Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses.

Despite the discovery of animal coronaviruses related to SARS-CoV-2, the evolutionary origins of this virus are elusive. We describe a meta-transcriptomic study of 411 bat samples collected from a small geographical region in Yunnan province, China, between May 2019 and November 2020. We identified 24 full-length coronavirus genomes, including four novel SARS-CoV-2-related and three SARS-CoV-related viruses. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome, although it possessed a more divergent spike gene. The other three SARS-CoV-2-related coronaviruses carried a genetically distinct spike gene that could weakly bind to the hACE2 receptor in vitro. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species, with the largest contiguous hotspots extending from South Laos and Vietnam to southern China. Our study highlights the remarkable diversity of bat coronaviruses at the local scale, including close relatives of both SARS-CoV-2 and SARS-CoV.

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.

SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP.

The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host's innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin-proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK-STAT signaling pathway via the ubiquitin-proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK-STAT signaling pathway and the strategies of SADS-CoV in evading the host's innate immune system.

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.

Cleavage of HDAC6 to dampen its antiviral activity by nsp5 is a common strategy of swine enteric coronaviruses.

HDAC6, a structurally and functionally unique member of the histone deacetylase (HDAC) family, is an important host factor that restricts viral infection. The broad-spectrum antiviral activity of HDAC6 makes it a potent antiviral agent. Previously, we found that HDAC6 functions to antagonize porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. However, the final outcome is typically a productive infection that materializes as cells succumb to viral infection, indicating that the virus has evolved sophisticated mechanisms to combat the antiviral effect of HDAC6. Here, we demonstrate that PDCoV nonstructural protein 5 (nsp5) can cleave HDAC6 at glutamine 519 (Q519), and cleavage of HDAC6 was also detected in the context of PDCoV infection. More importantly, the anti-PDCoV activity of HDAC6 was damaged by nsp5 cleavage. Mechanistically, the cleaved HDAC6 fragments (amino acids 1-519 and 520-1159) lost the ability to degrade PDCoV nsp8 due to their impaired deacetylase activity. Furthermore, nsp5-mediated cleavage impaired the ability of HDAC6 to activate RIG-I-mediated interferon responses. We also tested three other swine enteric coronaviruses (transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome-coronavirus) and found that all these coronaviruses have adopted similar mechanisms to cleave HDAC6 in both an overexpression system and virus-infected cells, suggesting that cleavage of HDAC6 is a common strategy utilized by swine enteric coronaviruses to antagonize the host's antiviral capacity. Together, these data illustrate how swine enteric coronaviruses antagonize the antiviral function of HDAC6 to maintain their infection, providing new insights to the interaction between virus and host.IMPORTANCEViral infections and host defenses are in constant opposition. Once viruses combat or evade host restriction, productive infection is achieved. HDAC6 is a broad-spectrum antiviral protein that has been demonstrated to inhibit many viruses, including porcine deltacoronavirus (PDCoV). However, whether HDAC6 is reciprocally targeted and disabled by viruses remains unclear. In this study, we used PDCoV as a model and found that HDAC6 is targeted and cleaved by nsp5, a viral 3C-like protease. The cleaved HDAC6 loses its deacetylase activity as well as its ability to degrade viral proteins and activate interferon responses. Furthermore, this cleavage mechanism is shared among other swine enteric coronaviruses. These findings shed light on the intricate interplay between viruses and HDAC6, highlighting the strategies employed by viruses to evade host antiviral defenses.

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.

Two amino acid residues in the N-terminal region of the polymerase acidic protein determine the virulence of Eurasian avian-like H1N1 swine influenza viruses in mice.

Reassortant Eurasian avian-like H1N1 (rEA H1N1) viruses carrying the internal genes of H1N1/2009 virus have been circulating in pigs for more than 10 years and have caused sporadic human infections. The enhanced virulence phenotype of the rEA H1N1 viruses highlights potential risks to public health. However, the molecular mechanism underlying the viral pathogenicity of the currently circulating rEA H1N1 viruses remains unclear. In this study, we found that two naturally isolated rEA H1N1 swine influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), possessed similar genetic characteristics but exhibited significantly different pathogenicity in a mouse model. Using reverse genetics, we demonstrated that amino acid mutations at positions 100 and 122 in the polymerase acidic (PA) protein had individual and synergistic effects on the polymerase activity and viral replication capacity in vitro, as well as the viral pathogenicity in mice. Furthermore, we revealed that amino acid residue 100 in PA influenced the transcription of viral RNA (vRNA) by altering the endonuclease activity, and amino acid residue 122 affected the synthesis of complementary RNA and messenger RNA by altering the RNA-binding ability and endonuclease activity of the PA protein. Taken together, we identified that two naturally occurring amino acid mutations in PA derived from H1N1/2009 virus are crucial determinants of the virulence of rEA H1N1 viruses and revealed the differential mechanism by which these two mutations affect the transcription and replication of vRNA. These findings will extend our understanding of the roles of PA in the virulence of influenza A viruses.IMPORTANCEMultiple genetic determinants are involved in the virulence of influenza A viruses. In this study, we identified two naturally occurring amino acid mutations, located at residues 100 and 122 in the polymerase acidic (PA) protein, which are associated with viral polymerase activity, replication competence, and pathogenicity in mice. In particular, we clarified the specific mechanism by which the two residues play an important role in viral transcription and replication. These findings will help to improve understanding the functions of amino acid residues in the N-terminal region of the PA protein involved in the pathogenicity of influenza A viruses.

Classical swine fever virus non-structural protein 4A recruits dihydroorotate dehydrogenase to facilitate viral replication.

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.

PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

IDO1 promotes CSFV replication by mediating tryptophan metabolism to inhibit NF-κB signaling.

