Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

African swine fever virus infection regulates pyroptosis by cleaving gasdermin A via active caspase-3 and caspase-4.

African swine fever, caused by the African swine fever virus (ASFV), is a viral hemorrhagic disease that affects domestic pigs and wild boars. ASFV infection causes extensive tissue damage, and the associated mechanism is poorly understood. Pyroptosis is characterized by the activation of inflammatory caspases and pore formation in the cellular plasma membrane, resulting in the release of inflammatory cytokines and cell damage. How ASFV infection regulates pyroptosis remains unclear. Here, using siRNA assay and overexpression methods, we report that ASFV infection regulated pyroptosis by cleaving the pyroptosis execution protein gasdermin A (GSDMA). ASFV infection activated caspase-3 and caspase-4, which specifically cleaved GSDMA at D75-P76 and D241-V242 to produce GSDMA into five fragments, including GSDMA-N1-75, GSDMA-N1-241, and GSDMA-N76-241 fragments at the N-terminal end of GSDMA. Only GSDMA-N1-241, which was produced in the late stage of ASFV infection, triggered pyroptosis and inhibited ASFV replication. The fragments, GSDMA-N1-75 and GSDMA-N76-241, lose the ability to induce pyroptosis. Overall ASFV infection differentially regulates pyroptosis by GSDMA in the indicated phase, which may be conducive to its own replication. Our findings reveal a novel molecular mechanism for the regulation of pyroptosis.

Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target.

Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

African swine fever virus pB475L evades host antiviral innate immunity via targeting STAT2 to inhibit IFN-I signaling.

African swine fever virus (ASFV) causes severe disease in domestic pigs and wild boars, seriously threatening the development of the global pig industry. Type I interferon (IFN-I) is an important component of innate immunity, inducing the transcription and expression of antiviral cytokines by activating Janus-activated kinase-signal transducer and activator of transcription (STAT). However, the underlying molecular mechanisms by which ASFV antagonizes IFN-I signaling have not been fully elucidated. Therefore, using coimmunoprecipitation, confocal microscopy, and dual luciferase reporter assay methods, we investigated these mechanisms and identified a novel ASFV immunosuppressive protein, pB475L, which interacts with the C-terminal domain of STAT2. Consequently, pB475L inhibited IFN-I signaling by inhibiting STAT1 and STAT2 heterodimerization and nuclear translocation. Furthermore, we constructed an ASFV-B475L7PM mutant strain by homologous recombination, finding that ASFV-B475L7PM attenuated the inhibitory effects on IFN-I signaling compared to ASFV-WT. In summary, this study reveals a new mechanism by which ASFV impairs host innate immunity.

Viroporin-like activity of the hairpin transmembrane domain of African swine fever virus B169L protein.

African swine fever virus (ASFV) is the causative agent of a contagious disease affecting wild and domestic swine. The function of B169L protein, as a potential integral structural membrane protein, remains to be experimentally characterized. Using state-of-the-art bioinformatics tools, we confirm here earlier predictions indicating the presence of an integral membrane helical hairpin, and further suggest anchoring of this protein to the ER membrane, with both terminal ends facing the lumen of the organelle. Our evolutionary analysis confirmed the importance of purifying selection in the preservation of the identified domains during the evolution of B169L in nature. Also, we address the possible function of this hairpin transmembrane domain (HTMD) as a class IIA viroporin. Expression of GFP fusion proteins in the absence of a signal peptide supported B169L insertion into the ER as a Type III membrane protein and the formation of oligomers therein. Overlapping peptides that spanned the B169L HTMD were reconstituted into ER-like membranes and the adopted structures analyzed by infrared spectroscopy. Consistent with the predictions, B169L transmembrane sequences adopted α-helical conformations in lipid bilayers. Moreover, single vesicle permeability assays demonstrated the assembly of lytic pores in ER-like membranes by B169L transmembrane helices, a capacity confirmed by ion-channel activity measurements in planar bilayers. Emphasizing the relevance of these observations, pore-forming activities were not observed in the case of transmembrane helices derived from EP84R, another ASFV protein predicted to anchor to membranes through a α-helical HTMD. Overall, our results support predictions of viroporin-like function for the B169L HTMD.IMPORTANCEAfrican swine fever (ASF), a devastating disease affecting domestic swine, is widely spread in Eurasia, producing significant economic problems in the pork industry. Approaches to prevent/cure the disease are mainly restricted to the limited information concerning the role of most of the genes encoded by the large (160-170 kba) virus genome. In this report, we present the experimental data on the functional characterization of the African swine fever virus (ASFV) gene B169L. Data presented here indicates that the B169L gene encodes for an essential membrane-associated protein with a viroporin function.

