Classical swine fever virus (CSFV), a highly virulent member of the Pestivirus genus, is one of the most significant pathogens within this group. Although uncoating is a prerequisite for productive infection, the molecular determinants orchestrating this process remain obscure. The Core protein, functioning as the viral nucleocapsid, plays a pivotal role in the uncoating cascade. This study delineates a SUMOylation-dependent, ubiquitin-independent proteolytic mechanism essential for CSFV uncoating. The valosin-containing protein (VCP/p97) preferentially associates with SUMO1-modified Core, directing it toward degradation via the 26S proteasome, specifically through engagement with PSMB2 and PSMD2 subunits. Site-directed mutagenesis of the SUMOylation motif abolishes VCP-mediated degradation, substantiating its functional indispensability. Fluorescent tracking of CSFV virions using molecular beacon and quantum dot labeling further reveals that VCP governs the endosomal trafficking of viral particles from early to late endosomes—an essential step for capsid disassembly and genome release. Moreover, VCP operates in concert with NPL4 and UFD1, enabling the translocation of SUMOylated Core toward the proteasomal machinery. Collectively, these findings uncover a previously uncharacterized SUMO1-VCP-PSMB2/PSMD2 axis that couples intracellular trafficking with proteasomal disassembly of the CSFV Core, providing mechanistic insights into Pestivirus uncoating and nominating host factors as promising antiviral targets. IMPORTANCE: The fusion of the viral membrane and genome release are hallmark events of enveloped virus infections. However, the related dynamic mechanisms of most viruses remain poorly understood. Here, we demonstrate that VCP directly interacts with the CSFV core protein, and the core protein undergoes proteasomal degradation mediated by the PSMD2 and PSMB2 subunits, with VCP acting as a critical mediator. Surprisingly, this degradation process is independent of ubiquitination but exhibits a strong correlation with the SUMOylation of the nucleocapsid protein. In addition, we found that CSFV genome uncoating occurred in late endosomes, a process regulated by the host VCP. Depletion of VCP prevents viral trafficking to late endosomes and thereby disrupts uncoating efficiency. This is the first evidence implicating SUMOylation in viral uncoating. Deciphering the molecular intricacies governing viral uncoating is pivotal for propelling the development of broad-spectrum antiviral therapeutics aimed at the Pestivirus genus.

Classical swine fever virus (CSFV) is a highly contagious and lethal pathogen that poses a major threat to the global swine industry. Despite its economic impact, no specific antiviral therapies are currently available, underscoring the urgent need to elucidate virus-host interactions for therapeutic innovation. In this study, we screened a glucose metabolism-targeted small-molecule library and identified Vps34-IN-1, a selective inhibitor of phosphatidylinositol 3-kinase class III (VPS34/PIK3C3), as a potent suppressor of CSFV replication in a dose-dependent manner. Time-of-addition experiments demonstrate that Vps34-IN-1 predominantly interferes with the late stage of the viral life cycle. Consistently, siRNA-mediated knockdown of VPS34 significantly impairs viral replication, confirming its role as a critical host dependency factor. Mechanistically, pharmacological inhibition or genetic silencing of VPS34 disrupts CSFV-induced autophagic flux. Notably, the CSFV non-structural protein p7 engages in a specific interaction with UVRAG, a pivotal constituent of the VPS34 complex II, and appears to potentiate VPS34-UVRAG complex assembly, thereby facilitating autophagosome-lysosome fusion. Collectively, these findings uncover an unappreciated role of VPS34 in sustaining CSFV replication and highlight its potential as a viable target for host-oriented antiviral intervention. IMPORTANCE: CSFV remains a major pathogen of global concern, causing severe disease in swine and incurring substantial economic losses in the pig industry. The absence of effective antiviral agents underscores the pressing need for host-targeted therapeutic strategies. In this study, we identified Vps34-IN-1, a selective inhibitor of VPS34, as a potent suppressor of CSFV replication in a dose-dependent manner. Remarkably, Vps34-IN-1 also exhibits potent inhibitory activity against other economically important swine viruses, including BVDV, PRV, and PEDV, demonstrating its potential as a broad-spectrum antiviral agent. Knockdown experiments further validated VPS34 as an essential host factor required for CSFV propagation. Mechanistically, the viral p7 protein engages in a specific interaction with UVRAG, a pivotal constituent of the VPS34 complex II, thereby potentially augmenting VPS34-UVRAG complex assembly and facilitating autophagosome-lysosome fusion. These findings delineate VPS34 as a compelling host-oriented antiviral target and open new therapeutic avenues for the control of CSF and other economically significant swine viral diseases.

