Jue Liu

Porcine circovirus type 2 (PCV2), a major immunosuppressive pathogen, causes substantial economic losses to the global swine industry. PCV2d is the dominant genotype in multiple regions, but its immunopathological mechanisms, particularly virus‒host immune crosstalk, remain unclear. Here, we evaluated immune responses to a rescued replication-competent PCV2d in C57BL/6 mice, focusing on lymphocyte activation, thymic T-cell apoptosis and dynamics, T-cell immunophenotype shifts, and splenic regulatory T (Treg) cell dynamics. CD69-based assessment of lymphocyte activation revealed time-specific patterns: CD8⁺ T cells were activated throughout infection, with stronger activation at 7–14 days post-infection (dpi); γδ T cells were activated only early; CD4⁺ T cells and natural killer (NK) cells were activated late (14–28 dpi); and B cells were persistently activated. Thymic analysis revealed subset-specific apoptosis; for example, CD8⁺ T cells underwent early apoptosis across all stages, immature CD4⁻CD8⁻ T cells were vulnerable late, and altered dynamics were detected: CD4⁺ and CD8⁺ T cells first increased and then decreased from early to late infection, while CD4⁺CD8⁺ T cells showed the opposite trend. T-cell immunophenotyping with CD44 and CD62L revealed reduced numbers of naive CD4⁺/CD8⁺ T cells, increased numbers of memory T cells, and upregulated the expression of PD-1, a marker of T-cell exhaustion. Splenic Treg cells (CD4⁺CD25⁺Foxp3⁺) expanded, with a peak at 21 dpi. These findings demonstrate the multifaceted immunomodulatory effects of PCV2d, including innate activation, adaptive immune suppression, thymic dysfunction, T-cell exhaustion, and Treg-mediated immunosuppression. This work improves our understanding of PCV2d pathogenesis, aiding the development of targeted control strategies and vaccines for swine PCV2.

Co-adaptation between viruses and autophagy has equipped viruses with diverse strategies to regulate host redox homeostasis, thereby facilitating viral replication. However, the mechanisms by which viruses manipulate PRDX1 (peroxiredoxin 1), a key antioxidative enzyme, via autophagy remain poorly understood. Here, we demonstrate that infection by Senecavirus A (SVA), an emerging picornavirus, induces PRDX1 degradation, and that PRDX1 negatively regulates viral replication. Decreased PRDX1 expression impairs cellular antioxidant defenses, leading to enhanced reactive oxygen species generation that facilitates SVA replication. Screening of viral proteins revealed that SVA VP1, VP2, and 3A induce PRDX1 degradation through vesicle formation-dependent macroautophagy. Notably, viral VP2 can also recruit HSPA8/HSC70 to specifically target PRDX1, directing it for degradation via LAMP2A-mediated chaperone-mediated autophagy (CMA). Collectively, these findings demonstrate that the SVA VP2 protein plays a central role in orchestrating both macroautophagy- and CMA-mediated PRDX1 degradation, establishing PRDX1 as a potential intervention target for countering SVA infection.Abbreviations: AKT/protein kinase B: AKT serine/threonine kinase; ATP: adenosine triphosphate; BHK-21: baby hamster kidney-21; CAT: catalase; CCCP: BMDMs: bone marrow-derived macrophages; CMA: chaperone-mediated autophagy; co-IP: co-immunoprecipitation; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CQ: chloroquine; DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; GPX: glutathione peroxidase; GSH: glutathione; HEK-293T: human embryonic kidney 293T; hpi: hours post-infection; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KO: knockout; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mdivi-1: mitochondrial division inhibitor-1; mM: millimole; MMP: mitochondrial membrane potential; mPTP: mitochondrial permeability transition pore; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetylcysteine; PI3K: phosphoinositide 3-kinase; PRDX1: peroxiredoxin 1; RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction; ROS: reactive oxygen species; SD: standard deviation; SOD: superoxide dismutase; SQSTM1: sequestosome 1; SVA: Senecavirus A; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; μg: microgram; μm: micrometer; μM: micromolar.

