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The African swine fever virus (ASFV) is spreading worldwide and causing huge economic losses to the global pig industry. The ASFV genome is 170-193 kb in length, contains approximately 150 open reading frames, and encodes more than 200 proteins, most of which have unknown functions. Owing to the unique viral structure, replication strategy, large number of genes of unknown function, and complicated pathogenesis, vaccine development research is challenging. Several naturally attenuated ASFV isolates have been extensively investigated and many genetically manipulated, gene-deleted, and cell-adapted ASFVs have been reported. Currently, live attenuated viruses prepared from weakly virulent strains are an efficient method to provide effective protection in vaccinated pigs; however, these have seldom been widely approved for vaccine use, except in Vietnam. Herein, we summarize the attenuated isolates or vaccine candidates for live vaccines derived from different sources, including naturally mutated, attenuated, cell-adapted, and genetically modified recombinant ASFVs. This will help to understand the gene function and immunogenicity of attenuated live ASFV, as well as the shortcomings of these viruses as vaccine candidates, and provide clues to prepare live, efficient, and safe vaccines for African swine fever.IMPORTANCEOutbreaks of African swine fever (ASF) have caused devastating losses to the global pig industry. Pigs immunized with ASFV attenuated virus can resist the lethal challenge of a strongly virulent virus. Here, we summarize the virulence of naturally mutated, cell-adapted, and genetically recombinant ASFV for pigs, and the protective effect after facing an attack challenge. We also analyze the advantages and disadvantages of ASFV attenuated viruses as vaccine candidates to provide clues for the preparation of efficient and safe live African swine fever vaccines., Competing Interests: The authors declare no conflict of interest.

To understand the clinicopathological forms of African swine fever (ASF) in wild boar, it is crucial to possess a basic knowledge of the biological characteristics of the currently circulating ASF virus isolates. The aim of this work is to establish an accurate and comprehensive histopathologic grading system to standardize the assessment of the ASF lesions in wild boar. The study evaluated the differences between animals infected with a high virulence genotype II isolate (Arm07) (HVI) through intramuscular (IM) (n = 6) and contact-infected (n = 12) routes, alongside those orally infected with a low virulence isolate (Lv17/WB/Riel) (LVI) (n = 6). The assessment included clinical (CS), macroscopic (MS), and histopathologic (HS) scores, as well as viral loads in blood and tissues by real-time quantitative polymerase chain reaction (qPCR). Tissues examined included skin, lymph nodes, bone marrow, palatine tonsil, lungs, spleen, liver, kidneys, thymus, heart, adrenal glands, pancreas, urinary bladder, brain, and gastrointestinal and reproductive tracts. The HVI group exhibited a 100% mortality rate with elevated CS, MS, and HS values. Animals infected by contact (CS = 12; MS = 58.5; HS = 112) and those intramuscularly infected (CS = 14.8; MS = 47; HS = 104) demonstrated similar values, indicating that the route of infection does not decisively influence the severity of clinical and pathological signs. The LVI group showed a 0% mortality rate, an inconspicuous clinical form, minimal lesions (CS = 0; MS = 12; HS = 29), and a lower viral load. Histopathologic evaluation has proven valuable in advancing our comprehension of ASF pathogenesis in wild boar and paves the groundwork for further research investigating protective mechanisms in vaccinated animals., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cell-passage-adapted strains of African swine fever virus (ASFV) typically exhibit substantial genomic alterations and attenuated virulence in pigs. We have indicated that the human embryonic kidney (HEK293T) cells-adapted ASFV strain underwent genetic alterations and the I7L gene in the right variable region was deleted compared with the ASFV HLJ/2018 strain (ASFV-WT). A recent study has revealed that the deletion of the I7L-I11L genes results in attenuation of virulent ASFV in vivo, but the underlying mechanism remains largely unknown. Therefore, we hypothesized that the deletion of the I7L gene may be related to the pathogenicity of ASFV in pigs. We generated the I7L gene-deleted ASFV mutant (ASFV-ΔI7L) and found that the I7L gene deletion does not influence the replication of ASFV in primary porcine alveolar macrophages (PAMs). Using transcriptome sequencing analysis, we identified that the differentially expressed genes in the PAMs infected with ASFV-ΔI7L were mainly involved in antiviral immune responses induced by interferon gamma (IFN-γ) compared with those in the ASFV-WT-infected PAMs. Meanwhile, we further confirmed that the I7L protein (pI7L) suppressed the IFN-γ-triggered JAK-STAT signaling pathway. Mechanistically, pI7L interacts with STAT1 and inhibits its phosphorylation and homodimerization, which depends on the tyrosine at position 98 (Y98) of pI7L, thereby preventing the nuclear translocation of STAT1 and leading to the decreased production of IFN-γ-stimulated genes. Importantly, ASFV-ΔI7L exhibited reduced replication and virulence compared with ASFV-WT in pigs, likely due to the increased production of IFN-γ-stimulated genes, indicating that pI7L is involved in the virulence of ASFV. Taken together, our findings demonstrate that pI7L is associated with pathogenicity and antagonizes the IFN-γ-triggered JAK-STAT signaling pathway via inhibiting the phosphorylation and homodimerization of STAT1 depending on the Y98 residue of pI7L and the Src homology 2 domain of STAT1, which provides more information for understanding the immunoevasion strategies and designing the live attenuated vaccines against ASFV infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: African swine fever (ASF) is a contagious viral disease that affects pigs and wild boars, with a mortality rate of up to 100% in susceptible animals. The virus has been circulating in Europe and Asia since its introduction in 2007. Initially, all studied isolates were identified as genotype II, but in 2021 genotype I was reported in China. Later in 2023, the first recombinant virus of genotype I and II was identified in China, with an isolate dating back to 2021, this was followed by the detection of 6 recombinant isolates in Vietnam., Methods: In this study, an ASFV isolate from the Primorsky Region of Russia obtained from a domestic pig was analyzed by sequencing several genome markers as well as the full genome. Eight pigs were infected with the isolate to assess its virulence., Results: Virus replication in cell culture showed hemadsorption, while sequencing of genome markers clustered the isolate into both genotype I and genotype II. The whole-genome sequence showed that the Russian isolate shared a 99.99% identity with recombinant isolates described earlier in China. Experimental animals developed ASF disease after the introduction of a low dose of the virus (10 HAU50) and died within 7 days post-infection, presenting an acute form of the disease., Conclusion: This is the first report on recombinant ASFV in Russia's territory. The results once again confirm the transboundary nature of the disease, demonstrating the vulnerability of the global pig industry underscoring the need for developing new ASF vaccines effective against recombinant strains and emphasizing the importance of continuous molecular monitoring to detect emerging threats promptly., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

