF-α, IL-12p40, and IL-10, which coincide with the manifestation of clinical disease and viral DNAemia. Lymphocytes and natural killer (NK) cells progressively declined, with fluctuations in B cell, CD8+ T cell, and CD4+/CD8+ T cell populations. Blood monocytes and macrophages were highly variable throughout infection, with an abrupt spike in CD203+ mature macrophages immediately prior to death. Transcriptomic analysis of blood showed downregulation of cellular translation as early as 1 day post-challenge (DPC) and significant upregulation of antiviral immune processes at 5 DPC and 7 DPC, which overlapped with the onset of clinical disease. Together, these results present a detailed delineation of fatal ASF which involves an initial infection and damage of susceptible myeloid cells prior to symptomatic disease characterized by pro-inflammatory immune responses, lymphoid depletion, and clinical deterioration. Full article

Background/Objectives: African swine fever (ASF) is a disease of domestic pigs and wild boar caused by African swine fever virus (ASFV), in which infection often leads to high morbidity and mortality. Although subunit and mRNA vaccines based on protective antigens have been explored for ASFV, their protective efficacy remains insufficient for practical ASF control, highlighting the need to identify new potential antigens capable of inducing more potent and broadly protective immune responses. Previously, we found that the antibodies against the ASFV E120R protein (pE120R) could significantly inhibit virus replication in primary porcine alveolar macrophages (PAMs). However, it is not yet known whether anti-pE120R antibodies can induce antibody-dependent cellular cytotoxicity (ADCC). Methods: In this study, we analyzed the conservation and immunogenic features of pE120R and established an HEK293T cell line with stable expression of pE120R as target cells (HEK293T-pE120R). Additionally, a co-culture system comprising target cells and peripheral blood mononuclear cells (PBMCs) was established to evaluate the ability of the anti-pE120R antibodies to induce ADCC as measured by lactate dehydrogenase (LDH) release assays. Results: The results showed that pE120R is highly conserved among different ASFV genotypes and contains multiple B-cell and T-cell epitopes. Importantly, LDH release assays demonstrated that anti-pE120R antibodies triggered NK cell-mediated ADCC. Notably, ASFV replication in HEK293T-pE120R cells was not promoted. Conclusions: In summary, pE120R was associated with antibody production in a cytotoxicity assay. The ability of this antigen to induce protective immunity, if any, requires further evaluation in vivo. Full article

Background/Objectives: The African swine fever virus (ASFV) multi-gene family (MGF) 360 proteins play critical roles in immune evasion, replication regulation, and virulence determination. Despite substantial advances in this field, the functional roles of many members within this gene family remain to be fully characterized. Methods: In this study, Transcriptional kinetics analysis indicated that the expression profile of MGF 360-2L was consistent with that of the late marker gene B646L (p72). Transcriptomic profiling identified 13 and 171 differentially expressed genes (DEGs) at 12 and 24 h post-infection (hpi) with ΔMGF 360-2L, respectively. Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that these DEGs were predominantly enriched in Type I interferon (IFN-I) signaling pathways. It is noteworthy that transcriptome analysis further demonstrates that the absence of MGF 360-2L specifically results in the dysregulation of expression of the replication-essential genes E199L and E301R. These findings indicate that MG F360-2L is essential for maintaining the stable expression of these proteins. Conclusions:MGF 360-2L is a late gene that contributes to the precise regulation of viral protein expression and modulates the host immune response during infection. Full article

Wild boars are recognized reservoirs of numerous viral pathogens, posing a significant risk to domestic pig populations, particularly in areas with poor biosecurity. This study assessed the prevalence and co-infection patterns of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine cytomegalovirus (PCMV), African swine fever virus (ASFV), classical swine fever virus (CSFV), and pseudorabies virus (PRV) in wild boars from western Serbia and the Republic of Srpska (Bosnia and Herzegovina). Sixty-six spleen samples from legally hunted wild boars were analyzed by qPCR. All animals were negative for ASFV, CSFV, and PRV. The cumulative prevalence of infection with at least one of the other three viruses was 86.4% (95% CI: 76.2–92.8%). PCMV was detected in 74.2% of samples, PCV2 in 50%, and PPV in 28.8%. Co-infections were common: 42.4% of animals were positive for two viruses, and 12.1% for all three. A statistically significant association was observed between triple co-infection and sex, with higher rates in males. Subadult wild boars showed the highest PCV2 + PCMV co-infection rate (p = 0.0547). These findings highlight the need to expand molecular surveillance, particularly for PCMV, in both wild and domestic pigs, especially in regions reliant on low-biosecurity backyard farming. Full article

