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 pH240R enhances viral replication via inhibition of the type I IFN signaling pathway.

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by ASF virus (ASFV) infection. At present, there are still no safe and effective drugs and vaccines to prevent ASF. Mining the important proteins encoded by ASFV that affect the virulence and replication of ASFV is the key to developing effective vaccines and drugs. In this study, ASFV pH240R, a capsid protein of ASFV, was found to inhibit the type I interferon (IFN) signaling pathway. Mechanistically, pH240R interacted with IFNAR1 and IFNAR2 to disrupt the interaction of IFNAR1-TYK2 and IFNAR2-JAK1. Additionally, pH240R inhibited the phosphorylation of IFNAR1, TYK2, and JAK1 induced by IFN-α, resulting in the suppression of the nuclear import of STAT1 and STAT2 and the expression of IFN-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induced more ISGs in porcine alveolar macrophages compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs expression. Taken together, our results clarify that pH240R enhances ASFV replication by inhibiting the JAK-STAT signaling pathway, which highlights the possibility of pH240R as a potential drug target.IMPORTANCEThe innate immune response is the host's first line of defense against pathogen infection, which has been reported to affect the replication and virulence of African swine fever virus (ASFV) isolates. Identification of ASFV-encoded proteins that affect the virulence and replication of ASFV is the key step in developing more effective vaccines and drugs. In this study, we found that pH240R interacted with IFNAR1 and IFNAR2 by disrupting the interaction of IFNAR1-TYK2 and IFNAR2-JAK1, resulting in the suppression of the expression of interferon (IFN)-stimulated genes (ISGs). Consistent with these results, H240R-deficient ASFV (ASFV-∆H240R) infection induces more ISGs' expression compared with its parental ASFV HLJ/18. We also found that pH240R enhanced viral replication via inhibition of ISGs' expression. Taken together, our findings showed that pH240R enhances ASFV replication by inhibiting the IFN-JAK-STAT axis, which highlights the possibility of pH240R as a potential drug target.

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.

African swine fever virus transmembrane protein pEP84R guides core assembly.

African swine fever virus (ASFV) causes a devastating hemorrhagic disease with worldwide circulation and no widely available therapeutic prevention. The infectious particle has a multilayered architecture that is articulated upon an endoplasmic reticulum (ER)-derived inner envelope. This membrane acts as docking platform for the assembly of the outer icosahedral capsid and the underlying core shell, a bridging layer required for the formation of the central genome-containing nucleoid. While the details of outer capsid assembly are relatively well understood, those of core formation remain unclear. Here we report the functional characterization of pEP84R, a transmembrane polypeptide embedded in the inner envelope that surrounds the viral core. Using an ASFV recombinant inducibly expressing the EP84R gene, we show that absence of pEP84R results in the formation of non-infectious core-less icosahedral particles displaying a significant DNA-packaging defect. Concomitantly, aberrant core shell-like structures formed by co-assembly of viral polyproteins pp220 and pp62 are mistargeted to non-ER membranes, as also occurs when these are co-expressed in the absence of other viral proteins. Interestingly, co-expression of both polyproteins with pEP84R led to the formation of ER-targeted core shell-like assemblies and co-immunoprecipitation assays showed that pEP84R binds to the N-terminal region of pp220. Altogether, these results indicate that pEP84R plays a crucial role in core assembly by targeting the core shell polyproteins to the inner viral envelope, which enables subsequent genome packaging and nucleoid formation. These findings unveil a key regulatory mechanism for ASFV morphogenesis and identify a relevant novel target for the development of therapeutic tools against this re-emerging threat.

Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

Structural Insights into the Assembly of the African Swine Fever Virus Inner Capsid.