Senecavirus A (SVA), an emerging vesicular pathogen, poses a significant threat to the global pig industry. STIP1 homology and U-box-containing protein 1 (STUB1), a chaperone-dependent E3 ubiquitin ligase, plays a pivotal role in protein quality control by mediating target protein degradation. However, its precise role of STUB1 in regulating SVA replication remains undefined. In this study, we combined liquid chromatography-mass spectrometry, confocal imaging, and Western blotting to demonstrate that STUB1 interacts with the SVA VP1 protein and negatively regulates SVA replication. Mechanistically, STUB1 promotes the ubiquitination-dependent degradation of VP1 by specifically targeting lysine residues at positions 177 and 260 (K177 and K260). This degradation process is significantly enhanced by heat shock protein 70 (HSP70) and heat shock cognate protein 70 (HSC70), which strengthen the STUB1-VP1 interaction. Notably, the SVA 3C protease (3Cpro) counteracts this antiviral defense by enzymatically reducing STUB1 expression. In vivo studies using a mouse model showed that a VP1 mutant virus lacking STUB1-targeted ubiquitination sites replicates more efficiently than the wild-type strain, resulting in significantly higher viral loads across multiple tissues and more severe pulmonary pathology. Together, these findings reveal that STUB1 inhibits SVA replication through ubiquitination-dependent degradation of VP1, a process that is antagonized by viral 3C protease via suppression of STUB1 expression. IMPORTANCE: Viruses have evolved diverse strategies to enhance their replication efficiency. Senecavirus A (SVA), an emerging porcine pathogen associated with vesicular disease outbreaks, has become increasingly prevalent in swine populations worldwide. As a chaperone-dependent E3 ubiquitin ligase, STUB1 plays a crucial role in maintaining cellular protein homeostasis. In this study, we elucidated the functional interplay between STUB1 and SVA replication. Our results demonstrate that STUB1 directly interacts with the viral protein VP1 and mediates its ubiquitination-dependent degradation through specific targeting of lysine residues at positions 177 and 260 (K177 and K260), thereby significantly inhibiting viral replication. However, SVA has evolved a countermeasure, whereby its 3C protease (3Cpro) downregulates STUB1 expression, effectively blocking VP1 degradation and subverting this host antiviral defense to promote viral propagation. These findings not only reveal novel host-virus interaction mechanisms but also provide valuable molecular targets for developing innovative strategies to control SVA infection.

Senecavirus A (SVA) is an emerging pathogen responsible for vesicular lesions and neonatal mortality in swine. In the absence of effective vaccines or therapeutics, early and accurate diagnosis is essential for controlling SVA outbreaks. Although nucleic acid-based detection methods are commonly employed, there remains a pressing need for rapid, convenient, highly sensitive, and specific diagnostic tools. Here, we developed a two-pot assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a containing crRNA targeting canonical protospacer adjacent motifs (PAMs) for simple, rapid, and visual identification of SVA in clinical samples. Subsequently, we successfully streamlined this system into a one-pot assay by selecting a specially designed crRNA targeting suboptimal PAM and integrating RPA amplification reagents and CRISPR/Cas12a detection components into a single reaction system in one tube. The developed methods exhibited diagnostic specificity, showing no cross-reactivity with four major swine viruses, while showing remarkable sensitivity with a lower detection limit of just two copies. Clinical validation in field samples using these two methods revealed perfect agreement (100% concordance) with conventional quantitative PCR (qPCR) results (sample size, n = 28), with both assays completing detection within 30 min. These results demonstrate that both the one-pot and two-pot RPA-CRISPR/Cas12a assays offer a reliable and efficient method for detecting SVA in this pilot study. Despite the limited sample size, the assays combine rapid reaction time with high sensitivity and specificity, showing great potential for future diagnostic applications.