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control. Full article

African swine fever virus (ASFV) causes a highly contagious and lethal hemorrhagic disease and significantly threatens the pig industry. There is no commercially effective vaccine available currently, making the detection of ASFV critical for control and prevention. Previously, we established the CRISPR-LbCas12a and LwCRSIRP-Cas13a visual detections of ASFV, separately, targeting the structural p17 gene D117L. In this study, we performed the parallel detections of ASFV based on the conserved viral protease gene S273R using CRISPR-LbCas12a and CRISPR-LbuCas13a systems. Our results showed that both systems are able to specifically detect ASFV as low as two copies of the S273R gene, and effectively detect clinical samples with minimal DNA purification. The work promotes CRISPR-Cas systems for the application of on-site detection in the field. Full article

Background: African swine fever (ASF) is a crucial socioeconomic setback to South Korea’s swine industry. This study aimed to determine seropositivity for ASF virus (ASFV) in pigs that appeared to be infected on farms with reported ASF outbreaks. Methods: A total of 2232 sera from ASF outbreaks (2019–2024) in South Korea were collected. Two enzyme-linked immunosorbent assay (ELISA) kits were used to detect ASFV antibodies, and an immunoperoxidase test (IPT) was used as a confirmatory test following the method recommended by the World Organisation for Animal Health in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Also, spatial clustering was identified using the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) model to understand ASF hotspots in the wild boar population and assess the spatial relationship between the hotspots and ASF antibody-positive domestic pig farms. Results: Antibodies were first detected in Hwacheon in 2020, but by 2024, only 1.43% of pigs had detectable antibodies against ASFV. Although this percentage is still low, the number of antibody-positive pigs is gradually increasing. Additionally, 32 positive samples were found from nine pig farms with outbreaks, and these samples were confirmed positive in both the two ELISA tests and the IPT. The highest seropositivity was recorded at the finishing stage of pig production. When compared to the confirmatory IPT, both blocking and competition ELISA demonstrated high diagnostic sensitivities. The statistical association between ASF antibody-positive farms and wild boars were analyzed using Fisher’s exact test, yielding a significant p-value of 0.007. This indicates a strong correlation, as eight out of nine ASF-seropositive farms were located within hotspots that were significantly associated. Conclusions: Our findings provide valuable insights into ASFV antibody detection in South Korea and demonstrate a statistical association between farms housing pigs with ASFV antibodies and hotspots of ASFV-infected wild boars. Confirmatory tests, such as the IPT, are needed. These insights will contribute to the improvement of surveillance and biosecurity measures for swine farms. Full article

African swine fever virus (ASFV) is a highly virulent pathogen that causes nearly 100% mortality in acute infections and poses persistent risks. Effective containment of ASFV outbreaks requires rapid and reliable diagnostic tools. The p54 protein, a key structural component of ASFV, has emerged as an important target for serological detection. Herein, the recombinant p54 protein (amino acids 53–184) was expressed in Escherichia coli, and three mouse monoclonal antibodies (mAbs) (IgG1/kappa subtype) were developed. Among these mAbs, the mAb 1F9 specifically recognized the B-cell epitope ⁶⁶IQFINPYQDQQ⁷⁶, which is conserved across different genotypes of ASFV, suggesting that the epitope may serve as a valuable target for serological detection of ASFV. Structural modeling analysis revealed that this epitope is surface-exposed on the p54 protein, with ⁶⁷Gln and ⁶⁸Phe identified as critical residues for 1F9 binding. Moreover, a blocking ELISA based on the mAb 1F9 was established for detecting ASFV-specific antibodies in clinical serum samples, achieving a coincidence rate exceeding 95%. These findings demonstrate that mAb 1F9, targeting a conserved and accessible region of p54, represents a valuable tool for ASFV serodiagnosis, surveillance, and outbreak management. Full article