African swine fever virus (ASFV), the cause of a highly contagious hemorrhagic and fatal disease of domestic pigs, has a complex multilayer structure. The inner capsid of ASFV located underneath the inner membrane enwraps the genome-containing nucleoid and is likely the assembly of proteolytic products from the virally encoded polyproteins pp220 and pp62. Here, we report the crystal structure of ASFV p150△NC, a major middle fragment of the pp220 proteolytic product p150. The structure of ASFV p150△NC contains mainly helices and has a triangular plate-like shape. The triangular plate is approximately 38 Šin thickness, and the edge of the triangular plate is approximately 90 Šlong. The structure of ASFV p150△NC is not homologous to any of the known viral capsid proteins. Further analysis of the cryo-electron microscopy maps of the ASFV and the homologous faustovirus inner capsids revealed that p150 or the p150-like protein of faustovirus assembles to form screwed propeller-shaped hexametric and pentametric capsomeres of the icosahedral inner capsids. Complexes of the C terminus of p150 and other proteolytic products of pp220 likely mediate interactions between the capsomeres. Together, these findings provide new insights into the assembling of ASFV inner capsid and provide a reference for understanding the assembly of the inner capsids of nucleocytoplasmic large DNA viruses (NCLDV). IMPORTANCE African swine fever virus has caused catastrophic destruction to the pork industry worldwide since it was first discovered in Kenya in 1921. The architecture of ASFV is complicated, with two protein shells and two membrane envelopes. Currently, mechanisms involved in the assembly of the ASFV inner core shell are less understood. The structural studies of the ASFV inner capsid protein p150 performed in this research enable the building of a partial model of the icosahedral ASFV inner capsid, which provides a structural basis for understanding the structure and assembly of this complex virion. Furthermore, the structure of ASFV p150△NC represents a new type of fold for viral capsid assembly, which could be a common fold for the inner capsid assembly of nucleocytoplasmic large DNA viruses (NCLDV) and would facilitate the development of vaccine and antivirus drugs against these complex viruses.

African Swine Fever Virus Gene B117L Encodes a Small Protein Endowed with Low-pH-Dependent Membrane Permeabilizing Activity.

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine in Central Europe to East Asia, resulting in economic losses for the swine industry. The virus contains a large double-stranded DNA genome that contains more than 150 genes, most with no experimentally characterized function. In this study, we evaluate the potential function of the product of ASFV gene B117L, a 115-amino-acid integral membrane protein transcribed at late times during the virus replication cycle and showing no homology to any previously published protein. Hydrophobicity distribution along B117L confirmed the presence of a single transmembrane helix, which, in combination with flanking amphipathic sequences, composes a potential membrane-associated C-terminal domain of ca. 50 amino acids. Ectopic transient cell expression of the B117L gene as a green fluorescent protein (GFP) fusion protein revealed the colocalization with markers of the endoplasmic reticulum (ER). Intracellular localization of various B117L constructs also displayed a pattern for the formation of organized smooth ER (OSER) structures compatible with the presence of a single transmembrane helix with a cytoplasmic carboxy terminus. Using partially overlapping peptides, we further demonstrated that the B117L transmembrane helix has the capacity to establish spores and ion channels in membranes at low pH. Furthermore, our evolutionary analysis showed the high conservation of the transmembrane domain during the evolution of the B117L gene, indicating that the integrity of this domain is preserved by the action of the purifying selection. Collectively our data support a viroporin-like assistant role for the B117L gene-encoded product in ASFV entry. IMPORTANCE ASFV is responsible for an extensively distributed pandemic causing important economic losses in the pork industry in Eurasia. The development of countermeasures is partially limited by the insufficient knowledge regarding the function of the majority of the more than 150 genes present on the virus genome. Here, we provide data regarding the functional experimental evaluation of a previously uncharacterized ASFV gene, B117L. Our data suggest that the B117L gene encodes a small membrane protein that assists in the permeabilization of the ER-derived envelope during ASFV infection.

New insights into the role of endosomal proteins for African swine fever virus infection.

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.

Aloperine Inhibits ASFV via Regulating PRLR/JAK2 Signaling Pathway In Vitro.