African swine fever virus (ASFV) is the causative agent of an often lethal disease in domestic pigs, African swine fever (ASF). ASF is currently a pandemic disease challenging pig production in Eurasia. While the ASFV genome encodes for over 160 proteins, the function of most of them are still not characterized. Among those ASF genes with unknown functions is the E111R gene. It has been recently reported that the deletion of the E111R gene from the genome of the virulent Chinese field isolate SY18 strain produced a reduction of virus virulence when pigs were inoculated at relatively low doses. Conversely, we report here that deletion of the ASFV gene E111R in the Georgia 2010 isolate does not alter the virulence of the parental virus in experimentally inoculated pigs. A recombinant virus lacking the E111R gene, ASFV-G-∆E111R was intramuscularly (IM) inoculated in domestic pigs at a dose of 10 2 HAD 50 of ASFV-G-∆E111R and compared with animals that received a similar dose of virulent ASFV-G. Both, animals inoculated with either the recombinant ASFV-G-∆E111R or the parental virus developed a fatal form of the disease and were euthanized around the 6th-7th day post-inoculation (dpi).

African swine fever (ASF) is an acute infectious disease with a high mortality rate in both domestic and wild boars. Commercial vaccines or antiviral drugs for ASF were not available due to the complex diversity of the structure and genome of its pathogen African swine fever virus (ASFV). In recent years, there have been many reports on candidate strains of attenuated vaccines for ASFV. In this study, we obtained a recombinant virus named SY18ΔL60LΔCD2v by simultaneously deleting the L60L gene and CD2v gene from highly virulent strain SY18. In vitro, SY18ΔL60LΔCD2v displayed a decreased growth kinetic compared to that of parental SY18. In vivo, high doses (10 5 TCID 50 ) of SY18ΔL60LΔCD2v can protect pigs (5/5) from attacks by the parental SY18 strain (10 2 TCID 50 ). Low doses (10 2 TCID 50 ) of SY18ΔL60LΔCD2v only protected 20% of pigs (1/5) from attacks by the parental SY18 strain (10 2 TCID 50 ). The results indicated that the absence of these two genes in SY18 could induce protection against the homologous parental strain, and there were no obvious clinical symptoms or viremia. These results indicate that the SY18ΔL60LΔCD2v strain can serve as a new live attenuated vaccine candidate for the prevention and control of ASFV infection.

Background: African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry., Methods and Results: In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1 L-5-6 L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24 h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96 h, except for Bcl-2 and NF-kB, which peaked at 6 h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values., Conclusion: The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96 h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1 L-5-6 L) contributes to ASFV attenuation without affecting virus replication kinetics., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

African swine fever (ASF) is a devastating disease with a high impact on the pork industry worldwide. ASF virus (ASFV) is a very complex pathogen, the sole member of the family Asfaviridae , which induces a state of immune suppression in the host through infection of myeloid cells and apoptosis of lymphocytes. Moreover, haemorrhages are the other main pathogenic effect of ASFV infection in pigs, related to the infection of endothelial cells, as well as the activation and structural changes of this cell population by proinflammatory cytokine upregulation within bystander monocytes and macrophages. There are still many gaps in the knowledge of the role of proteins produced by the ASFV, which is related to the difficulty in producing a safe and effective vaccine to combat the disease, although few candidates have been approved for use in Southeast Asia in the past couple of years.

Wild boar population dynamics promote the increase in numbers and distribution of the species in Eurasia, leading to a rise in the interaction with human activities, as well as generating problems with the management of certain infectious diseases, most notably African swine fever (ASF). ASF virus possesses high stability in several contaminated pork and pork products that can be a source of indirect transmission to susceptible hosts habituated to anthropogenic food waste. This transmission route is a concerning threat for the dispersion of the disease, primarily into unaffected areas given the worldwide widespread distribution of the disease and the increase of wild boar contact with humans. Thus, in this study, a straightforward tool to assess the relative risk of wild boar natural populations potentially consuming food waste is presented using synthetic data. Three risk groups were defined related to urban areas, travel, and leisure. The surrounding quality of habitat of wild boar was used to obtain the relative risk of wild boar potentially consuming anthropogenic food waste. To assign the relative risk to the corresponding risk unit, we also included the population for the urban areas group, and traffic volume for the travel risk group. The leisure group had higher scaled risk scores, followed by the urban areas group. Higher risk was found in the edges of the study area where more natural landscapes are found. The implications of this risk are discussed focusing on the context of ASF transmission. The outputs can help prioritize decision-making in terms of the improvement of preventive measures against the habituation of wild boar to anthropogenic food waste and ASFV introduction in a given study area., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Aguilar-Vega et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Since the confirmation of African swine fever (ASF) in South Korea in 2019, its spread, predominantly in wild boars, has been a significant concern. A key factor in this situation is the lack of identification of risk factors by surveillance bias. The unique orography, characterized by high mountains, complicates search efforts, leading to overlooked or delayed case detection and posing risks to the swine industry. Additionally, shared rivers with neighboring country present a continual threat of virus entry. This study employs geospatial analysis and statistical methods to 1) identify areas at high risk of ASF occurrence but possibly under-surveilled, and 2) indicate strategic surveillance points for monitoring the risk of ASF virus entry through water bodies and basin influences. Pearson's rho test indicated that elevation (rho = -0.908, p-value < 0.001) and distance from roads (rho = -0.979, p-value < 0.001) may have a significant impact on limiting surveillance activities. A map of potential under-surveilled areas was created considering these results and was validated by a chi-square goodness-of-fit test (X-square = 208.03, df = 1, p-value < 0.001). The strong negative correlation (rho = -0.997, p-value <0.001) between ASF-positive wild boars and distance from water sources emphasizes that areas surrounding rivers are one of the priority areas for monitoring. The subsequent hydrological analyses provided important points for monitoring the risk of virus entry via water from the neighboring country. This research aims to facilitate early detection and prevent further spread of ASF., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 10 5 TCID 50 /pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 10 3 TCID 50 /pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications., Importance: African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF., Competing Interests: The authors declare no conflict of interest.