Six adenoviral vectored African swine fever virus genes protect against fatal disease caused by genotype I challenge.

African swine fever virus causes a lethal hemorrhagic disease in domestic swine and wild boar for which currently licensed commercial vaccines are only available in Vietnam. Development of subunit vaccines is complicated by the lack of information on protective antigens as well as suitable delivery systems. Our previous work showed that a pool of eight African swine fever virus genes vectored using an adenovirus prime and modified vaccinia virus boost could prevent fatal disease after challenge with a virulent genotype I isolate of the virus. Here, we identify antigens within this pool of eight that are essential for the observed protection and demonstrate that adenovirus-prime followed by adenovirus-boost can also induce protective immune responses against genotype I African swine fever virus. Immunization with a pool of adenoviruses expressing individual African swine fever virus genes partially tailored to genotype II virus did not protect against challenge with genotype II Georgia 2007/1 strain, suggesting that different antigens may be required to induce cross-protection for genetically distinct viruses. IMPORTANCE: African swine fever virus causes a lethal hemorrhagic disease in domestic pigs and has killed millions of animals across Europe and Asia since 2007. Development of safe and effective subunit vaccines against African swine fever has been problematic due to the complexity of the virus and a poor understanding of protective immunity. In a previous study, we demonstrated that a complex combination of eight different virus genes delivered using two different viral vector vaccine platforms protected domestic pigs from fatal disease. In this study, we show that three of the eight genes are required for protection and that one viral vector is sufficient, significantly reducing the complexity of the vaccine. Unfortunately, this combination did not protect against the current outbreak strain of African swine fever virus, suggesting that more work to identify immunogenic and protective viral proteins is required to develop a truly effective African swine fever vaccine.

Bis-benzylisoquinoline alkaloids inhibit African swine fever virus internalization and replication by impairing late endosomal/lysosomal function.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly infectious disease afflicting domestic pigs and wild boars. It exhibits an alarming acute infection fatality rate of up to 100%. Regrettably, no commercial vaccines or specific drugs for combating this disease are currently available. This study evaluated the anti-ASFV activities in porcine alveolar macrophages, 3D4/21 cells, and PK-15 cells of four bis-benzylisoquinoline alkaloids (BBAs): cepharanthine (CEP), tetrandrine, fangchinoline, and iso-tetrandrine. Furthermore, we demonstrated that CEP, which exhibited the highest selectivity index (SI = 81.31), alkalized late endosomes/lysosomes, hindered ASFV endosomal transport, disrupted virus uncoating signals, and thereby inhibited ASFV internalization. Additionally, CEP disrupted ASFV DNA synthesis, leading to the inhibition of viral replication. Moreover, berbamine was labeled with NBD to synthesize a fluorescent probe to study the cellular location of these BBAs. By co-staining with Lyso-Tracker and lysosome-associated membrane protein 1, we demonstrated that BBAs target the endolysosomal compartments for the first time. Our data together indicated that BBAs are a class of natural products with significant inhibitory effects against ASFV infection. These findings suggest their potential efficacy as agents for the prevention and control of ASF, offering valuable references for the identification of potential drug targets.IMPORTANCEThe urgency and severity of African swine fever (ASF) underscore the critical need for effective interventions against this highly infectious disease, which poses a grave threat to domestic pigs and wild boars. Our study reveals the potent anti-African swine fever virus (ASFV) efficacy of bis-benzylisoquinoline alkaloids (BBAs), particularly evident in the absence of progeny virus production under a 5 µM concentration treatment. The structural similarity among cepharanthine, tetrandrine, fangchinoline, and iso-tetrandrine, coupled with their analogous inhibitory stages and comparable selectivity indexes, strongly suggests a shared antiviral mechanism within this drug category. Further investigation revealed that BBAs localize to lysosomes and inhibit the internalization and replication of ASFV by disrupting the endosomal/lysosomal function. These collective results have profound implications for ASF prevention and control, suggesting the potential of the investigated agents as prophylactic and therapeutic measures. Furthermore, our study offers crucial insights into identifying drug targets and laying the groundwork for innovative interventions.

African swine fever virus A137R assembles into a dodecahedron cage.

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.

Transcriptome profiling in swine macrophages infected with African swine fever virus at single-cell resolution.