African swine fever (ASF) has become a global pandemic due to inadequate prevention and control measures, posing a significant threat to the swine industry. Despite the approval of a single vaccine in Vietnam, no antiviral drugs against the ASF virus (ASFV) are currently available. Aloperine (ALO), a quinolizidine alkaloid extracted from the seeds and leaves of bitter beans, exhibits various biological functions, including anti-inflammatory, anti-cancer, and antiviral activities. In this study, we found that ALO could inhibit ASFV replication in MA-104, PK-15, 3D4/21, and WSL cells in a dose-dependent manner without cytotoxicity at 100 µM. Furthermore, it was verified that ALO acted on the co- and post-infection stages of ASFV by time-of-addition assay, and inhibited viral internalization rather than directly inactivating the virus. Notably, RT-qPCR analysis indicated that ALO did not exert anti-inflammatory activity during ASFV infection. Additionally, gene ontology (GO) and KEGG pathway enrichment analyses of transcriptomic data revealed that ALO could inhibit ASFV replication via the PRLR/JAK2 signaling pathway. Together, these findings suggest that ALO effectively inhibits ASFV replication in vitro and provides a potential new target for developing anti-ASFV drugs.

African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D.

African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N1-107). Interestingly, unlike the effect of GSDMD-N1-279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N1-107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.

The H240R Protein of African Swine Fever Virus Inhibits Interleukin 1ß Production by Inhibiting NEMO Expression and NLRP3 Oligomerization.

The H240R protein (pH240R), encoded by the H240R gene of African swine fever virus (ASFV), is a 241-amino-acid capsid protein. We previously showed that the deletion of H240R from the ASFV genome, creating ASFV-ΔH240R, resulted in an approximately 2-log decrease in infectious virus production compared with the wild-type ASFV strain (ASFV-WT), and ASFV-ΔH240R induced higher interleukin 1ß (IL-1ß) production in porcine alveolar macrophages (PAMs) than did ASFV-WT, but the underlying mechanism remains to be elucidated. Here, we demonstrate that the activation of the NF-κB signaling and NLRP3 inflammasome was markedly induced in PAMs upon ASFV-ΔH240R infection compared with ASFV-WT. Moreover, pH240R inhibited NF-κB activation by interacting with NEMO and promoting the autophagy-mediated lysosomal degradation of NEMO, resulting in reduced pro-IL-1ß transcription. Strikingly, NLRP3 deficiency in PAMs inhibited the ASFV-ΔH240R-induced IL-1ß secretion and caspase 1 activation, indicating an essential role of NLRP3 inflammasome activation during ASFV-ΔH240R replication. Mechanistically, pH240R interacted with NLRP3 to inhibit its oligomerization, leading to decreased IL-1ß production. Furthermore, the inhibition of the NF-κB signaling and NLRP3 inflammasome activation promoted ASFV-ΔH240R replication in PAMs. Taken together, the results of this study reveal an antagonistic mechanism by which pH240R suppresses the host immune response by manipulating activation of the NF-κB signaling and NLRP3 inflammasome, which might guide the rational design of live attenuated vaccines or therapeutic strategies against ASF in the future. IMPORTANCE African swine fever (ASF), a lethal hemorrhagic disease, is caused by African swine fever virus (ASFV). There are no commercially available vaccines or antivirals for the disease. Here, we showed that ASFV with a deletion of the H240R gene exhibits high-level expression of interleukin 1ß (IL-1ß), a proinflammatory cytokine, in porcine alveolar macrophages and that the H240R protein (pH240R) exhibits robust inhibitory effects on IL-1ß transcription and production. More specifically, pH240R inhibited NF-κB activation via the autophagy-mediated lysosomal degradation of NEMO, leading to the decrease of pro-IL-1ß transcription. In addition, pH240R interacted with NLRP3 to inhibit its oligomerization, leading to decreased IL-1ß production. Our results indicate that pH240R is involved in the evasion of host innate immunity and provide a novel target for the development of a live attenuated vaccine against ASF.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

African swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2',3'-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2',3'-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2',3'-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2',3'-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2',3'-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2',3'-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2',3'-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2',3'-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2',3'-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

An overview of the role of Niemann-pick C1 (NPC1) in viral infections and inhibition of viral infections through NPC1 inhibitor.

