Understanding neurotropic enteric viruses: routes of infection and mechanisms of attenuation.

The intricate connection between the gut and the brain involves multiple routes. Several viral families begin their infection cycle in the intestinal tract. However, amongst the long list of viral intestinal pathogens, picornaviruses, and astroviruses stand out for their ability to transition from the intestinal epithelia to central or peripheral nervous system cells. In immunocompromised, neonates and young children, these viral infections can manifest as severe diseases, such as encephalitis, meningitis, and acute flaccid paralysis. What confers this remarkable plasticity and makes them efficient in infecting cells of the gut and the brain axes? Here, we review the current understanding of the virus infection along the gut-brain axis for some enteric viruses and discuss the molecular mechanisms of their attenuation.

RSAD2 suppresses viral replication by interacting with the Senecavirus A 2 C protein.

Senecavirus A (SVA), an emerging virus that causes blisters on the nose and hooves, reduces the production performance of pigs. RSAD2 is a radical S-adenosylmethionine (SAM) enzyme, and its expression can suppress various viruses due to its broad antiviral activity. However, the regulatory relationship between SVA and RSAD2 and the mechanism of action remain unclear. Here, we demonstrated that SVA infection increased RSAD2 mRNA levels, whereas RSAD2 expression negatively regulated viral replication, as evidenced by decreased viral VP1 protein expression, viral titres, and infected cell numbers. Viral proteins that interact with RSAD2 were screened, and the interaction between the 2 C protein and RSAD2 was found to be stronger than that between other proteins. Additionally, amino acids (aa) 43-70 of RSAD2 were crucial for interacting with the 2 C protein and played an important role in its anti-SVA activity. RSAD2 was induced by type I interferon (IFN-I) via Janus kinase signal transducer and activator of transcription (JAK-STAT), and had antiviral activity. Ruxolitinib, a JAK-STAT pathway inhibitor, and the knockdown of JAK1 expression substantially reduced RSAD2 expression levels and antiviral activity. Taken together, these results revealed that RSAD2 blocked SVA infection by interacting with the viral 2 C protein and provide a strategy for preventing and controlling SVA infection.

TRIM5 inhibits the replication of Senecavirus A by promoting the RIG-I-mediated type I interferon antiviral response.

Senecavirus A (SVA) is an emerging virus that poses a threat to swine herds worldwide. To date, the role of tripartite motif 5 (TRIM5) in the replication of viruses has not been evaluated. Here, TRIM5 was reported to inhibit SVA replication by promoting the type I interferon (IFN) antiviral response mediated by retinoic acid-inducible gene I (RIG-I). TRIM5 expression was significantly upregulated in SVA-infected cells, and TRIM5 overexpression inhibited viral replication and promoted IFN-α, IFN-ß, interleukin-1beta (IL-1ß), IL-6, and IL-18 expression. Conversely, interfering with the expression of TRIM5 had the opposite effect. Viral adsorption and entry assays showed that TRIM5 did not affect the adsorption of SVA but inhibited its entry. In addition, TRIM5 promoted the expression of RIG-I and RIG-I-mediated IFNs and proinflammatory cytokines, and this effect was also proven by inhibiting the expression of TRIM5. These findings expand the scope of knowledge on host factors inhibiting the replication of SVA and indicate that targeting TRIM5 may aid in the development of new agents against SVA.

Seneca Valley virus circumvents Gasdermin A-mediated inflammation by targeting the pore-formation domain for cleavage.