DP96R of African swine fever virus (ASFV), also known as uridine kinase ( UK ), encodes a virulence-associated protein. Previous studies have examined DP96R along with other genes in an effort to create live attenuated vaccines. While experiments in pigs have explored the impact of DP96R on the pathogenicity of ASFV, the precise molecular mechanism underlying this phenomenon remains unknown. Here, we describe a novel molecular mechanism by which DP96R suppresses interferon regulator factor-3 (IRF3)-mediated antiviral immune responses. DP96R interacts with a crucial karyopherin (KPNA) binding site within IRF3, disrupting the KPNA-IRF3 interaction and consequently impeding the translocation of IRF3 to the nucleus. Under this mechanistic basis, the ectopic expression of DP96R enhances the replication of DNA and RNA viruses by inhibiting the production of IFNs, whereas DP96R knock-down resulted in higher IFNs and IFN-stimulated gene (ISG) transcription during ASFV infection. Collectively, these findings underscore the pivotal role of DP96R in inhibiting IFN responses and increase our understanding of the relationship between DP96R and the virulence of ASFV.

Importance: African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines., Competing Interests: The authors declare no conflict of interest.

Importance: African swine fever (ASF) caused by ASF virus (ASFV) is a highly contagious and acute hemorrhagic viral disease in domestic pigs. Until now, no effective commercial vaccine and antiviral drugs are available for ASF control. Here, we generated a new live-attenuated vaccine candidate (ASFV-ΔH240R-Δ7R) by deleting H240R and MGF505-7R genes from the highly pathogenic ASFV HLJ/18 genome. Piglets immunized with ASFV-ΔH240R-Δ7R were safe without any ASF-related signs and produced specific antibodies against p30. Challenged with a virulent ASFV HLJ/18, the piglets immunized with high-dose group (105 HAD50) exhibited 100% protection without clinical symptoms, showing that low levels of virus replication with no observed pathogenicity by postmortem and histological analysis. Overall, our results provided a new strategy by designing live-attenuated vaccine candidate, resulting in protection against ASFV infection., Competing Interests: The authors declare no conflict of interest.

Importance: African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines., Competing Interests: The authors declare no conflict of interest.

African swine fever, caused by a large icosahedral DNA virus (African swine fever virus, ASFV), is a highly contagious disease in domestic and feral swine, thus posing a significant economic threat to the global swine industry. Currently, there are no effective vaccines or the available methods to control ASFV infection. Attenuated live viruses with deleted virulence factors are considered to be the most promising vaccine candidates; however, the mechanism by which these attenuated viruses confer protection is unclear. Here, we used the Chinese ASFV CN/GS/2018 as a backbone and used homologous recombination to generate a virus in which MGF110-9L and MGF360-9L, two genes antagonize host innate antiviral immune response, were deleted (ASFV-ΔMGF110/360-9L). This genetically modified virus was highly attenuated in pigs and provided effective protection of pigs against parental ASFV challenge. Importantly, we found ASFV-ΔMGF110/360-9L infection induced higher expression of Toll-like receptor 2 (TLR2) mRNA compared with parental ASFV as determined by RNA-Seq and RT-PCR analysis. Further immunoblotting results showed that parental ASFV and ASFV-ΔMGF110/360-9L infection inhibited Pam3CSK4-triggered activating phosphorylation of proinflammatory transcription factor NF-κB subunit p65 and phosphorylation of NF-κB inhibitor IκBα levels, although NF-κB activation was higher in ASFV-ΔMGF110/360-9L-infected cells compared with parental ASFV-infected cells. Additionally, we show overexpression of TLR2 inhibited ASFV replication and the expression of ASFV p72 protein, whereas knockdown of TLR2 had the opposite effect. Our findings suggest that the attenuated virulence of ASFV-ΔMGF110/360-9L might be mediated by increased NF-κB and TLR2 signaling., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023. Published by Elsevier Inc.)

The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R , DP71L , and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 10 4 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene ( H3.1 ) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro . These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R ( MGF360-18R ), NL ( DP71L ), and UK ( DP96R ). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R , UK , and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.

African swine fever (ASF) is a highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV), with a mortality rate of up to 100%. In order to replicate efficiently in macrophages and monocytes, ASFV has evolved multiple strategies to evade host antiviral responses. However, the underlying molecular mechanisms by which ASFV-encoded proteins execute immune evasion are not fully understood. In this study, we found that ASFV pH240R strongly inhibits transcription, maturation, and secretion of interleukin-1β (IL-1β). Importantly, pH240R not only targeted NF-κB signaling but also impaired NLRP3 inflammasome activation. In this mechanism, pH240R interacted with NF-kappa-B essential modulator (NEMO), a component of inhibitor of kappa B kinase (IKK) complex and subsequently reduced phosphorylation of IκBα and p65. In addition, pH240R bonded to NLRP3 to inhibit NLRP3 inflammasome activation, resulting in reduced IL-1β production. As expected, infection with H240R -deficient ASFV (ASFV-ΔH240R) induced more inflammatory cytokine expression both in vitro and in vivo than its parental ASFV HLJ/18 strain. Consistently, H240R deficiency reduced the viral pathogenicity in pigs compared with its parental strain. These findings reveal that the H240R gene is an essential virulence factor, and deletion of the H240R gene affects the pathogenicity of ASFV HLJ/18 by enhancing antiviral inflammatory responses, which provides insights for ASFV immune evasion mechanisms and development of attenuated live vaccines and drugs for prevention and control of ASF. IMPORTANCE African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease of domestic pigs, with a high mortality approaching 100%. ASFV has spread rapidly worldwide and caused huge economic losses and ecological consequences. However, the pathogenesis and immune evasion mechanisms of ASFV are not fully understood, which limits the development of safe and effective ASF attenuated live vaccines. Therefore, investigations are urgently needed to identify virulence factors that are responsible for escaping the host antiviral innate immune responses and provide a new target for development of ASFV live-attenuated vaccine. In this study, we determined that the H240R gene is an essential virulence factor, and its depletion affects the pathogenicity of ASFV by enhancing NLRP3-mediated inflammatory responses, which provides theoretical support for the development of an ASFV attenuated live vaccine.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a devastating disease affecting domestic and wild swine and currently causing a global pandemic, severely affecting swine production. Here, we demonstrate that the deletion of the previously uncharacterized ASFV gene, H108R from the highly virulent ASFV-Georgia2007 (ASFV-G) genome strain, reduces virulence in domestic swine. ASFV-G-ΔH108R, a recombinant virus with the H108R gene deleted, was used to evaluate the involvement of the H108R gene for ASFV replication and virulence in swine. ASFV-G-ΔH108R showed a delayed replication in swine macrophage cultures. A group of five pigs, intramuscularly inoculated with 10 2 HAD 50 of ASFV-G-ΔH108R, was observed over a 28-day period and compared with a similar group of animals inoculated with similar doses of the parental virulent virus. While all animals inoculated with ASFV-G developed an acute fatal disease, ASFV-G-ΔH108R inoculated animals, with the exception of one animal showing a protracted but fatal form of the disease, all survived the infection, remaining clinically healthy during the observational period. The surviving animals presented protracted viremias with lower virus titers compared with those of animals inoculated with the parental virus, and all of them developed a strong virus-specific antibody response. Importantly, all animals surviving ASFV-G-ΔAH108R infection were protected when challenged with the virulent parental strain, ASFV-G. This report constitutes the first evidence that the H108R gene is involved in ASFV virulence in swine and that the deletion of this gene may be used as a tool to increase the attenuation of currently experimental vaccines to improve their safety profiles. IMPORTANCE Currently, there is no commercial vaccine available to prevent ASF. ASFV-Georgia2007 (ASFV-G) and its field isolate derivatives are producing a large pandemic which is drastically affecting pork production in Eurasia. We present here the discovery of a novel virus determinant of virulence, the H108R gene, which, when deleted from the ASFV-G genome, significantly reduces virus virulence in domestic swine. Additionally, animals that survive the inoculation with a recombinant virus harboring a deletion of the H108R gene, ASFV-G-ΔH108R, are protected against a challenge with the virulent parental virus. Although presenting residual virulence, ASFV-G-ΔH108R confers protection even at low doses (10 2 HAD 50 ), demonstrating its potential to be used as an additional gene deletion to increase the safety profile of the preexisting vaccine candidate.