Viruses communicate with their hosts through interactions with proteins, lipids, and carbohydrate moieties on the plasma membrane (PM), often resulting in viral absorption via receptor-mediated endocytosis. Many viruses cannot multiply unless the host's cholesterol level remains steady. The large endo/lysosomal membrane protein (MP) Niemann-Pick C1 (NPC1), which is involved in cellular cholesterol transport, is a crucial intracellular receptor for viral infection. NPC1 is a ubiquitous housekeeping protein essential for the controlled cholesterol efflux from lysosomes. Its human absence results in Niemann-Pick type C disease, a deadly lysosomal storage disorder. NPC1 is a crucial viral receptor and an essential host component for filovirus entrance, infection, and pathogenesis. For filovirus entrance, NPC1's cellular function is unnecessary. Furthermore, blocking NPC1 limits the entry and replication of the African swine fever virus by disrupting cholesterol homeostasis. Cell entrance of quasi-enveloped variants of hepatitis A virus and hepatitis E virus has also been linked to NPC1. By controlling cholesterol levels, NPC1 is also necessary for the effective release of reovirus cores into the cytoplasm. Drugs that limit NPC1's activity are effective against several viruses, including SARS-CoV and Type I Feline Coronavirus (F-CoV). These findings reveal NPC1 as a potential therapeutic target for treating viral illnesses and demonstrate its significance for several viral infections. This article provides a synopsis of NPC1's function in viral infections and a review of NPC1 inhibitors that may be used to counteract viral infections. Video Abstract.

Differential infection behavior of African swine fever virus (ASFV) genotype I and II in the upper respiratory tract.

African swine fever virus (ASFV) is a substantial threat to pig populations worldwide, contributing to economic disruption and food security challenges. Its spread is attributed to the oronasal transmission route, particularly in animals with acute ASF. Our study addresses the understudied role of nasal mucosa in ASFV infection, using a nasal explant model. The explants remained viable and revealed a discernible ASFV infection in nasal septum and turbinates post-inoculation. Interestingly, more infected cells were found in the turbinates despite its thinner structure. Further analyses showed (i) a higher replication of genotype II strain BEL18 than genotype I strain E70 in the epithelial cell layer, (ii) a preference of ASFV infection for the lamina propria and a tropism of ASFV for various susceptible cell types in different areas in the nasal mucosa, including epithelial cells, macrophages, and endothelial cells. Using porcine respiratory epithelial cells (PoRECs), isolated from nasal tissue, we found a difference in infection mechanism between the two genotypes, with genotype I favoring the basolateral surface and genotype II preferring the apical surface. Moreover, disruption of intercellular junctions enhanced infection for genotype I. This study demonstrated that ASFV may use the respiratory mucosa for entry using different cell types for replication with a genotype difference in their infection of respiratory epithelial cells.

African Swine Fever Virus MGF-505-7R Negatively Regulates cGAS-STING-Mediated Signaling Pathway.

African swine fever virus (ASFV) is a devastating infectious disease in pigs, severely threatening the global pig industry. To efficiently infect animals, ASFV must evade or inhibit fundamental elements of the innate immune system, namely the type I IFN response. In this study, we identified that ASFV MGF-505-7R protein exerts a negative regulatory effect on STING-dependent antiviral responses. MGF-505-7R interacted with STING and inhibited the cGAS-STING signaling pathway at STING level. MGF-505-7R overexpression either degraded STING or STING expression was reduced in ASFV-infected cells via autophagy, whereas STING expression was elevated in MGF-505-7R-deficient ASFV-infected cells. We further found that MGF-505-7R promoted the expression of the autophagy-related protein ULK1 to degrade STING, whereas ULK1 was elevated in MGF-505-7R-deficient ASFV-infected cells. Moreover, MGF-505-7R-deficient ASFV induced more IFN-ß production than wild-type ASFV and was attenuated in replication compared with wild-type ASFV. The replicative ability of MGF-505-7R-deficient ASFV was also attenuated compared with wild-type. Importantly, MGF-505-7R-deficient ASFV was fully attenuated in pigs. Our results showed for the first time, to our knowledge, a relationship involving the cGAS-STING pathway and ASFV MGF-505-7R, contributing to uncover the molecular mechanisms of ASFV virulence and to the rational development of ASFV vaccines.