African swine fever virus (ASFV) is the causative agent of African swine fever, a highly contagious and usually fatal disease in pigs. The pathogenesis of ASFV infection has not been clearly elucidated. Here, we used single-cell RNA-sequencing technology to survey the transcriptomic landscape of ASFV-infected primary porcine alveolar macrophages. The temporal dynamic analysis of viral genes revealed increased expression of viral transmembrane genes. Molecular characteristics in the ASFV-exposed cells exhibited the activation of antiviral signaling pathways with increased expression levels of interferon-stimulated genes and inflammatory- and cytokine-related genes. By comparing infected cells with unexposed cells, we showed that the unfolded protein response (UPR) pathway was activated in low viral load cells, while the expression level of UPR-related genes in high viral load cells was less than that in unexposed cells. Cells infected with various viral loads showed signature transcriptomic changes at the median progression of infection. Within the infected cells, differential expression analysis and coregulated virus­host analysis both demonstrated that ASFV promoted metabolic pathways but inhibited interferon and UPR signaling, implying the regulation pathway of viral replication in host cells. Furthermore, our results revealed that the cell apoptosis pathway was activated upon ASFV infection. Mechanistically, the production of tumor necrosis factor alpha (TNF-α) induced by ASFV infection is necessary for cell apoptosis, highlighting the importance of TNF-α in ASFV pathogenesis. Collectively, the data provide insights into the comprehensive host responses and complex virus­host interactions during ASFV infection, which may instruct future research on antiviral strategies.

African swine fever virus pS273R antagonizes stress granule formation by cleaving the nucleating protein G3BP1 to facilitate viral replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.

Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge.

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.

Crystal structure of African swine fever virus pE301R reveals a ring-shaped trimeric DNA sliding clamp.

African swine fever virus (ASFV) is an important animal pathogen that is causing a current African swine fever pandemic and affecting pork industry globally. ASFV encodes at least 150 proteins, and the functions of many of them remain to be clarified. The ASFV protein E301R (pE301R) was predicted to be a DNA sliding clamp protein homolog working as a DNA replication processivity factor. However, structural evidence was lacking to support the existence of a ring-shaped sliding clamp in large eukaryotic DNA viruses. Here, we have solved a high-resolution crystal structure of pE301R and identified a canonical ring-shaped clamp comprising a pE301R trimer. Interestingly, this complete-toroidal structure is different from those of the monomeric clamp protein homolog, herpes simplex virus UL42, and the C-shaped dimeric human cytomegalovirus UL44, but highly homologous to that of the eukaryotic clamp homolog proliferating cell nuclear antigen. Moreover, pE301R has a unique N-terminal extension that is important in maintaining the trimeric form of the protein in solution, while specific features in length and surface electrostatic potential of its interdomain connector implies specificity in interactions with binding partners such as the viral DNA polymerase. Thus, our data pave the way for further dissection of the processivity clamp protein structural and functional diversity and ASFV DNA replication mechanisms.

Detection of Recombinant African Swine Fever Virus Strains of p72 Genotypes I and II in Domestic Pigs, Vietnam, 2023.

African swine fever virus (ASFV) genotype II is endemic to Vietnam. We detected recombinant ASFV genotypes I and II (rASFV I/II) strains in domestic pigs from 6 northern provinces in Vietnam. The introduction of rASFV I/II strains could complicate ongoing ASFV control measures in the region.

Coreceptor AXL Facilitates African Swine Fever Virus Entry via Apoptotic Mimicry.