Tryptophan metabolism plays a crucial role in facilitating various cellular processes essential for maintaining normal cellular function. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn), thereby initiating the degradation of Trp. The resulting Kyn metabolites have been implicated in the modulation of immune responses. Currently, the role of IDO1-mediated tryptophan metabolism in the process of viral infection remains relatively unknown. In this study, we discovered that classical swine fever virus (CSFV) infection of PK-15 cells can induce the expression of IDO1, thereby promoting tryptophan metabolism. IDO1 can negatively regulate the NF-κB signaling by mediating tryptophan metabolism, thereby facilitating CSFV replication. We found that silencing the IDO1 gene enhances the expression of IFN-α, IFN-ß, and IL-6 by activating the NF-κB signaling pathway. Furthermore, our observations indicate that both silencing the IDO1 gene and administering exogenous tryptophan can inhibit CSFV replication by counteracting the cellular autophagy induced by Rapamycin. This study reveals a novel mechanism of IDO1-mediated tryptophan metabolism in CSFV infection, providing new insights and a theoretical basis for the treatment and control of CSFV.IMPORTANCEIt is well known that due to the widespread use of vaccines, the prevalence of classical swine fever (CSF) is shifting towards atypical and invisible infections. CSF can disrupt host metabolism, leading to persistent immune suppression in the host and causing significant harm when co-infected with other diseases. Changes in the host's metabolic profiles, such as increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, can also influence virus replication. Mammals utilize various pathways to modulate immune responses through amino acid utilization, including increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, thereby limiting viral replication. Therefore, this study proposes that targeting the modulation of tryptophan metabolism may represent an effective approach to control the progression of CSF.

Foot-and-mouth disease virus (FMDV) negatively regulates ZFP36 protein expression to alleviate its antiviral activity.

Zinc finger protein 36 (ZFP36) is a key regulator of inflammatory and cytokine production. However, the interplay between swine zinc-finger protein 36 (sZFP36) and foot-and-mouth disease virus (FMDV) has not yet been reported. Here, we demonstrate that overexpression of sZFP36 restricted FMDV replication, while the knockdown of sZFP36 facilitated FMDV replication. To subvert the antagonism of sZFP36, FMDV decreased sZFP36 protein expression through its non-structural protein 3C protease (3Cpro). Our results also suggested that 3Cpro-mediated sZFP36 degradation was dependent on its protease activity. Further investigation revealed that both N-terminal and C-terminal-sZFP36 could be degraded by FMDV and FMDV 3Cpro. In addition, both N-terminal and C-terminal-sZFP36 decreased FMDV replication. Moreover, sZFP36 promotes the degradation of FMDV structural proteins VP3 and VP4 via the CCCH-type zinc finger and NES domains of sZFP36. Together, our results confirm that sZFP36 is a host restriction factor that negatively regulates FMDV replication.IMPORTANCEFoot-and-mouth disease (FMD) is an infectious disease of animals caused by the pathogen foot-and-mouth disease virus (FMDV). FMD is difficult to prevent and control because there is no cross-protection between its serotypes. Thus, we designed this study to investigate virus-host interactions. We first demonstrate that swine zinc-finger protein 36 (sZFP36) impaired FMDV structural proteins VP3 and VP4 to suppress viral replication. To subvert the antagonism of sZFP36, FMDV and FMDV 3Cpro downregulate sZFP36 expression to facilitate FMDV replication. Taken together, the present study reveals a previously unrecognized antiviral mechanism for ZFP36 and elucidates the role of FMDV in counteracting host antiviral activity.

NS7a of SADS-CoV promotes viral infection via inducing apoptosis to suppress type III interferon production.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro, and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo. These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection.

Androgenic steroids induce pathologic scarring in a preclinical porcine model via dysfunctional extracellular matrix deposition.

Hypertrophic scarring is a major source of morbidity. Sex hormones are not classically considered modulators of scarring. However, based on increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical porcine model to explore these effects. Mini-swine underwent castration, received no testosterone (noT) or biweekly testosterone therapy (+T), and underwent excisional wounding. To create a delayed wound healing model, a subset of wounds were re-excised at 2 weeks. Scars from postoperative day 42 (POD42) and delayed wounds (POD28) were harvested 6 weeks after initial wounding for analysis via histology, bulk RNA-seq, and mechanical testing. Histologic analysis of scars from +T animals showed increased mean fibrosis area (16 mm2noT, 28 mm2+T; p = .007) and thickness (0.246 mm2noT, 0.406 mm2+T; p < .001) compared to noT. XX+T and XY+T scars had greater tensile burst strength (p = .024 and p = .013, respectively) compared to noT swine. Color deconvolution analysis revealed greater deposition of type I and type III collagen as well as increased collagen type I:III ratio in +T scars. Dermatopathologist histology scoring showed that +T exposure was associated with worse overall scarring (p < .05). Gene ontology analysis found that testosterone exposure was associated with upregulation of cellular metabolism and immune response gene sets, while testosterone upregulated pathways related to keratinization and laminin formation on pathway analysis. In conclusion, we developed a preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces increased scar tissue deposition and appears to increase physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increased burst strength seen in both XX and XY animals suggests that hormone administration has a strong influence on scar mechanical properties independent of chromosomal sex. Anti-androgen topical therapies may be a promising future area of research.

Passive immunotherapies for the next influenza pandemic.

Influenzavirus is among the most relevant candidates for a next pandemic. We review here the phylogeny of former influenza pandemics, and discuss candidate lineages. After briefly reviewing the other existing antiviral options, we discuss in detail the evidences supporting the efficacy of passive immunotherapies against influenzavirus, with a focus on convalescent plasma.