Flaviviruses intricately rewire host metabolic networks to establish a replication-permissive environment; however, the role of glucose transporter-mediated uptake, particularly via glucose transporter 4 (GLUT4), remains insufficiently defined. Japanese encephalitis virus (JEV) infection induces extensive remodeling of glucose metabolism, exemplified by the coordinated upregulation of critical metabolic effectors. Pharmacological blockade of glucose metabolic pathways markedly attenuates JEV replication, whereas exogenous glucose supplementation enhances viral propagation in a concentration-dependent manner. A targeted screen of 111 metabolism-oriented compounds identified selective GLUT4 inhibitors with potent antiviral efficacy. Notably, GLUT4 expression is consistently upregulated during JEV infection across multiple cell types, albeit to varying degrees, and is similarly induced by duck Tembusu virus (DTMUV), suggesting a potentially conserved mechanism shared by these two flaviviruses. However, broader validation across additional members of the Flavivirus genus remains warranted. Mechanistically, the viral nonstructural protein 3 (NS3) engages insulin receptor substrate 1 (IRS1), thereby activating the IRS1-PI3K-Akt-mTORC1-SREBP-1c signaling axis to transcriptionally drive GLUT4 expression. Concurrently, JEV infection induces PI3K-Akt-dependent phosphorylation of AS160, promoting GLUT4 vesicular trafficking via the coordinated action of Rab8 and Rab10. Collectively, these findings delineate a previously unrecognized mechanism whereby JEV commandeers host insulin signaling to orchestrate GLUT4 biosynthesis and membrane translocation, thereby ensuring continuous metabolic substrate availability to sustain replication. This GLUT4-centric metabolic circuitry represents a mechanistically tractable target for host-directed antiviral strategies against Flavivirus .

Although commercial vaccines against porcine circovirus type 2 (PCV2) have been widely implemented globally, PCV2 remains endemic in swine populations, accompanied by ongoing genotype replacement. Meanwhile, the emergence of novel porcine circoviruses (PCVs), including PCV3 and PCV4, has further complicated the prevention and control of porcine circovirus-associated diseases (PCVAD). This study systematically characterized the epidemiological patterns and genetic diversity of PCVs circulating in Eastern China. A total of 739 clinical samples collected between 2010 and 2016 were screened for PCV2. Additionally, 653 samples obtained during 2023–2024 were analyzed using a triplex real-time quantitative PCR (qPCR) assay for the simultaneous detection of PCV2, PCV3, and PCV4. Full-genome amplification and sequencing were subsequently performed on all PCR-positive samples. Epidemiological analysis revealed an overall PCV2 positivity rate of 37.62% during 2010–2016. In the 2023–2024 cohort, the positivity rates for PCV2 and PCV3 were 35.99% and 16.39%, respectively, with a co-infection rate of 10.26%. Notably, no PCV4-positive samples were detected. Phylogenetic analysis demonstrated that PCV2d is the predominant genotype in Eastern China. Furthermore, PCV2g strains were identified in clinical samples for the first time in mainland China, while PCV3b were determined to be the dominant circulating subtype of PCV3. Multiple critical amino acid substitutions were identified within the neutralizing epitopes of the PCV2 Cap protein, and a recombination event involving a PCV2d vaccine strain and a PCV2c reference strain was detected. In contrast, the PCV3 genome exhibited a high degree of genetic conservation. Collectively, these findings expand the molecular epidemiological landscape of PCVs in Eastern China and elucidate the evolutionary dynamics of circulating PCV strains, providing important insights for the development of next-generation vaccines and region-specific PCVAD control strategies.

Porcine circovirus 3 (PCV3) is an emerging swine pathogen associated with reproductive disorders, multisystemic inflammation, and other clinical syndromes. The capsid protein (Cap), the principal structural and immunogenic component, is an ideal antigen for serological detection. However, its poor solubility in prokaryotic expression systems limits diagnostic assay development. In this study, the N-terminal 32 amino acids of Cap were truncated, and the protein was expressed in a soluble form using the SHuffle T7 Express strain. An indirect enzyme-linked immunosorbent assay (ELISA) using purified soluble Cap as a coating antigen was established and optimized. The assay exhibited excellent specificity, with no cross-reactivity against other common swine pathogens, high sensitivity, and good repeatability, with intra- and inter-assay coefficients of variation below 10%. The cut-off value was determined, and testing of 144 porcine serum samples showed a 78.47% concordance rate with PCR, indicating good diagnostic performance. This Cap-based indirect ELISA provides a reliable, cost-effective, and high-throughput tool for large-scale serological surveillance and epidemiological monitoring of PCV3, supporting improved disease control strategies in pig populations.