African swine fever (ASF) is an acute and hemorrhagic infectious disease caused by African swine fever virus (ASFV), which is listed as an animal epidemic disease that must be reported by The World Organization for Animal Health and that causes serious economic losses to China and even the whole world. Currently, the entry mechanism of ASFV is not fully understood. Especially in the early stages of virus entry, the host factors required for ASFV entry have not yet been identified and characterized. In this study, we demonstrated that ASFV externalized phosphatidylserine (PS) on the envelope functioned as viral apoptotic mimicry, which interacts with AXL, a tyrosine kinase receptor, to mediate ASFV entry into porcine alveolar macrophages (PAMs). We found that AXL was the most pronounced phosphatidylserine receptor (PSR) affecting ASFV entry in PAMs by RNA interference screening. Knockout AXL gene expression remarkably decreased ASFV internalization and replication in MA104 cells. Furthermore, the antibody against AXL extracellular domains effectively inhibited the ASFV entry. Consistent with these results, the deletion of the intracellular kinase domain of AXL and the treatment of the AXL inhibitor, R428, significantly inhibited the internalization of ASFV. Mechanistically, AXL facilitated the internalization of ASFV virions via macropinocytosis. Collectively, we provide evidence that AXL is a coreceptor for ASFV entry into PAMs, which expands our knowledge of ASFV entry and provides a theoretical basis for identifying new antiviral targets. IMPORTANCE African swine fever (ASF) is a highly contagious infectious disease caused by the ASF virus (ASFV), with a mortality rate of up to 100%. ASFV has caused huge economic losses to pig farming worldwide. Specific cellular surface receptors are considered crucial determinants of ASFV tropism. However, the host factors required for ASFV entry have not yet been identified, and the molecular mechanism of its entry remains unclear. Here, we found that ASFV utilized phosphatidylserine (PS) on the surface of virions to masquerade as apoptotic mimicry and facilitated virus entry by interacting with host factor AXL. We found that knockout of AXL remarkably decreased ASFV internalization and replication. The antibody against AXL extracellular domains and AXL inhibitor R428 significantly inhibited the internalization of ASFV via macropinocytosis. The current work deepens our understanding of ASFV entry and provides clues for the development of antiviral drugs to control ASFV infection.

African Swine Fever Virus MGF505-7R Interacts with Interferon Regulatory Factor 9 to Evade the Type I Interferon Signaling Pathway and Promote Viral Replication.

African swine fever (ASF) is an acute and severe infectious disease caused by the ASF virus (ASFV). The mortality rate of ASF in pigs can reach 100%, causing huge economic losses to the pig industry. Here, we found that ASFV protein MGF505-7R inhibited the beta interferon (IFN-ß)-mediated Janus-activated kinase-signal transducer and activation of transcription (JAK-STAT) signaling. Our results demonstrate that MGF505-7R inhibited interferon-stimulated gene factor 3 (ISGF3)-mediated IFN-stimulated response element (ISRE) promoter activity. Importantly, we observed that MGF505-7R inhibits ISGF3 heterotrimer formation by interacting with interferon regulatory factor 9 (IRF9) and inhibits the nuclear translocation of ISGF3. Moreover, to demonstrate the role of MGF505-7R in IFN-I signal transduction during ASFV infection, we constructed and evaluated ASFV-ΔMGF505-7R recombinant viruses. ASFV-ΔMGF505-7R restored STAT2 and STAT1 phosphorylation, alleviated the inhibition of ISGF3 nuclear translocation, and showed increased susceptibility to IFN-ß, unlike the parental GZ201801 strain. In conclusion, our study shows that ASFV protein MGF505-7R plays a key role in evading IFN-I-mediated innate immunity, revealing a new mode of evasion for ASFV. IMPORTANCE ASF, caused by ASFV, is currently prevalent in Eurasia, with mortality rates reaching 100% in pigs. At present, there are no safe or effective vaccines against ASFV. In this study, we found that the ASFV protein MGF505-7R hinders IFN-ß signaling by interacting with IRF9 and inhibiting the formation of ISGF3 heterotrimers. Of note, we demonstrated that MGF505-7R plays a role in the immune evasion of ASFV in infected hosts and that recombinant viruses alleviated the effect on type I IFN (IFN-I) signaling and exhibited increased susceptibility to IFN-ß. This study provides a theoretical basis for developing vaccines against ASFV using strains with MGF505-7R gene deletions.

African Swine Fever Virus L83L Negatively Regulates the cGAS-STING-Mediated IFN-I Pathway by Recruiting Tollip To Promote STING Autophagic Degradation.