Members of the gasdermin (GSDM) family are critical for inducing programmable pyroptosis by forming pores on the cell membrane. GSDMB, GSDMC, GSDMD, and GSDME are activated by caspases or granzyme, leading to the release of their autoinhibitory domains. The protease SpeB from group A Streptococcus has been shown to cleave and activate GSDMA-mediated pyroptosis. Meanwhile, African Swine Fever Virus infection regulates pyroptosis by cleaving porcine GSDMA (pGSDMA) via active caspase-3 and caspase-4. However, it is not known whether virus-encoded proteases also target GSDMA. Here, we show that residues 1-252 of pGSDMA (pGSDMA1-252) is the pore-forming fragment that induces lytic cell death and pyroptosis. Interestingly, Seneca Valley Virus (SVV) infection induces the cleavage of both pGSDMA and human GSDMA and suppresses GSDMA-mediated cell death. Mechanistically, SVV protease 3C cleaves pGSDMA between Q187 and G188 to generate a shorter fragment, pGSDMA1-186, which fails to induce lytic cell death and lactate dehydrogenase release. Furthermore, pGSDMA1-186 does not localize to the plasma membrane and does not induce cell death, thereby promoting viral replication by suppressing host immune responses. These studies reveal a sophisticated evolutionary adaptation of SVV to bypass GSDMA-mediated pyroptosis, allowing it to overcome host inflammatory defenses. IMPORTANCE: Gasdermin A (GSDMA) remains a protein shrouded in mystery, particularly regarding its regulation by virus-encoded proteases. Previous studies have identified human GSDMA (hGSDMA) as a sensor and substrate of the SpeB from group A Streptococcus, which initiates pyroptosis. However, it is not clear if viral proteases also cleave GSDMA. In this study, we show that a fragment of porcine GSDMA (pGSDMA) containing the first 252 residues constitutes the pore-forming domain responsible for inducing lytic cell death and pyroptosis. Interestingly, picornavirus Seneca Valley Virus (SVV) protease 3C cleaves both pGSDMA and hGSDMA, generating a shorter fragment that fails to associate with the plasma membrane and does not induce pyroptosis. This cleavage by SVV 3C suppresses GSDMA-mediated lactate dehydrogenase release, bactericidal activity, and lytic cell death. This study reveals how SVV subverts host inflammatory defense by disrupting GSDMA-induced pyroptosis, thereby advancing our understanding of antiviral immunity and opening avenues for treating GSDMA-associated autoimmune diseases.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

ZBP1 inhibits the replication of Senecavirus A by enhancing NF-κB signaling pathway mediated antiviral response in porcine alveolar macrophage 3D4/21 cells.

BACKGROUND: Senecavirus A (SVA) caused porcine idiopathic vesicular disease (PIVD) showing worldwide spread with economic losses in swine industry. Although some progress has been made on host factors regulating the replication of SVA, the role of Z-DNA binding protein 1 (ZBP1) remains unclear. METHODS: The expression of ZBP1 in SVA-infected 3D/421 cells was analyzed by quantitative real-time PCR (qRT-PCR) and western blot. Western blot and qRT-PCR were used to detect the effects of over and interference expression of ZBP1 on SVA VP2 gene and protein. Viral growth curves were prepared to measure the viral proliferation. The effect on type I interferons (IFNs), interferon-stimulated genes (ISGs), and pro-inflammatory cytokines in SVA infection was analyzed by qRT-PCR. Western blot was used to analysis the effect of ZBP1 on NF-κB signaling pathway and inhibitor are used to confirm. RESULTS: ZBP1 is shown to inhibit the replication of SVA by enhancing NF-κB signaling pathway mediated antiviral response. SVA infection significantly up-regulated the expression of ZBP1 in 3D4/21 cells. Infection of cells with overexpression of ZBP1 showed that the replication of SVA was inhibited with the enhanced expression of IFNs (IFN-α, IFN-ß), ISGs (ISG15, PKR, and IFIT1) and pro-inflammatory cytokines (IL-6, IL-8, and TNF-α), while, infected-cells with interference expression of ZBP1 showed opposite effects. Further results showed that antiviral effect of ZBP1 is achieved by activation the NF-κB signaling pathway and specific inhibitor of NF-κB also confirmed this. CONCLUSIONS: ZBP1 is an important host antiviral factor in SVA infection and indicates that ZBP1 may be a novel target against SVA.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases.

The discovery that extracellular vesicles (EVs) serve as carriers of virus particles calls for a reevaluation of the release strategies of non-enveloped viruses. Little is currently known about the molecular mechanisms that determine the release and composition of EVs produced by virus-infected cells, as well as conservation of these mechanisms among viruses. We previously described an important role for the Leader protein of the picornavirus encephalomyocarditis virus (EMCV) in the induction of virus-carrying EV subsets with distinct molecular and physical properties. EMCV L acts as a 'viral security protein' by suppressing host antiviral stress and type-I interferon (IFN) responses. Here, we tested the ability of functionally related picornavirus proteins of Theilers murine encephalitis virus (TMEV L), Saffold virus (SAFV L), and coxsackievirus B3 (CVB3 2Apro), to rescue EV and EV-enclosed virus release when introduced in Leader-deficient EMCV. We show that all viral security proteins tested were able to promote virus packaging in EVs, but that only the expression of EMCV L and CVB3 2Apro increased overall EV production. We provide evidence that one of the main antiviral pathways counteracted by this class of picornaviral proteins, i.e. the inhibition of PKR-mediated stress responses, affected EV and EV-enclosed virus release during infection. Moreover, we show that the enhanced capacity of the viral proteins EMCV L and CVB3 2Apro to promote EV-enclosed virus release is linked to their ability to simultaneously promote the activation of the stress kinase P38 MAPK. Taken together, we demonstrate that cellular stress pathways involving the kinases PKR and P38 are modulated by the activity of non-structural viral proteins to increase the release EV-enclosed viruses during picornavirus infections. These data shed new light on the molecular regulation of EV production in response to virus infection.

Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection.

Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.

SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy.

IMPORTANCE: Senecavirus A (SVA) is an emerging picornavirus associated with vesicular disease, which wide spreads around the world. It has evolved multiple strategies to evade host immune surveillance. The mechanism and pathogenesis of the virus infection remain unclear. In this study, we show that SERPINB1, a member of the SERPINB family, promotes SVA replication, and regulates both innate immunity and the autophagy pathway. SERPINB1 catalyzes K48-linked polyubiquitination of IκB kinase epsilon (IKBKE) and degrades IKBKE through the proteasome pathway. Inhibition of IKBKE expression by SERPINB1 induces autophagy to decrease type I interferon signaling, and ultimately promotes SVA proliferation. These results provide importantly the theoretical basis of SVA replication and pathogenesis. SERPINB1 could be a potential therapeutic target for the control of viral infection.

Species-specific cleavage of cGAS by picornavirus protease 3C disrupts mitochondria DNA-mediated immune sensing.

RNA viruses cause numerous infectious diseases in humans and animals. The crosstalk between RNA viruses and the innate DNA sensing pathways attracts increasing attention. Recent studies showed that the cGAS-STING pathway plays an important role in restricting RNA viruses via mitochondria DNA (mtDNA) mediated activation. However, the mechanisms of cGAS mediated innate immune evasion by RNA viruses remain unknown. Here, we report that seneca valley virus (SVV) protease 3C disrupts mtDNA mediated innate immune sensing by cleaving porcine cGAS (pcGAS) in a species-specific manner. Mechanistically, a W/Q motif within the N-terminal domain of pcGAS is a unique cleavage site recognized by SVV 3C. Three conserved catalytic residues of SVV 3C cooperatively contribute to the cleavage of pcGAS, but not human cGAS (hcGAS) or mouse cGAS (mcGAS). Additionally, upon SVV infection and poly(dA:dT) transfection, pcGAS and SVV 3C colocalizes in the cells. Furthermore, SVV 3C disrupts pcGAS-mediated DNA binding, cGAMP synthesis and interferon induction by specifically cleaving pcGAS. This work uncovers a novel mechanism by which the viral protease cleaves the DNA sensor cGAS to evade innate immune response, suggesting a new antiviral approach against picornaviruses.

Seneca Valley Virus Enters PK-15 Cells via Caveolae-Mediated Endocytosis and Macropinocytosis Dependent on Low-pH, Dynamin, Rab5, and Rab7.

Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. IMPORTANCE Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family Picornaviridae enter host cells, and provides new insights for preventing and controlling SVV infection.

Seneca Valley Virus Induces DHX30 Cleavage to Antagonize Its Antiviral Effects.

Seneca Valley virus (SVV) is a new pathogen associated with porcine idiopathic vesicular disease (PIVD) in recent years. However, SVV-host interaction is still unclear. In this study, through LC-MS/MS analysis and coimmunoprecipitation analysis, DHX30 was identified as a 3Cpro-interacting protein. 3Cpro mediated the cleavage of DHX30 at a specific site, which depends on its protease activity. Further study showed that DHX30 was an intrinsic antiviral factor against SVV that was dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of viral infection. RIP-seq showed comparatively higher coverage depth at SVV 5'UTR, but the distribution across SVV RNA suggested that the interaction had low specificity. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. Interestingly, DHX30 was determined to interact with 3D in an SVV RNA-dependent manner. Thus, DHX30 negatively regulated SVV propagation by blocking viral RNA synthesis, presumably by participating in the viral replication complex. IMPORTANCE DHX30, an RNA helicase, is identified as a 3Cpro-interacting protein regulating Seneca Valley virus (SVV) replication dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of virus infection. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. In addition, 3Cpro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.

Isolation, Characterization, and Molecular Detection of Porcine Sapelovirus.

