Zhanyong Wei

Porcine deltacoronavirus (PDCoV) infection induces severe intestinal inflammation and acute diarrhea in piglets, resulting substantial economic losses. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, a key component of the innate immune system, drives interleukin (IL)‐1β maturation, and its excessive activation is closely linked to viral pathogenesis. However, its role in PDCoV infection remains unclear. Here, we report that PDCoV infection induces IL‐1β maturation through NLRP3 inflammasome activation both in vivo and in vitro , and the M protein promotes NLRP3 inflammasome assembly and activation. Mechanistically, M protein directly bound the LRR domain of the NLRP3 protein. Seven residues (M116, H201, T205, K207, R212, Y214, and M217) in the β‐sheet core of the M protein formed hydrogen bonds with eight residues (Y861, R920, E949, E1007, Y1009, K1015, N1011, and E1033) in the α‐helix groove of the LRR domain, serving as critical determinants of binding. Moreover, the M protein protects NLRP3 from proteasomal degradation by removing tripartite motif‐containing 31‐mediated K48‐linked polyubiquitin chains on NLRP3, thereby stabilizing NLRP3 protein. Collectively, these findings reveal a novel mechanism of NLRP3 inflammasome activation during PDCoV infection, offering valuable insights into PDCoV pathogenesis that may facilitate the design of antiviral agents against PDCoV.

To establish effective infection, viral pathogens employ diverse strategies through encoded proteins to interfere with host antiviral responses. While previous studies have predominantly focused on elucidating the mechanisms by which individual viral proteins regulate type I interferon (IFN) responses, this study presents the first demonstration that Japanese encephalitis virus (JEV)-encoded NS1 and NS4B proteins cooperatively target the TLR3 receptor signaling pathway to suppress IFN production. Here, we first discovered JEV-encoded multifunctional glycoprotein NS1, which inhibits TLR3-mediated IFN-β production by targeting TLR3 and TRIF. Mechanistically, NS1 interacts with these host factors and may induce their degradation via the autophagy pathway. Building on these findings, we further demonstrated that NS1 and NS4B act synergistically to suppress type I IFN production by enhancing TLR3 and TRIF degradation, thereby facilitating JEV replication. Structural simulation of the NS1-NS4B-TLR3 complex unveiled a dynamic mechanism that NS4B binding induces conformational changes in dimerized NS1, which leads to a tighter binding posture between NS1 and TLR3. Notably, NS4B binding expands the interface between NS1 and TLR3 by 1.5-fold that results in enhanced TLR3 degradation and downstream IFN-β suppression. Functional analysis confirmed that the C291/K293/R314 triple mutation in NS1 synergistically impairs TLR3 degradation. Collectively, using JEV as a model, this study reveals a novel mechanism by which two viral components can act synergistically to evade the host antiviral response. Given the common coexistence and functional interplay of viral-encoded proteins in naturally infected cells, this study establishes a framework for investigating cooperative interactions among multiple viral proteins. IMPORTANCE: Viruses evade host immunity through encoded viral proteins, while previous research has predominantly focused on single protein mechanisms. This study reveals a novel cooperative immune evasion strategy, demonstrating for the first time that Japanese encephalitis virus NS1 and NS4B proteins act synergistically to degrade the host’s TLR3 and TRIF adaptor, thereby suppressing type I interferon production. Structural simulations show that NS4B induces conformational changes in NS1, enhancing its binding to TLR3 and accelerating its degradation. This work establishes a new paradigm for how multiple viral components can function cooperatively to subvert antiviral defenses. Given that viral proteins naturally coexist and interact, this study provides a crucial framework for investigating complex viral protein interplay, which is fundamental to understanding viral pathogenesis.

Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen causing acute diarrhea and high mortality in neonatal piglets, yet the interplay between TGEV infection and host immune responses, particularly swine leukocyte antigen class I (SLA-I) mediated antigen presentation and type I interferon (IFN) signaling, remains poorly defined. This study aimed to clarify how TGEV modulates SLA-I expression through NOD-like receptor family CARD domain containing 5 (NLRC5) and the underlying signaling pathways. Since TGEV infection was shown to induce SLA-I upregulation in porcine intestinal tissues, we used swine testicular (ST) cells to investigate the expression dynamics of retinoic acid inducible gene I (RIG-I)/IFN-β/signal transducer and activator of transcription 1 (STAT1) axis. TGEV infection significantly upregulated SLA-I and NLRC5 in ST cells, with high expression coinciding with active viral replication, while NLRC5 inhibited TGEV proliferation. Furthermore, TGEV upregulated SLA-I and NLRC5 expression via RIG-I activation, which triggered downstream IFN-β secretion and subsequent STAT1 phosphorylation, thereby facilitating the induction of SLA-I and NLRC5. Moreover, the TGEV open reading frame 7 (ORF7) protein was identified as a critical viral effector contributing to SLA-I and NLRC5 upregulation. Collectively, TGEV infection activates the RIG-I/IFN-β/STAT1 axis to promote NLRC5 mediated SLA-I upregulation, which inhibits TGEV proliferation, elucidating a novel immune regulatory mechanism that balances viral replication and host antiviral defense and providing insights for targeted management strategies against porcine enteric coronaviruses.

Red blood cells (RBCs) are essential for transporting oxygen from lungs to peripheral tissues. However, the impact of transmissible gastroenteritis virus (TGEV) infection on RBCs and its potential pathophysiological significance during disease progression remain largely unexplored. In this study, hematological analysis of TGEV-infected piglets revealed significant reduction in both RBC distribution width–coefficient of variation and RBC distribution width–standard deviation, alongside elevated pCO₂ levels. Viral detection confirmed the presence of TGEV within RBCs from infected piglets. Further investigation demonstrated that TGEV could bind to, but not replicate in, RBCs. TGEV-bound RBCs exhibited crenated and impaired deformability, which were associated with reduced oxygen-carrying capacity. Additionally, TGEV infection promoted macrophage-mediated phagocytosis of RBCs and led to decreased serum iron levels, factors that might enhance TGEV infection. Collectively, these results demonstrated the involvement of RBCs in the progression of TGEV infection, providing new insights for the development of diagnostic and therapeutic strategies.

Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.

Introduction: Coinfection with porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) is a major cause of acute diarrhea in piglets, which poses a significant challenge to the swine industry. The early detection and control of these two viruses require highly sensitive diagnostic tools. We developed a novel chip digital PCR (cdPCR) assay that uses two probes for the simultaneous quantitative detection of both PDCoV and PEDV in clinical samples.Methods: In this study, the dual cdPCR reaction system, including the annealing temperature and primer–probe concentration ratio, was systematically optimized. Additionally, we validated the developed method for specificity, sensitivity, linearity, and repeatability. Finally, the method was applied to assess the biological samples with low viral loads.Results: The dual cdPCR assay demonstrated exceptional sensitivity, with limits of detection (LoD) of 1.83 ± 0.15 copies/μL for PDCoV and 0.99 ± 0.07 copies/μL for PEDV, high specificity (no cross-reactivity with TGEV, PSV, or PRV), outstanding linearity (R² = 0.9972 for PDCoV and R² = 0.9969 for PEDV) and reproducibility (intra- and inter-assay CV < 6%). Validation across 148 clinical samples indicates that our dual cdPCR is more sensitive than qPCR for detecting both single and mixed infections. Notably, this assay can effectively quantify PDCoV and PEDV in environmental aerosol samples.Discussion: Our results demonstrate that this dual cdPCR assay offers a highly sensitive, stable, and accurate platform for the simultaneous quantification of both PDCoV and PEDV. It represents a valuable tool for early disease monitoring (particularly in aerosol surveillance and mixed-infection scenarios with low viral loads), thereby supporting the effective prevention of porcine viral diarrhea and the sustainable growth of the swine industry.