African swine fever virus (ASFV) is responsible for an ongoing pandemic that is affecting central Europe, Asia, and recently the Dominican Republic, the first report of the disease in the Western Hemisphere in over 40 years. ASFV is a large, complex virus with a double-stranded DNA (dsDNA) genome that carries more than 150 genes, most of which have not been studied. Here, we assessed the role of the MGF110-5L-6L gene during virus replication in cell cultures and experimental infection in swine. A recombinant virus with MGF110-5L-6L deleted (ASFV-G-ΔMGF110-5L-6L) was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. ASFV-G-ΔMGF110-5L-6L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, indicating that the MGF110-5L-6L gene is nonessential for virus replication. Similarly, domestic pigs inoculated with ASFV-G-ΔMGF110-5L-6L presented with a clinical disease undistinguishable from that caused by the parental ASFV-G, confirming that the MGF110-5L-6L gene is not involved in producing disease in swine. Sera from animals inoculated with an efficacious vaccine candidate, ASFV-G-ΔMGF, strongly recognized the protein encoded by the MGF110-5L-6L gene as a potential target for the development of an antigenic marker differentiation of infected from vaccinated animals (DIVA) vaccine. To test this hypothesis, the MGF110-5L-6L gene was deleted from the highly efficacious ASFV vaccine candidate ASFV-G-ΔI177L, generating the recombinant ASFV-G-ΔI177L/ΔMGF110-5L-6L. Animals inoculated with ASFV-G-ΔI177L/ΔMGF110-5L-6L developed an ASFV-specific antibody response detected by enzyme-linked immunosorbent assay (ELISA). The sera strongly recognized ASFV p30 expressed in eukaryotic cells but did not recognize ASFV MGF110-5L-6L protein, demonstrating that deletion of the MGF110-5L-6L gene can enable DIVA capabilities in preexisting vaccine candidates. IMPORTANCE Currently, there are no African swine fever (ASF) commercial vaccines that can be used to prevent or control the spread of ASF. The only effective experimental vaccines against ASF are live-attenuated vaccines. However, these experimental vaccines, which rely on a deletion of a specific gene of the current circulating strain of ASF, make it hard to tell the difference between a vaccinated and an infected animal. In our search for a serological marker, we identified that the virus protein encoded by the MGF110-5L-6L gene induced an immune response, making a virus lacking this gene a vaccine candidate that allows the differentiation of infected from vaccinated animals (DIVA). Here, we show that deletion of MGF110-5L-6L does not affect virulence or virus replication. However, when the deletion of MGF110-5L-6L was added to vaccine candidate ASFV-G-ΔI177L, a reduction in the effectiveness of the vaccine occurred.

African swine fever is one of the most serious viral diseases caused by African swine fever virus (ASFV). The metabolic changes induced by ASFV infection remain unknown. Here, porcine alveolar macrophages (PAMs) infected with ASFV was analyzed by ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 90 metabolites were significantly changed after ASFV infection, and most of them were amino acids and tricarboxylic acid (TCA) cycle intermediates. ASFV infection induced an increase in most of amino acids in the host during the early stages of infection, and amino acids decreased in the late stages of infection. ASFV infection did not significantly affect the glycolysis pathway, whereas it induced increases in citrate, succinate, α-ketoglutarate, and oxaloacetate levels in the TCA cycle, suggesting that ASFV infection promoted the TCA cycle. The activities of aspartate aminotransferase and glutamate production were significantly elevated in ASFV-infected cells and pigs, resulting in reversible transition between TCA cycle and amino acid synthesis. Aspartate, glutamate, and TCA cycle were essential for ASFV replication. In addition, ASFV infection induced an increase in lactate level using lactate dehydrogenase, which led to low expression of beta interferon (IFN-β) and increased ASFV replication. Our data, for the first time, indicate that ASFV infection controls IFN-β production through RIG-I-mediated signaling pathways. These data identified a novel mechanism evolved by ASFV to inhibit host innate immune responses and provide insights for development of new preventive or therapeutic strategies targeting the altered metabolic pathways. IMPORTANCE In order to promote viral replication, viruses often cause severe immunosuppression and seize organelles to synthesize a large number of metabolites required for self-replication. African swine fever virus (ASFV) has developed many strategies to evade host innate immune responses. However, the impact of ASFV infection on host cellular metabolism remains unknown. Here, for the first time, we analyzed the metabolomic profiles of ASFV-infected PAMs. ASFV infection increased host TCA cycle and amino acid metabolism. Aspartate, glutamate, and TCA cycle promoted ASFV replication. ASFV infection also induced the increase of lactate production to inhibit innate immune responses for self-replication. This study identified novel immune evasion mechanisms utilized by ASFV and provided insights into ASFV-host interactions, which is critical for guiding the design of new prevention strategies against ASFV targeting the altered metabolic pathways.

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., Competing Interests: The authors have declared that no competing interests exist.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection., Competing Interests: The authors have declared that no competing interests exist.