The Seneca Valley virus (SVV), a newly emerged virus within the Picornaviridae family, causes porcine idiopathic vesicular disease, imposing substantial economic costs to the global pork industry. However, the molecular mechanisms underlying its pathogenesis remain poorly understood. In this study, we identified TRIM32 as a novel antiviral factor against SVV and found that SVV infection led to the cleavage and degradation of TRIM32. Knocking down TRIM32 increased viral replication, while overexpressing it reduced viral titers. Further study showed that TRIM32 restricts SVV replication by selectively targeting viral VP3 protein for ubiquitination and proteasomal degradation. To counteract this, SVV 3C protease (3Cpro) targets TRIM32 for cleavage at E332. This cleavage renders TRIM32 unable to inhibit SVV replication or block the degradation of VP3. Additionally, the cleaved TRIM32 products weaken E3 ubiquitin ligase activity and reduce activation of the type I interferon (IFN) pathway. Taken together, our results indicate that SVV 3C ^pro -mediated cleavage of TRIM32 impairs its function in the ubiquitination and degradation of viral VP3 and type I IFN signaling. These findings offer novel insights into the strategies viruses use to evade the host’s antiviral immune responses, thereby contributing to efficient viral replication. IMPORTANCE: Seneca Valley virus (SVV) is an emerging pathogen that causes vesicular diseases in pigs, posing a significant threat to the global swine industry. Tripartite motif-containing protein (TRIM) family members are recognized as intrinsic antiviral effectors that provide a frontline shield against viruses prior to the transcription of interferon (IFN) and interferon-stimulated genes (ISGs). In this study, we uncovered the antiviral mechanism, which promotes the K48-linked ubiquitination of viral VP3 protein, leading to the degradation of VP3 via the proteasome pathway. SVV 3C ^pro abolished the antiviral effects of TRIM32 by inducing its cleavage. The cleaved TRIM32 fragment attenuates its E3 ubiquitin ligase activity and weakens the activation of IFN signaling. Our results reveal a potential mechanism of viral immune evasion, which is crucial for understanding how SVV has evolved a novel strategy to evade the intrinsic cellular restrictions against viral infection.

Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes porcine dermatitis and nephropathy syndrome-like symptoms, multisystemic inflammation, and reproductive failure. The PCV3 capsid (Cap) protein interacts with DEAD-box RNA helicase 10 (DDX10), a protein that functions primarily through regulating interferon (IFN)-β production to exert its antiviral activity. However, how the interaction between DDX10 and PCV3 Cap regulates viral replication remains unknown. We used Western blotting, interaction assays, and knockdown analyses to observe impaired PCV3 proliferation in transiently DDX10-overexpressing cells, as indicated by decreased viral protein expression levels and virus production. In contrast, PCV3 replication increased upon small interfering RNA-mediated DDX10 depletion. Furthermore, DDX10 positively regulated IFN-β production and interferon-stimulated gene expression, inhibiting PCV3 replication. Mechanistically, PCV3 Cap co-localized and interacted with DDX10, and the N-terminal nuclear localization signal of PCV3 Cap and the helicase domain of DDX10 were essential for the Cap-DDX10 interaction. Furthermore, PCV3 infection decreases DDX10 expression to antagonize its antiviral activity. These results show that DDX10 antagonizes PCV3 replication by interacting with the PCV3 Cap protein and activating IFN-β responses, which provides important insight into the prevention and control of PCV3 infection. IMPORTANCE: Clarifying how host factors contribute to infection with PCV3, a newly discovered pathogen associated with multiple clinicopathological signs in swine, helps elucidate viral pathogenesis. The PCV3 Cap protein has been shown to interact with DDX10, a crucial protein that regulates RNA virus replication. Herein, we further demonstrated that DDX10 expression is downregulated in PCV3-infected cells and antagonizes the replication of PCV3 and that DDX10 increases interferon-β and interferon-stimulated gene levels to inhibit PCV3 replication by binding to the PCV3 Cap. In addition, PCV3 infection decreases DDX10 expression to antagonize its antiviral activity. These results reveal a molecular mechanism by which DDX10 antagonizes PCV3 replication by binding to the PCV3 Cap protein and activating IFN signals, thereby providing important targets for preventing and controlling PCV3 infection.