Entropy-driven dynamic self-assembled DNA dendrimers for colorimetric detection of African swine fever virus.

Herein, we subtly engineered an amplified colorimetric biosensor for the cyclic detection of African swine fever virus DNA (ASFV-DNA), which associated the branched catalytic hairpin assembly (bCHA) amplification with G-quadruplex DNAzyme activity through triplex DNA formation. Firstly, a Y-shaped hairpin trimer was constructed for the dynamic self-assembly of DNA dendrimers. Then, in the presence of ASFV-DNA, the signal strand CP was opened, exposing the toehold regions, which would trigger the CHA cascade reaction between hairpin trimers. In the CHA cascade reaction, H1, H2, and H3 opened and bound in sequence, eventually forming the structure of DNA dendrimers. Subsequently, the obtained bCHA product was specifically recognized by the GGG repeat sequences of L1 and L2, then amplified by the synergistic effect of triplex DNA and the formation of asymmetric split G-quadruplex. Benefiting from the amplification properties of bCHA and the high peroxidase-like catalytic activity of asymmetrically split G-quadruplex DNAzymes, it could achieve effective colorimetric signal output in the presence of ASFV-DNA by means of triplex DNA formation. Under the optimal experimental conditions, this biosensor exhibited excellent sensitivity with a detection limit of 1.8 pM. Further, it was applied to the content detection of simulated samples of African swine fever, and the recoveries were 98.9 ~ 103.2%. This method has the advantages of simple operation, good selectivity, and high sensitivity, which is expected to be used for highly sensitive detection of actual samples of African swine fever virus.

African swine fever virus protein MGF-505-7R promotes virulence and pathogenesis by inhibiting JAK1- and JAK2-mediated signaling.

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.

African swine fever virus MGF360-11L negatively regulates cGAS-STING-mediated inhibition of type I interferon production.

The type I interferon (IFN-I) signaling pathway is an important part of the innate immune response and plays a vital role in controlling and eliminating pathogens. African swine fever virus (ASFV) encodes various proteins to evade the host's natural immunity. However, the molecular mechanism by which the ASFV-encoded proteins inhibit interferon production remains poorly understood. In the present study, ASFV MGF360-11L inhibited cGAS, STING, TBK1, IKKε, IRF7 and IRF3-5D mediated activation of the IFN-ß and ISRE promoters, accompanied by decreases in IFN-ß, ISG15 and ISG56 mRNA expression. ASFV MGF360-11L interacted with TBK1 and IRF7, degrading TBK1 and IRF7 through the cysteine, ubiquitin-proteasome and autophagy pathways. Moreover, ASFV MGF360-11L also inhibited the phosphorylation of TBK1 and IRF3 stimulated by cGAS-STING overexpression. Truncation mutation analysis revealed that aa 167-353 of ASFV MGF360-11L could inhibit cGAS-STING-mediated activation of the IFN-ß and ISRE promoters. Finally, the results indicated that ASFV MGF360-11L plays a significant role in inhibiting IL-1ß, IL-6 and IFN-ß production in PAM cells (PAMs) infected with ASFV. In short, these results demonstrated that ASFV MGF360-11L was involved in regulating IFN-I expression by negatively regulating the cGAS signaling pathway. In summary, this study preliminarily clarified the molecular mechanism by which the ASFV MGF360-11L protein antagonizes IFN-I-mediated antiviral activity, which will help to provide new strategies for the treatment and prevention of ASF.

One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10-6 ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10-5 ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. KEY POINTS: • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.