African swine fever (ASF) is a devastating infectious disease caused by African swine fever virus (ASFV). The ASFV genome encodes multiple structural and non-structural proteins that contribute to evasion of host immunity. In this study, we determined that the viral non-structural protein MGF360-14L inhibits interferon-β (IFN-β) promoter activity induced by cGAS-STING signaling. MGF360-14L was also found to downregulate expression of the IRF3 protein and promote its degradation through ubiquitin-meditated proteolysis. Moreover, MGF360-14L was shown to interact with and destabilize IRF3 by facilitating E3 ligase TRIM21-mediated K63-linked ubiquitination of IRF3. Overall, our study revealed that MGF360-14L promotes degradation of IRF3 through TRIM21, thereby inhibiting type I interferon production. These findings provide new insights into the mechanisms underlying ASFV immune evasion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Cui, Xin, Wang, Yu, Chen, Jiang, Gao, Jiang, Guo, Jia and Zhu.)

African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.

African swine fever virus (ASFV) infection can result in lethal disease in pigs. ASFV encodes 150-167 proteins, of which only approximately 50 encoded viral structure proteins are functionally known. ASFV also encodes some nonstructural proteins that are involved in the regulation of viral transcription, viral replication and evasion from host defense. However, the understanding of the molecular correlates of the severity of these infections is still limited. The purpose of this study was to compare host and viral gene expression differences and perform functional analysis in acutely infected, dead and cohabiting asymptomatic pigs infected with ASFV by using RNA-Seq technique; healthy pigs were used as controls. A total of 3,760 and 2,874 upregulated genes and 4,176 and 2,899 downregulated genes were found in healthy pigs vs. acutely infected, dead pigs or asymptomatic pigs, respectively. Additionally, 941 upregulated genes and 956 downregulated genes were identified in asymptomatic vs. acutely infected, dead pigs. Different alternative splicing (AS) events were also analyzed, as were gene chromosome locations, and protein-protein interaction (PPI) network prediction analysis was performed for significantly differentially expressed genes (DEGs). In addition, 30 DEGs were validated by RT-qPCR, and the results were consistent with the RNA-Seq results. We further analyzed the interaction between ASFV and its host at the molecular level and predicted the mechanisms responsible for asymptomatic pigs based on the selected DEGs. Interestingly, we found that some viral genes in cohabiting asymptomatic pigs might integrate into host genes (DP96R, I73R and L83L) or remain in the tissues of cohabiting asymptomatic pigs. In conclusion, the data obtained in the present study provide new evidence for further elucidating ASFV-host interactions and the ASFV infection mechanism and will facilitate the implementation of integrated strategies for controlling ASF spread., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sun, Niu, Yang, Zhao, Tian, Fan, Zhang, Wang, Geng, Zhang, Guan, Williams, Luo, Yin and Liu.)

The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-haemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.

African swine fever (ASF) is a severe hemorrhagic disease in swine characterized by massive lymphocyte depletion and cell death, with apoptosis and necrosis in infected lymphoid tissues. However, the molecular mechanism regarding ASFV-induced cell death remains largely unknown. In this study, 94 ASFV-encoded proteins were screened to determine the viral proteins involved in cell death in vitro, and pE199L showed the most significant effect. Ectopic expression of pE199L in porcine cells (CRL-2843) and human cells (HEK293T and HeLa cells) induced cell death remarkably, showing obvious shrinking, blistering, apoptotic bodies, and nuclear DNA fragments. Meanwhile, cell death was markedly alleviated when the expression of pE199L was knocked down during ASFV infection. Additionally, the expression of pE199L caused a loss of mitochondrial membrane potential, release of cytochrome C, and caspase-9 and -3/7 activation, indicating that the mitochondrial apoptotic pathway was involved in pE199L-induced apoptosis. Further investigations showed that pE199L interacted with several anti-apoptotic BCL-2 subfamily members (such as BCL-X L , MCL-1, BCL-W, and BCL-2A1) and competed with BAK for BCL-X L , which promoted BAK and BAX activation. Taken together, ASFV pE199L induces the mitochondrial-dependent apoptosis, which may provide clues for a comprehensive understanding of ASFV pathogenesis.

African swine fever virus (ASFV) is a large DNA virus that is highly contagious and pathogenic in domestic pigs with a mortality rate up to 100%. However, how ASFV suppresses JAK-STAT1 signaling to evade the immune response remains unclear. In this study, we found that the ASFV-encoded protein MGF-505-7R inhibited proinflammatory IFN-γ-mediated JAK-STAT1 signaling. Mechanistically, MGF-505-7R was found to interact with JAK1 and JAK2 and mediate their degradation. Further study indicated that MGF-505-7R promoted degradation of JAK1 and JAK2 by upregulating the E3 ubiquitin ligase RNF125 expression and inhibiting expression of Hes5, respectively. Consistently, MGF-505-7R-deficient ASFV induced high levels of IRF1 expression and displayed compromised replication both in primary porcine alveolar macrophages and pigs compared with wild-type ASFV. Furthermore, MGF-505-7R deficiency attenuated the virulence of the ASFV and pathogenesis of ASF in pigs. These findings suggest that the JAK-STAT1 axis mediates the innate immune response to the ASFV and that MGF-505-7R plays a critical role in the virulence of the ASFV and pathogenesis of ASF by antagonizing this axis. Thus, we conclude that deletion of MGF-505-7R may serve as a strategy to develop attenuated vaccines against the ASFV., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia, resulting in economic losses for the regional swine industry. There are no commercial vaccines; therefore, disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-ΔA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 10 2 50% hemadsorption doses (HAD 50 ) of ASFV-G-ΔA137R remained clinically healthy during the 28-day observational period. All animals inoculated with ASFV-G-ΔA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-ΔA137R-inoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-G-ΔA137R-inoculated animals. Therefore, ASFV-G-ΔA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic. IMPORTANCE No commercial vaccine is available to prevent African swine fever. The ASF pandemic caused by ASFV Georgia2007 strain (ASFV-G) is seriously affecting pork production in a contiguous area from Central Europe to East Asia. Here we report the rational development of a potential live attenuated vaccine strain by deleting a virus-specific gene, A137R, from the genome of ASFV-G. The resulting virus presented a completely attenuated phenotype and, importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. ASFV-G-ΔA137R confers protection even at low doses (10 2 HAD 50 ), demonstrating its potential as a vaccine candidate. Therefore, ASFV-G-ΔA137R is a novel experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

African swine fever (ASF) is mainly an acute hemorrhagic disease which is highly contagious and lethal to domestic pigs and wild boars. The global pig industry has suffered significant economic losses due to the lack of an effective vaccine and treatment. The African swine fever virus (ASFV) has a large genome of 170-190 kb, encoding more than 150 proteins. During infection, ASFV evades host innate immunity via multiple viral proteins. A528R is a very important member of the polygene family of ASFV, which was shown to inhibit IFN-β production by targeting NF-κB, but its mechanism is not clear. This study has shown that A528R can suppress the TLR8-NF-κB signaling pathway, including the inhibition of downstream promoter activity, NF-κB p65 phosphorylation and nuclear translocation, and the antiviral and antibacterial activity. Further, we found the cellular co-localization and interaction between A528R and p65, and ANK repeat domains of A528R and RHD of p65 are involved in their interaction and the inhibition of p65 activity. Therefore, we conclude that A528R inhibits TLR8-NF-κB signaling by targeting p65 activation and nuclear translocation.

