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ASFV is a large DNA virus that is highly pathogenic in domestic pigs. How this virus is sensed by the innate immune system as well as why it is so virulent remains enigmatic. In this study, we show that the ASFV genome contains AT-rich regions that are recognized by the DNA-directed RNA polymerase III (Pol-III), leading to viral RNA sensor RIG-I-mediated innate immune responses. We further show that ASFV protein I267L inhibits RNA Pol-III-RIG-I-mediated innate antiviral responses. I267L interacts with the E3 ubiquitin ligase Riplet, disrupts Riplet-RIG-I interaction and impairs Riplet-mediated K63-polyubiquitination and activation of RIG-I. I267L-deficient ASFV induces higher levels of interferon-β, and displays compromised replication both in primary macrophages and pigs compared with wild-type ASFV. Furthermore, I267L-deficiency attenuates the virulence and pathogenesis of ASFV in pigs. These findings suggest that ASFV I267L is an important virulence factor by impairing innate immune responses mediated by the RNA Pol-III-RIG-I axis.

African swine fever (ASF) is a swine disease caused by a large, structurally complex, double-stranded DNA virus, African swine fever virus (ASFV). In domestic pigs, acute infection by highly virulent ASF viruses causes hemorrhagic fever and death. Previous work has suggested that ASFV pathogenesis is primarily mediated by host cytokines produced by infected monocytes and macrophages. To better understand molecular mechanisms mediating virus pathogenesis and immune evasion, we used transcriptome analysis to identify gene expression changes after ASFV infection in ex vivo swine macrophages. Our results suggest that the cytokines of TNF family including FASLG, LTA, LTB, TNF, TNFSF4, TNFSF10, TNFSF13B and TNFSF18 are the major causative cytokine factors in ASF pathogenesis via inducing apoptosis. Other up-regulated proinflammatory cytokines (IL17F and interferons) and down-regulated anti-inflammatory cytokine (IL10) may also significantly contribute to ASF pathogenesis and cause excessive tissue inflammatory responses. The differential expression of genes also indicates that ASFV could evade both the innate and adaptive immune responses by (i) inhibiting MHC Class II antigen processing and presentation, (ii) avoiding CD8+ T effector cells and neutrophil extracellular traps via decreasing expression of neutrophil/CD8+ T effector cell-recruiting chemokines, (iii) suppressing M1 activation of macrophages, (iv) inducing immune suppressive cytokines, and (v) inhibiting the processes of macrophage autophagy and apoptosis. These results provide novel information to further investigate and better understand the mechanism of pathogenesis and immune evasion of this devastating swine disease.

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

African swine fever (ASF) is a highly contagious and often fatal viral disease caused by African swine fever virus (ASFV), which poses a significant economic burden on the global pig industry. ASFV infection triggers a robust production of proinflammatory cytokines, leading to severe inflammation that contributes significantly to the high mortality rate associated with ASF. However, the underlying mechanisms remain incompletely understood. Here, we identified the ASFV B169L protein (pB169L) as a viroporin that exerts dual functions in viral replication and proinflammatory responses. We demonstrated that pB169L formed oligomeric calcium (Ca 2+ )-permeable channels in vitro by bilayer lipid membrane assay. The ectopically expressed pB169L significantly altered Ca 2+ homeostasis in cells and induced robust proinflammatory responses. Mutagenesis revealed critical residues—including P29, K55, and K57—that are indispensable for channel function and proinflammatory signaling. Importantly, the B169L gene knockdown during ASFV infection reduced inflammasome activation and viral replication, highlighting its dual role as both a structural component of virus and an inflammatory mediator. These findings provide the first direct evidence that ASFV encodes a functional viroporin and uncover a novel mechanism by which ASFV manipulates Ca 2+ homeostasis to drive inflammasome activation, offering new insights into ASFV pathogenesis and potential antiviral targets.

The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8(+) T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8(+) T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease.

•Immunised pigs with BeninΔMGF were protected against parental virulent ASFV strain.•To improve safety and efficacy new doses and routes of immunisation were tested.•Intramuscular immunisation of high doses showed the best percentage of protection.•A new ELISA detected specific IgM antibodies at early stages after ASFV infection.•Early induction of IFNγ and IL-10 in vaccinated pigs probably critical to protection. A live attenuated African swine fever virus (ASFV) vaccine candidate, produced by deletion of several genes belonging to multi-gene families MGF360 and 505 from virulent Benin 97/1 strain (BeninΔMGF), induces protection in pigs against parental virulent strain. In order to better define the safety and efficacy of this attenuated vaccine candidate and to understand protective mechanisms, we extended previous studies by intramuscular immunisation of pigs with the deletion mutant BeninΔMFG at different doses (102, 103, 104 TCID50), together with intranasal immunisation at the 103 dose. Results demonstrated a strong correlation between both doses and routes of immunisation of BeninΔMFG and the percentage of protection achieved, the onset of clinical signs, the viremia levels reached and the onset of death in non-protected pigs. The results show that the intramuscular route using high doses (104 TCID50) is the best option for immunisation. Only transient increase in temperature associated with a peak of virus genome levels was observed in most pigs after immunisation. Then, virus genome levels progressively decreased throughout the experiment until reaching low or undetectable levels in those protected pigs that survived after challenge. The IgM antibody responses following immunisation were detected between day 7–10 post-immunisation and remained at elevated levels for 10–18 days in most pigs before dropping. IgG was detected from day 15 to 21 post-immunisation and maintained at increased levels for the remainder of the experiment in most pigs. Induction of IFNγ and IL-10 was detected by ELISA in sera from some pigs immunised with 103 TCID50 by intramuscular or intranasal route at early times post-immunisation. IL-10 was also detected in serum from some non-protected pigs included in these groups after challenge.