African swine fever (ASF) is a devastating infectious disease of pigs caused by the African swine fever virus (ASFV), which poses a great danger to the global pig industry. Many viral proteins can suppress with interferon signaling to evade the host's innate immune responses. Therefore, the development of an effective vaccine against ASFV has been dampened. Recent studies have suggested that the L83L gene may be integrated into the host genome, weakening the host immune system, but the underlying mechanism is unknown. Our study found that L83L negatively regulates the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. Overexpression of L83L inhibited IFN-ß promoter and ISRE activity, and knockdown of L83L induced higher transcriptional levels of interferon-stimulated genes (ISGs) and phosphorylation levels of IRF3 in primary porcine alveolar macrophages. Mechanistically, L83L interacted with cGAS and STING to promote autophagy-lysosomal degradation of STING by recruiting Tollip, thereby blocking the phosphorylation of the downstream signaling molecules TBK1, IRF3, and IκBα and reducing IFN-I production. Altogether, our study reveals a negative regulatory mechanism involving the L83L-cGAS-STING-IFN-I axis and provides insights into an evasion strategy involving autophagy and innate signaling pathways employed by ASFV. IMPORTANCE African swine fever virus (ASFV) is a large double-stranded DNA virus that primarily infects porcine macrophages. The ASFV genome encodes a large number of immunosuppressive proteins. Current options for the prevention and control of this pathogen remain pretty limited. Our study showed that overexpression of L83L inhibited the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. In contrast, the knockdown of L83L during ASFV infection enhanced IFN-I production in porcine alveolar macrophages. Additional analysis revealed that L83L protein downregulated IFN-I signaling by recruiting Tollip to promote STING autophagic degradation. Although L83L deletion has been reported to have little effect on viral replication, its immune evade mechanism has not been elucidated. The present study extends our understanding of the functions of ASFV-encoded pL83L and its immune evasion strategy, which may provide a new basis for developing a live attenuated vaccine for ASF.

African Swine Fever Virus Cysteine Protease pS273R Inhibits Type I Interferon Signaling by Mediating STAT2 Degradation.

African swine fever virus (ASFV) is a large DNA virus that causes African swine fever (ASF), an acute and hemorrhagic disease in pigs with lethality rates of up to 100%. To date, how ASFV efficiently suppress the innate immune response remains enigmatic. In this study, we identified ASFV cysteine protease pS273R as an antagonist of type I interferon (IFN). Overexpression of pS273R inhibited JAK-STAT signaling triggered by type I IFNs. Mechanistically, pS273R interacted with STAT2 and recruited the E3 ubiquitin ligase DCST1, resulting in K48-linked polyubiquitination at K55 of STAT2 and subsequent proteasome-dependent degradation of STAT2. Furthermore, such a function of pS273R in JAK-STAT signaling is not dependent on its protease activity. These findings suggest that ASFV pS273R is important to evade host innate immunity. IMPORTANCE ASF is an acute disease in domestic pigs caused by infection with ASFV. ASF has become a global threat with devastating economic and ecological consequences. To date, there are no commercially available, safe, and efficacious vaccines to prevent ASFV infection. ASFV has evolved a series of strategies to evade host immune responses, facilitating its replication and transmission. Therefore, understanding the immune evasion mechanism of ASFV is helpful for the development of prevention and control measures for ASF. Here, we identified ASFV cysteine protease pS273R as an antagonist of type I IFNs. ASFV pS273R interacted with STAT2 and mediated degradation of STAT2, a transcription factor downstream of type I IFNs that is responsible for induction of various IFN-stimulated genes. pS273R recruited the E3 ubiquitin ligase DCST1 to enhance K48-linked polyubiquitination of STAT2 at K55 in a manner independent of its protease activity. These findings suggest that pS273R is important for ASFV to escape host innate immunity, which sheds new light on the mechanisms of ASFV immune evasion.

Deletion of the gene for the African swine fever virus BCL-2 family member A179L increases virus uptake and apoptosis but decreases virus spread in macrophages and reduces virulence in pigs.

IMPORTANCE: African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.

Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II.

IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologics or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.

OAS1 suppresses African swine fever virus replication by recruiting TRIM21 to degrade viral major capsid protein.

IMPORTANCE: African swine fever virus (ASFV) completes the replication process by resisting host antiviral response via inhibiting interferon (IFN) secretion and interferon-stimulated genes (ISGs) function. 2', 5'-Oligoadenylate synthetase gene 1 (OAS1) has been reported to inhibit the replication of various RNA and some DNA viruses. However, the regulatory mechanisms involved in the ASFV-induced IFN-related pathway still need to be fully elucidated. Here, we found that OAS1, as a critical host factor, inhibits ASFV replication in an RNaseL-dependent manner. Furthermore, overexpression of OAS1 can promote the activation of the JAK-STAT pathway promoting innate immune responses. In addition, OAS1 plays a new function, which could interact with ASFV P72 protein to suppress ASFV infection. Mechanistically, OAS1 enhances the proteasomal degradation of P72 by promoting TRIM21-mediated ubiquitination. Meanwhile, P72 inhibits the production of avSG and affects the interaction between OAS1 and DDX6. Our findings demonstrated OAS1 as an important target against ASFV replication and revealed the mechanisms and intrinsic regulatory relationships during ASFV infection.