African swine fever virus (ASFV) is the etiological agent of the devastating disease African swine fever (ASF), for which there is currently no licensed vaccine or treatment available. ASF is defined as one of the most serious animal diseases identified to date, due to its global spread in regions of Africa, Europe and Asia, causing massive economic losses. On the Italian island of Sardinia, the disease has been endemic since 1978, although the last control measures put in place achieved a significant reduction in ASF, and the virus has been absent from circulation since April 2019. Like many large DNA viruses, ASFV mutates at a relatively slow rate. However, the limited availability of whole-genome sequences from spatial-localized outbreaks makes it difficult to explore the small-scale genetic structure of these ASFV outbreaks. It is also unclear if the genetic variability within outbreaks can be captured in a handful of sequences, or if larger sequencing efforts can improve phylogenetic reconstruction and evolutionary or epidemiological inference. The aim of this study was to investigate the phylogenetic patterns of ASFV outbreaks between 1978 and 2018 in Sardinia, in order to characterize the epidemiological dynamics of the viral strains circulating in this Mediterranean island. To reach this goal, 58 new whole genomes of ASFV isolates were obtained, which represents the largest ASFV whole-genome sequencing effort to date. We provided a complete description of the genomic diversity of ASFV in terms of nucleotide mutations and small and large indels among the isolates collected during the outbreaks. The new sequences capture more than twice the genomic and phylogenetic diversity of all the previously published Sardinian sequences. The extra genomic diversity increases the resolution of the phylogenetic reconstruction, enabling us to dissect, for the first time, the genetic substructure of the outbreak. We found multiple ASFV subclusters within the phylogeny of the Sardinian epidemic, some of which coexisted in space and time.

African swine fever virus (ASFV) is a large nucleoplasmic DNA virus, in which the genome is around 170-198 kilobases (kb). More than 50 % genes have unknown functions. Here, MGF100-1R gene is chosen to study the primary function and sublocalization. The gene was located at the left variable region of the ASFV genome that belongs to MGF100 families. It located at the cytoplasm without cytotoxic activities. However, it related to induce the transcriptional levels of pro-inflammatory cytokines. A deletion mutant of MGF100-1R gene was constructed based on ASFV Chinese strain GZ201801. The recombinant deletion mutant (ASFV△MGF100-1R) was demonstrated in vitro that the gene is non-essential for virus replication with a similar replication kinetics in bone marrow-derived macrophages (BMDMs) cell cultures when compared to parental virus. In vivo evaluation, ASFV△MGF100-1R was inoculated intramuscularly and led to a similar pathogenesis that caused by the parental ASFV GZ201801, confirming that deletion of MGF100-1R gene from the ASFV genome does not impact virulence., (Copyright © 2021. Published by Elsevier B.V.)

African swine fever (ASF) is one of the most important viral diseases of pigs caused by the ASF virus (ASFV). The virus is highly stable over a wide range of temperatures and pH and can survive in meat and meat products for several months, leading to long-distance transmission of ASF. Whole blood, serum, and organs from infected pigs are used routinely as approved sample types in the laboratory diagnosis of ASF. However, these sample types may not always be available. Here, we investigated meat exudate as an alternative sample type for the detection of ASFV-specific nucleic acids and antibodies. Pigs were infected with various ASFV strains: the highly virulent ASFV Malawi LIL 18/2 strain, the moderately-virulent ASFV Estonia 2014 strain, or the low-virulent ASFV OURT/88/3 strain. The animals were euthanized on different days post-infection (dpi), and meat exudates were collected and tested for the presence of ASFV-specific nucleic acids and antibodies. Animals infected with the ASFV Malawi LIL 18/2 developed severe clinical signs and succumbed to the infection within seven dpi, while pigs infected with ASFV Estonia 2014 also developed clinical signs but survived longer, with a few animals seroconverting before succumbing to the ASFV infection or being euthanized as they reached humane endpoints. Pigs infected with ASFV OURT/88/3 developed transient fever and seroconverted without mortality. ASFV genomic material was detected in meat exudate from pigs infected with ASFV Malawi LIL 18/2 and ASFV Estonia 2014 at the onset of viremia but at a lower amount when compared to the corresponding whole blood samples. Low levels of ASFV genomic material were detected in the whole blood of ASFV OURT/88/3-infected pigs, and no ASFV genomic material was detected in the meat exudate of these animals. Anti-ASFV antibodies were detected in the serum and meat exudate derived from ASFV OURT/88/3-infected pigs and in some of the samples derived from the ASFV Estonia 2014-infected pigs. These results indicate that ASFV genomic material and anti-ASFV antibodies can be detected in meat exudate, indicating that this sample can be used as an alternative sample type for ASF surveillance when routine sample types are unavailable or are not easily accessible.

The introduction of genotype II African swine fever (ASF) virus, presumably from Africa into Georgia in 2007, and its continuous spread through Europe and Asia as a panzootic disease of suids, continues to have a huge socio-economic impact. ASF is characterized by hemorrhagic fever leading to a high case/fatality ratio in pigs. In Europe, wild boar are especially affected. This review summarizes the currently available knowledge on ASF in wild boar in Europe. The current ASF panzootic is characterized by self-sustaining cycles of infection in the wild boar population. Spill-over and spill-back events occur from wild boar to domestic pigs and vice versa. The social structure of wild boar populations and the spatial behavior of the animals, a variety of ASF virus (ASFV) transmission mechanisms and persistence in the environment complicate the modeling of the disease. Control measures focus on the detection and removal of wild boar carcasses, in which ASFV can remain infectious for months. Further measures include the reduction in wild boar density and the limitation of wild boar movements through fences. Using these measures, the Czech Republic and Belgium succeeded in eliminating ASF in their territories, while the disease spread in others. So far, no vaccine is available to protect wild boar or domestic pigs reliably against ASF.