Background: African swine fever (ASF) is a highly contagious acute febrile disease with a near 100% mortality rate. There are currently no safe and effective vaccines for this disease. Cellular immunity plays an important role in the process of anti-viral, activating an effective cellular immune response is a prerequisite for the effectiveness of the vaccine. Methods: To effectively activate cellular immune responses, 133 immunodominant T cell epitopes (TEPs) were identified and synthesized into ten recombinant multi-epitope proteins (MEPs). These MEPs were subsequently conjugated to porcine ferritin (pFTH1) to generate MEPs-pFTH1 nanoparticles. Animal experiments were conducted to evaluate their immunogenicity and biocompatibility. Results: Animal experiments demonstrated that both MEPs and MEPs-pFTH1 nanoparticles induced significant humoral and cellular immune responses. Compared to MEPs monomers, the MEPs-pFTH1 nanoparticles induced a 10- to 100-fold increase in IgG and IgG2a antibody titers (p < 0.05), as well as a significantly higher number of IFN-γ⁺ cells. Serum from pigs immunized with MEPs-pFTH1 nanoparticles can significantly inhibit ASFV replication. Conclusions: Our novel self-assembled porcine ferritin nanovaccine candidate can induce strong humoral and cellular immune responses in swine and mice that effectively inhibit ASFV replication. Therefore, the nanovaccine is a highly biocompatible and safe candidate vaccine for ASF that warrants further investigation, such as conducting animal challenge experiments to evaluate the effectiveness of the vaccine. Full article

African swine fever (ASF) is a disease that affects both domestic and wild swine. It was recently reported in the Dominican Republic and Haiti (2021), representing a substantial risk to America. The goal of this study was to produce a truncated form of the ASF-p72 recombinant protein based on the ASF strain genotype II (Georgia 2017) as well as to develop and validate a sensitive and specific ASF indirect-ELISA (iELISA) for early detection of ASF. The truncated ASF-p72 recombinant protein was successfully expressed in E. coli BL21/DE3 cells using the pET-SUMO plasmid. Bioinformatics analysis showed 100% homology among the new isolates of ASFV from genotype II. The ASF-p72-truncated protein was used to develop an iELISA, which had a high sensitivity (88%) and strong specificity (97%); the concordance index kappa was K = 0.872, indicating nearly perfect agreement compared to the WOAH confirmatory immunoperoxidase test. The validation results utilizing the reference sera panel from the OIE-ASF Reference Laboratory show the excellent detection capabilities of ASF antibodies up to a 1:1000 serum dilution. The inter-assay coefficient of variation (CV 10.4%) and intra-assay CV (2.8%) data show that the assay is precise and reproducible. This biotechnology advancement can be used to conduct future epidemiological research for ASF surveillance in ASF-free American countries. Full article

African swine fever virus (ASFV) is a highly virulent DNA virus that has spread globally since its introduction into Georgia in 2007, causing substantial economic losses in the swine industry. In the absence of an effective vaccine, chemical disinfection remains a key strategy for disease control. However, in cell-based disinfectant efficacy testing, distinguishing between disinfectant-induced cytotoxicity and virus-induced cytopathic effects (CPEs) remains a major challenge, leading to the potential misinterpretation of results. To address this, we developed a multi-step analytical framework to differentiate CPEs from cytotoxicity using a Vero cell-adapted ASFV strain. Virkon^® S was tested at three dilutions—375×, 275× (manufacturer-recommended), and 175×—and evaluated through CPE observation, lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and antigen detection via lateral flow immunoassay (p30) and immunofluorescence (p54). Notably, the 375× dilution achieved effective viral inactivation with significantly lower cytotoxicity, demonstrating that this framework can facilitate a more refined determination of disinfectant working dilutions. Furthermore, increased p30 signals after disinfection and the observation of lower cytotoxicity in virus-plus-disinfectant groups compared to disinfectant-only groups highlight the complexity of virus-disinfectant interactions and the potential for misinterpretation. This study provides a standardized and interpretable strategy for assessing ASFV disinfectant efficacy and offers a practical basis for evaluating other enveloped viruses in future disinfection studies. Full article