The African swine fever virus (ASFV) has the ability to infect pigs and cause a highly contagious acute fever that can result in a mortality rate as high as 100%. Due to the viral epidemic, the pig industry worldwide has suffered significant financial setbacks. The absence of a proven vaccine for ASFV necessitates the development of a sensitive and reliable serological diagnostic method, enabling laboratories to effectively and expeditiously detect ASFV infection. In this study, four strains of monoclonal antibodies (mAbs) against p72, namely, 5A1, 4C4, 8A9, and 5E10, were generated through recombinant expression of p72, the main capsid protein of ASFV, and immunized mice with it. Epitope localization was performed by truncated overlapping polypeptides. The results indicate that 5A1 and 4C4 recognized the amino acid 20–39 aa, 8A9 and 5E10 are recognized at 263–282 aa, which is consistent with the reported 265–280 aa epitopes. Conserved analysis revealed 20–39 aa is a high conservation of the epitopes in the ASFV genotypes. Moreover, a blocking ELISA assay for detection ASFV antibody based on 4C4 monoclonal antibody was developed and assessed. The receiver-operating characteristic (ROC) was performed to identify the best threshold value using 87 negative and 67 positive samples. The established test exhibited an area under the curve (AUC) of 0.9997, with a 95% confidence interval ranging from 99.87 to 100%. Furthermore, the test achieved a diagnostic sensitivity of 100% (with a 95% confidence interval of 95.72 to 100%) and a specificity of 98.51% (with a 95% confidence interval of 92.02 to 99.92%) when the threshold was set at 41.97%. The inter- and intra-batch coefficient of variation were below 10%, demonstrating the exceptional repeatability of the method. This method can detect the positive standard serum at a dilution as high as 1:512. Subsequently, an exceptional blocking ELISA assay was established with high diagnostic sensitivity and specificity, providing a novel tool for detecting ASFV antibodies. Key points • Four strains of ASFV monoclonal antibodies against p72 were prepared and their epitopes were identified. • Blocking ELISA method was established based on monoclonal antibody 4C4 with an identified conservative epitope. • The established blocking ELISA method has a good effect on the detection of ASFV antibody.

African swine fever (ASF) is a devastating haemorrhagic fever of pigs with mortality rates approaching 100 per cent. It causes major economic losses, threatens food security and limits pig production in affected countries. ASF is caused by a large DNA virus, African swine fever virus (ASFV). There is no vaccine against ASFV and this limits the options for disease control. ASF has been confined mainly to sub-Saharan Africa, where it is maintained in a sylvatic cycle and/or among domestic pigs. Wildlife hosts include wild suids and arthropod vectors. The relatively small numbers of incursions to other continents have proven to be very difficult to eradicate. Thus, ASF remained endemic in the Iberian peninsula until the mid-1990s following its introductions in 1957 and 1960 and the disease has remained endemic in Sardinia since its introduction in 1982. ASF has continued to spread within Africa to previously uninfected countries, including recently the Indian Ocean islands of Madagascar and Mauritius. Given the continued occurrence of ASF in sub-Saharan Africa and increasing global movements of people and products, it is not surprising that further transcontinental transmission has occurred. The introduction of ASF to Georgia in the Caucasus in 2007 and dissemination to neighbouring countries emphasizes the global threat posed by ASF and further increases the risks to other countries. We review the mechanisms by which ASFV is maintained within wildlife and domestic pig populations and how it can be transmitted. We then consider the risks for global spread of ASFV and discuss possibilities of how disease can be prevented.

African swine fever (ASF) is a contagious and fatal disease caused by the African swine fever virus (ASFV), which can infect pigs of all breeds and ages. Most infected pigs have poor prognosis, leading to substantial economic losses for the global pig industry. Therefore, it is imperative to develop a safe and efficient commercial vaccine against ASF. The development of ASF vaccine can be traced back to 1960. However, because of its large genome, numerous encoded proteins, and complex virus particle structure, currently, no effective commercial vaccine is available. Several strategies have been applied in vaccine design, some of which are potential candidates for vaccine development. This review provides a comprehensive analysis on the safety and effectiveness, suboptimal immunization effects at high doses, absence of standardized evaluation criteria, notable variations among strains of the same genotype, and the substantial impact of animal health on the protective efficacy against viral challenge. All the information will be helpful to the ASF vaccine development.