African swine fever is a devastating disease of domestic swine and wild boar caused by a large double-stranded DNA virus that encodes for more than 150 open reading frames. There is no licensed vaccine for the disease and the most promising current candidates are modified live viruses that have been attenuated by deletion of virulence factors. Like many viruses African swine fever virus significantly alters the host cell machinery to benefit its replication and viral genes that modify host pathways represent promising targets for development of gene deleted vaccines. Autophagy is an important cellular pathway that is involved in cellular homeostasis, innate and adaptive immunity and therefore is manipulated by a number of different viruses. Autophagy is regulated by a complex protein cascade and here we show that African swine fever virus can block formation of autophagosomes, a critical functional step of the autophagy pathway through at least two different mechanisms. Interestingly this does not require the A179L gene that has been shown to interact with Beclin-1, an important autophagy regulator.

African swine fever virus causes a frequently fatal disease of domestic pigs and wild boar that has a high economic impact across 3 continents. The large double-stranded DNA genome codes for approximately 160 proteins. Many of these have unknown functions and this hinders our understanding of the virus and host interactions. The purpose of the study was to evaluate the role of two virus proteins, K145R and DP148R, in virus replication in macrophages and virulence in pigs. To do this, the DP148R gene, alone or in combination with the K145R gene, was deleted from the virulent genotype II Georgia 2007/1 isolate. Neither of these deletions reduced the ability of the viruses to replicate in porcine macrophages compared to the parental wild-type virus. Pigs infected with GeorgiaΔDP148R developed clinical and post-mortem signs and high viremia, typical of acute African swine fever, and were culled on day 6 post-infection. The additional deletion of the K145R gene delayed the onset of clinical signs and viremia in pigs by 3 days, but pigs showed signs of acute African swine fever and were culled on days 10 or 13 post-infection. The results show that the deletion of DP148R did not attenuate the genotype II Georgia 2007/1 isolate, contrary to the results obtained with the genotype I Benin97/1 isolate. Additional deletion of the K145R gene delayed clinical signs, but infected pigs reached the humane endpoint. The deletion of additional genes would be required to attenuate the virus.

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF., Competing Interests: The authors have declared that no competing interests exist.

African swine fever (ASF) has been present in Lithuania since 2014. It is mainly the wild boar population that is affected. Currently, little is known about the epidemiological course of ASF in Lithuania. In the present study, ASF surveillance data from 2016-2021 were analyzed. The numbers of samples taken from hunted wild boar and wild boar found dead per year and month were recorded and the prevalence was estimated for each study month and administrative unit. A Bayesian space-time model was used to calculate the temporal trend of the prevalence estimates. In addition, population data were analyzed on a yearly basis. Most samples were investigated in 2016 and 2017 and originated from hunted animals. Prevalence estimates of ASF virus-positive wild boar decreased from May 2019 onwards. Seroprevalence estimates showed a slight decrease at the same time, but they increased again at the end of the study period. A significant decrease in the population density was observed over time. The results of the study show that ASF is still present in the Lithuanian wild boar population. A joint interdisciplinary effort is needed to identify weaknesses in the control of ASF in Lithuania and to combat the disease more successfully.

African swine fever (ASF) is an emerging disease threat to the swine industry worldwide. There is no vaccine against ASF, and progress is hindered by a lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. We have previously demonstrated that homologous ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin are required for protection against challenge with the virulent ASFV strain Congo (Genotype I, Serogroup 2), and we have identified T-cell epitopes on CD2v which may be associated with serotype-specific protection. Here, using a cell-culture adapted derivative of the ASFV strain Congo (Congo-a) with specific deletion of the EP402R gene (ΔCongoCD2v) in swine vaccination/challenge experiments, we demonstrated that deletion of the EP402R gene results in the failure of ΔCongoCD2v to induce protection against challenge with the virulent strain Congo (Congo-v). While ΔCongoCD2v growth kinetics in COS-1 cells and primary swine macrophage culture were almost identical to parental Congo-a, replication of ΔCongoCD2v in vivo was significantly reduced compared with parental Congo-a. Our data support the idea that the CD2v protein is important for the ability of homologous live-attenuated vaccines to induce protective immunity against the ASFV strain Congo challenge in vivo.

Prevention and control of African swine fever virus (ASFV) in Europe, Asia, and Africa seem to be extremely difficult in view of the ease with which it spreads, its high resistance to environmental conditions, and the many obstacles related to the introduction of effective specific immunoprophylaxis. Biological properties of ASFV indicate that the African swine fever (ASF) pandemic will continue to develop and that only the implementation of an effective and safe vaccine will ensure a reduction in the spread of ASFV. At present, vaccines against ASF are not available. The latest approaches to the ASFV vaccine's design concentrate on the development of either modified live vaccines by targeted gene deletion from different isolates or subunit vaccines. The construction of an effective vaccine is hindered by the complex structure of the virus, the lack of an effective continuous cell line for the isolation and propagation of ASFV, unpredictable and stain-specific phenotypes after the genetic modification of ASFV, a risk of reversion to virulence, and our current inability to differentiate infected animals from vaccinated ones. Moreover, the design of vaccines intended for wild boars and oral administration is desirable. Despite several obstacles, the design of a safe and effective vaccine against ASFV seems to be achievable.

African swine fever virus (ASFV) causes a devastating disease of swine that has caused outbreaks in Central Europe since 2007, spreading into Asia in 2018. ASFV is a large, structurally complex virus with a large dsDNA genome encoding for more than 160 genes, most of them still uncharacterized. p22, encoded by the ASFV gene KP177R, is an early transcribed, structural virus protein located in the ASFV particle. Although its exact function is unknown, p22 has recently been identified as an interacting partner of several host proteins. Here, we describe the development of a recombinant ASFV (ASFV-G-∆KP177R) lacking the KP177R gene as a tool to evaluate the role of p22 in virus replication and virulence in swine. The recombinant ASFV-G-∆KP177R demonstrated that the KP177R gene is non-essential for ASFV replication in primary swine macrophages, with virus yields similar to those of the parental, highly virulent field isolate Georgia2010 (ASFV-G). In addition, experimental infection of domestic pigs with ASFV-G-∆KP177R produced a clinical disease similar to that caused by the parental ASFV-G. Therefore, and surprisingly, p22 does not seem to be involved in virus replication or virulence in swine.

This study aimed to indicate the influence of infection caused by genotype II African swine fever virus (ASFV)-isolate Pol18&#95;28298&#95;O111, currently circulating in Poland, on blood counts, biochemical parameters, as well as inflammatory and immune responses. Blood and sera collected from 21 domestic pigs infected intranasally with different doses of virulent ASFV were analysed. The infection led to variable changes in blood counts depending on the stage of the disease with a tendency towards leukopenia and thrombocytopenia. The elevated C-reactive protein (CRP) concentrations and microscopic lesions in organs confirmed the development of the inflammation process, which also resulted in an increased level of biochemical markers such as: Aspartate transaminase (AST), creatine kinase (CK), creatinine, and urea. Antibodies could be detected from 9 to 18 days post infection (dpi). Two survivors presented the highest titer of antibodies (>5 log 10 /mL) with a simultaneous increase in the lymphocyte T (CD3+) percentage-revealed by flow cytometry. Results confirmed a progressive inflammatory process occurring during the ASFV infection, which may lead to multiple organs failure and death of the majority of affected animals.