African swine fever (ASF) is caused by the African swine fever virus (ASFV); infection in domestic pigs and wild boars leads to a highly contagious, hemorrhagic disease. The p54 protein is encoded by the ASFV E183L gene and is an important structural protein located on the inner envelope of the virus. It is involved in processes of virus assembly, apoptosis induction, and neutralizing antibody production. In this study, three specific monoclonal antibodies (mAbs) against ASFV p54 protein were generated, namely 6B11, 3E3, and 3C10, from mice who were immunized with recombinant prokaryotic p54-truncated protein. Three novel linear B cell epitopes, recognized by the mAbs, were revealed: ⁶⁰AAIEEEDIQFINP⁷², ¹²⁸MATGGPAAAPAAASAPAHPAE¹⁴⁸, and ¹⁶³MSAIENLRQRNTY¹⁷⁵. The epitopes ⁶⁰AAIEEEDIQFINP⁷² and ¹⁶³MSAIENLRQRNTY¹⁷⁵ were highly conserved in genotype I and II ASFV strains. In addition, the epitope peptide ELISA can be used for the detection of ASFV antibodies. Our work provides new insights for p54 antigenicity and an alternative tool for serological diagnosis of ASF. Full article

Background/Objectives: African swine fever (ASF), a fatal febrile hemorrhagic disease in domestic pigs and Eurasian wild boars, is caused by ASF virus (ASFV). ASF continues to spread across the globe, causing a significant impact on the world’s pig industry. Recently, highly virulent chimeric ASFV (chASFV) strains with recombined genomes of the p72 genotype I and II viruses have been reported in China, Vietnam and Russia. Methods: In order to understand the propensity of ASFV genome for recombination, we attempted to experimentally generate chASFVs both in vitro and in vivo employing two distinct attenuated ASFV strains: OUR T88/3 (genotype I) and AQSΔB119L (genotype II). Results: When IPKM cells were co-infected with ASFV OUR T88/3 and AQSΔB119L strains, three genetically distinct chASFV emerged. When pigs were inoculated with the individual chASFV isolates, all pigs developed acute ASF. When four pigs were co-infected with ASFV OUR T88/3 and AQSΔB119L, all of them developed acute ASF and died or were euthanized. Three chASFV strains were successfully isolated from splenic homogenates from each pig. Conclusions: Our research indicates that genotype I and II chASFV with diverse genomes can be easily generated experimentally both in vitro and in vivo. Full article

An African Swine Fever (ASF) outbreak was first recorded in the Philippines in July 2019. Since then, the disease has spread across provinces in Luzon, Visayas, and Mindanao, causing severe economic consequences for the country’s swine industry. Here, we report the genome sequencing of ASF virus strains from outbreaks in several provinces of the Philippines between 2021 and 2023, using a long-read tiled amplicon sequencing approach. The coding-complete genomes generated ranged from 187,609 to 189,540 bp in length, with GC contents of 38.4% to 38.5%. Notably, a strain from the Bataan province had a 1.9 kb deletion at the 5′-end, affecting several coding regions. The strains were characterized using 13 genes and regions; namely the B646L gene, the CD2v serogroup, the central variable region (CVR) of the B602L gene, the intergenic region (IGR) between the I73R and I329L genes, the IGR between A179L and A137R, O174L, K145R, Bt/Sj, J268L, and ECO2, the multigene family (MGF) 505-5R, and the MGF 505-9R and 10R regions. The ASFV strains were mostly related to Asian and European p72 genotype II strains. Genetic profiling provides valuable information on the diversity of local strains of ASFV in the Philippines, which are useful for epidemiology, diagnostics, and vaccine development. Full article

Background/Objectives: African Swine Fever (ASF) is one of the most significant infectious diseases affecting both domestic pig and wild boar populations, leading to substantial economic and biosanitary consequences. In Europe, disease management relies on stringent biosecurity measures and surveillance through diagnosis, highlighting the urgent need for an effective and safe vaccine for ASF control. In this context, the VACDIVA project has generated several promising vaccine candidates, including those with the EP153R gene deleted and replaced by the eGFP reporter gene. Methods: In this study, pEP153R and eGFP proteins were produced using recombinant technology and demonstrated their antigenicity and DIVA capability through indirect ELISA. Additionally, a prototype serological DIVA test was designed and developed. The assay is based on the detection of antibodies against both DIVA antigens and the well-established immunogenic p72 protein. Results: This preliminary DIVA diagnostic assay complements vaccine candidates based on a genotype II ASFV strain, featuring the deletion of the EP153R gene and/or the insertion of the eGFP reporter gene, exemplified by the Lv17/WB/Rie1-∆CD vaccine candidate. Conclusions: This approach could potentially improve surveillance during prospective vaccination campaigns. Full article