Porcine sapelovirus (PSV) is an important emerging pathogen associated with a wide variety of diseases in swine, including acute diarrhoea, respiratory distress, skin lesions, severe neurological disorders, and reproductive failure. Although PSV is widespread, serological assays for field-based epidemiological studies are not yet available. Here, four PSV strains were recovered from diarrheic piglets, and electron microscopy revealed virus particles with a diameter of ~32 nm. Analysis of the entire genome sequence revealed that the genomes of PSV isolates ranged 7569-7572 nucleotides in length. Phylogenetic analysis showed that the isolated viruses were classified together with strains from China. Additionally, monoclonal antibodies for the recombinant PSV-VP1 protein were developed to specifically detect PSV infection in cells, and we demonstrated that isolated PSVs could only replicate in cells of porcine origin. Using recombinant PSV-VP1 protein as the coating antigen, we developed an indirect ELISA for the first time for the detection of PSV antibodies in serum. A total of 516 swine serum samples were tested, and PSV positive rate was 79.3%. The virus isolates, monoclonal antibodies and indirect ELISA developed would be useful for further understanding the pathophysiology of PSV, developing new diagnostic assays, and investigating the epidemiology of the PSV.

Prolonged Viability of Senecavirus A in Exposed House Flies (Musca domestica).

House flies (Musca domestica) are often present in swine farms worldwide. These flies utilize animal secretions and waste as a food source. House flies may harbor and transport microbes and pathogens acting as mechanical vectors for diseases. Senecavirus A (SVA) infection in pigs occurs via oronasal route, and animals shed high virus titers to the environment. Additionally, SVA possesses increased environmental resistance. Due to these reasons, we investigated the tenacity of SVA in house flies. Five groups of flies, each composed of ten females and ten males, were exposed to SVA, titer of 109.3 tissue culture infectious dose (TCID50/mL). Groups of male and female flies were collected at 0, 6, 12, 24, and 48 h post-exposure. For comparison purposes, groups of flies were exposed to Swinepox virus (SwPV). Infectious SVA was identified in all tested groups. Successful isolation of SVA demonstrated the titers varied between 106.8 and 102.8 TCID50/mL in female groups and varied from 105.85 to 103.8 TCID50/mL in male groups. In contrast, infectious SwPV was only detected in the female group at 6 h. The significant SVA infectious titer for prolonged periods of time, up to 48 h, indicates a potential role of flies in SVA transmission.

Synergetic Contributions of Viral VP1, VP3, and 3C to Activation of the AKT-AMPK-MAPK-MTOR Signaling Pathway for Seneca Valley Virus-Induced Autophagy.

Seneca Valley virus (SVV), a member of the Picornaviridae family, can activate autophagy via the PERK and ATF6 unfolded protein response pathways and facilitate viral replication; however, the precise molecular mechanism that regulates SVV-induced autophagy remains unclear. Here, we revealed that SVV infection inhibited the phosphorylation of mechanistic target of rapamycin kinase (MTOR) and activated phosphorylation of the serine/threonine kinase AKT. We observed that activating AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), and p38 MAPK signaling by SVV infection promoted autophagy induction and viral replication; additionally, the SVV-induced autophagy was independent of the ULK1 complex. We further evaluated the role of viral protein(s) in the AKT-AMPK-MAPK-MTOR pathway during SVV-induced autophagy and found that VP1 induced autophagy, as evidenced by puncta colocalization with microtubule-associated protein 1 light chain 3 (LC3) in the cytoplasm and enhanced LC3-II levels. This might be associated with the interaction of VP1 with sequestosome 1 and promoting its degradation. In addition, the expression of VP1 enhanced AKT phosphorylation and AMPK phosphorylation, while MTOR phosphorylation was inhibited. These results indicate that VP1 induces autophagy by the AKT-AMPK-MTOR pathway. Additionally, expression of VP3 and 3C was found to activate autophagy induction via the ERK1/2 MAPK-MTOR and p38 MAPK-MTOR pathway. Taken together, our data suggest that SVV-induced autophagy has finely tuned molecular mechanisms in which VP1, VP3, and 3C contribute synergistically to the AKT-AMPK-MAPK-MTOR pathway. IMPORTANCE Autophagy is an essential cellular catabolic process to sustain normal physiological processes that are modulated by a variety of signaling pathways. Invading virus is a stimulus to induce autophagy that regulates viral replication. It has been demonstrated that Seneca Valley virus (SVV) induced autophagy via the PERK and ATF6 unfolded protein response pathways. However, the precise signaling pathway involved in autophagy is still poorly understood. In this study, our results demonstrated that viral proteins VP1, VP3, and 3C contribute synergistically to activation of the AKT-AMPK-MAPK-MTOR signaling pathway for SVV-induced autophagy. These findings reveal systemically the finely tuned molecular mechanism of SVV-induced autophagy, thereby facilitating deeper insight into the development of potential control strategies against SVV infection.

The Stem-Loop I of Senecavirus A IRES Is Essential for Cap-Independent Translation Activity and Virus Recovery.