African swine fever (ASF) is currently causing an epizootic, affecting pigs throughout Eurasia, and causing significant economic losses in the swine industry. ASF is caused by African swine fever virus (ASFV) that consists of a large dsDNA genome that encodes for more than 160 genes; few of these genes have been studied in detail. ASFV contains four multi-gene family (MGF) groups of genes that have been implicated in regulating the immune response and host specificity; however, the individual roles of most of these genes have not been well studied. Here, we describe the evaluation of the previously uncharacterized ASFV MGF110-1L open reading frame (ORF) using a deletion mutant of the ASFV currently circulating throughout Eurasia. The recombinant ASFV lacking the MGF110-1L gene (ASFV-G-ΔMGF110-1L) demonstrated in vitro that the MGF110-1L gene is non-essential, since ASFV-G-ΔMGF110-1L had similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔMGF110-1L produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the MGF110-1L gene from the ASFV genome does not affect viral virulence.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a major epidemic disease endangering the swine industry. Although a number of vaccine candidates have been reported, none are commercially available yet. To explore the effect of unknown genes on the biological characteristics of ASFV and the possibility of a gene-deleted isolate as a vaccine candidate, the strain SY18ΔL7-11, with deletions of L7L-L11L genes from ASFV SY18, was constructed, and its biological properties were analyzed. The results show that deletion of genes L7L-L11L did not affect replication of the virus in vitro. Virulence of SY18△L7-11 was significantly reduced, as 11 of the 12 pigs survived for 28 days after intramuscular inoculation with a low dose (10 3 TCID 50 ) or a high dose (10 6 TCID 50 ) of SY18ΔL7-11. All 11 surviving pigs were completely protected against challenge with the parental ASFV SY18 on 28 days postinoculation (dpi). Transient fever and/or irregularly low levels of genomic DNA in the blood were monitored in some pigs after inoculation. No ASF clinical signs or viremia were monitored after challenge. Antibodies to ASFV were induced in all pigs from 14 to 21 days postinoculation. IFN-γ was detected in most of the inoculated pigs, which is usually inhibited in ASFV-infected pigs. Overall, the results demonstrate that SY18ΔL7-11 is a candidate for further constructing safer vaccine(s), with better joint deletions of other gene(s) related to virulence.

African swine fever (ASF) is a major threat to domestic pigs and wild boars. Since 2018, ASF outbreaks have been ongoing in China. As of August 3, 2019, a total of 151 ASF clusters of outbreaks reported in China have caused severe economic losses for the industry, the pig farmers and pork producers, due to the lack of an efficacious vaccine. The present study aims to analyze the epidemiologic characteristics of ASF outbreak that occurred in several regions across China during the period August 2018- August 2019. Particular focus was on the epidemic distribution, main transmission routes, incidence/fatality, impact on pig production capacity, and the main preventive measures adopted to mitigate the risk of ASF spread in pig farming systems by Chinese government. Results show that anthropogenic factors, spatial distribution, efficient measures taken by China,and good response timely in implementation of preventive measures are important on the transmission of ASF and these suggest that effective ASF risk management in China will require a comprehensive and integrated approach linking science and implemented by all relevant stakeholders. This provides an empirical basis to optimize current interventions as well as develop new tools and strategies to reduce the risk transmission of African swine fever virus (ASFV) to domestic pigs and wild boars., Competing Interests: Declaration of Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Since African swine fever (ASF) first appeared in the Caucasus region in 2007, it has spread rapidly and is now present in numerous European and Asian countries. In Europe, mainly wild boar populations are affected and pose a risk for domestic pigs. In Asia, domestic pigs are almost exclusively affected. An effective and safe vaccine is not available, and correlates of protection are far from being understood. Therefore, research on immune responses, immune dysfunction and pathogenesis is mandatory. It is acknowledged that T cells play a pivotal role. Thus, we investigated T-cell responses of domestic pigs and wild boar upon infection with the highly virulent ASF virus (ASFV) strain 'Armenia08'. For this purpose, we used a flow cytometry-based multicolour analysis to identify T-cell subtypes (cytotoxic T cells, T-helper cells, γδ T cells) and their functional impairment in ASFV-infected pigs. Domestic pigs showed lymphopaenia, and neither in the blood nor in the lymphoid organs was a proliferation of CD8 + effector cells observed. Furthermore, a T-bet-dependent activation of the remaining CD8 T cells did not occur. In contrast, a T-cell response could be observed in wild boar at 5 days post-inoculation in the blood and in tendency also in some organs. However, this cytotoxic response was not beneficial as all wild boars showed a severe acute lethal disease and a higher proportion died spontaneously or was euthanized at the humane endpoint., (© 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

The African swine fever (ASF) pandemic is currently affecting pigs throughout Eurasia, resulting in significant swine production losses. The causative agent, ASF virus (ASFV), is a large, structurally complex virus with a genome encoding more than 160 genes. The function of most of those genes remains unknown. Here, we presented the previously uncharacterized ASFV gene MGF360-1L, the first gene in the genome. The kinetic studies of virus RNA transcription demonstrated that the MGF360-1L gene was transcribed as a late virus protein. The essentiality of MGF360-1L to virus replication was evaluated by developing a recombinant ASFV lacking the gene (ASFV-G-ΔMGF360-1L). In primary swine macrophage cell cultures, ASFV-G-ΔMGF360-1L showed similar replication kinetics as the parental highly virulent field isolate Georgia2007 (ASFV-G). Domestic pigs experimentally infected with ASFV-G-ΔMGF360-1L presented with a clinical disease indistinguishable from that caused by ASFV-G, demonstrating that MGF360-1L was not involved in virulence in swine, the natural host of ASFV.

African swine fever (ASF) is a viral hemorrhagic disease with exceptionally high lethality in domestic pigs and Eurasian wild boar. Over the last decade, ASF has emerged in several European and Asian countries and has now an unprecedented distribution. Against this background, the presented review focuses on current knowledge and advances in ASF virology, clinical disease upon infection with recent strains, epidemiology, diagnosis, and control. This review highlights knowledge gaps and controversial opinions related to ASF., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)