Senecavirus A (SVA) is a picornavirus that causes vesicular disease in swine and the only member of the Senecavirus genus. Like in all members of Picornaviridae, the 5' untranslated region (5'UTR) of SVA contains an internal ribosome entry site (IRES) that initiates cap-independent translation. For example, the replacement of the IRES of foot-and-mouth disease virus (FMDV) with its relative bovine rhinitis B virus (BRBV) affects the viral translation efficiency and virulence. Structurally, the IRES from SVA resembles that of hepatitis C virus (HCV), a flavivirus. Given the roles of the IRES in cap-independent translation for picornaviruses, we sought to functionally characterize the IRES of this genus by studying chimeric viruses generated by exchanging the native SVA IRES with that of HCV either entirely or individual domains. First, the results showed that a chimeric SVA virus harboring the IRES from HCV, H-SVA, is viable and replicated normally in rodent-derived BHK-21 cells but displays replication defects in porcine-derived ST cells. In the generation of chimeric viruses in which domain-specific elements from SVA were replaced with those of HCV, we identified an essential role for the stem-loop I element for IRES activity and recombinant virus recovery. Furthermore, a series of stem-loop I mutants allowed us to functionally characterize discrete IRES regions and correlate impaired IRES activities, using reporter systems with our inability to recover recombinant viruses in two different cell types. Interestingly, mutant viruses harboring partially defective IRES were viable. However, no discernable replication differences were observed, relative to the wild-type virus, suggesting the cooperation of additional factors, such as intermolecular viral RNA interactions, act in concert in regulating IRES-dependent translation during infection. Altogether, we found that the stem-loop I of SVA is an essential element for IRES-dependent translation activity and viral replication.

The Insufficient Activation of RIG-I-Like Signaling Pathway Contributes to Highly Efficient Replication of Porcine Picornaviruses in IBRS-2 Cells.

Seneca Valley virus (SVV) or commonly known as senecavirus A, is one of the picornavirus that is associated with vesicular disease and neonatal mortality in swine herds. Our previous study found that SVV replicates extremely faster in porcine Instituto Biologico-Rim Suino-2 (IBRS-2) cells than that in porcine kidney-15 (PK-15) cells. However, the underlying mechanism remains unknown. In this study, we comprehensively compared the expression features between IBRS-2 cells and PK-15 cells in response to SVV infection by an unbiased high-throughput quantitative proteomic analysis. We found that the innate immune response-related pathways were efficiently activated in PK-15 cells but not in IBRS-2 cells during SVV infection. A large amount of interferon (IFN)-stimulated genes were induced in PK-15 cells. In contrast, no IFN-stimulated genes were induced in IBRS-2 cells. Besides, we determined similar results in the two cell lines infected by another porcine picornavirus foot-and-mouth disease virus. Further study demonstrated that the Janus kinase signal transducer and activator of transcription signaling pathway was functioning properly in both IBRS-2 and PK-15 cells. A systematic screening study revealed that the aberrant signal transduction from TANK-binding kinase 1 to IFN regulatory factor 3 in the retinoic acid-inducible gene I-like receptor signaling pathway in IBRS-2 cells was the fundamental cause of the different innate immune response manifestation and different viral replication rate in the two cell lines. Together, our findings determined the different features of IBRS-2 and PK-15 cell lines, which will help for clarification of the pathogenesis of SVV. Besides, identification of the underlying mechanisms will provide new targets and an insight for decreasing the viral clearance rate and probably improve the oncolytic effect by SVV in cancer cells.

CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination.

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9-/- and corresponding C57BL/6 wild-type control mice were infected with Theiler's murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9-/- mice showed an increased loss of neuronal nuclear protein+ mature neurons and doublecortin+ neuronal precursor cells and an increase in ß-amyloid precursor protein+ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8+ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

Cholesterol 25-hydroxylase inhibits Senecavirus A replication by enzyme activity-dependent and independent mechanisms.

Cholesterol 25-hydroxylase (CH25 H), as a host restriction factor, has been reported to take a broad-spectrum antiviral effect. However, the role of CH25H in Senecavirus A (SVA) infection remains unknown. In this study, we first demonstrate that overexpression of CH25H inhibits SVA replication. Consistently, knockdown or knockout of the endogens CH25H promotes SVA infection. Further, the anti-SVA effect of 25-hydroxycholesterol (25HC), which is the product of CH25H, operates via inhibition of viral attachment and replication. On the other hand, the CH25H mutant (CH25H-M) lacking hydroxylase activity still restricts SVA infection, which can selectively interact and degrade SVA 3A protein via the ubiquitin-proteasome manner. Altogether, these results suggest that CH25H has an antiviral function in SVA infection and provides an alternative manner to control SVA.