The global emergence of multiple viral zoonoses underscores the substantial threats of viral infections to human health. Given the dynamic and complex mechanisms underlying viral pathogenesis, sophisticated approaches are requisite to advance viral research. Here, we present a systematic review of single-cell RNA sequencing (scRNA-seq), a high-throughput technology enabling transcriptomic profiling at the individual cell level, focusing on its pivotal role in elucidating heterogeneous host cellular responses to viral infection and deciphering underlying pathogenic mechanisms. We summarize scRNA-seq’s developmental milestones, compare characteristics of various platforms, and outline its key applications in viral infection research: identifying infection-induced novel cell types/subpopulations, characterizing virus-specific host cell gene expression changes, defining viral target cells, elucidating antiviral immune mechanisms, and clarifying in vivo viral distribution and pathogenesis. By synthesizing these information, this review offers novel research and technical perspectives for dissecting the dynamic and complex virus-host interactions, aiding future advancements in viral infection research.

The widespread dissemination of New Delhi metallo-β-lactamase-1 (NDM-1) has severely compromised the clinical efficacy of carbapenem antibiotics, highlighting the need for strategies to restore meropenem activity. SKQ1 was identified through surface plasmon resonance (SPR)-based screening. Broth microdilution checkerboard assays demonstrated strong synergy between SKQ1 and meropenem (FICI = 0.25-0.5). Enzyme kinetic analyses revealed that SKQ1 acts as a noncompetitive inhibitor of NDM-1 (IC50 = 34.99 ± 3.13 μg/mL). Molecular docking, molecular dynamics simulations, microscale thermophoresis (MST) and thermal stability assays collectively supported the direct binding of SKQ1 to NDM-1, resulting in the inhibition of its hydrolytic activity. Further analyses showed that SKQ1 affected bacterial envelope physiology, including membrane integrity, membrane potential, ATP distribution and oxidative stress, suggesting additional envelope-associated effects beyond NDM-1 inhibition. Both in vitro and in vivo models confirmed the efficacy of the SKQ1-meropenem combination. SKQ1 enhanced the antibiofilm activity and therapeutic efficacy of meropenem while attenuating lipopolysaccharide (LPS)-induced inflammatory responses. Together, these findings support SKQ1 as a repurposed meropenem potentiator that acts through NDM-1 inhibition and envelope-associated effects, offering a potential strategy for combating NDM-mediated carbapenem resistance.

Background Pseudorabies virus (PRV), an alphaherpesvirus endemic in swine, has recently emerged as a causative agent of fatal human encephalitis, yet its neuropathogenic mechanisms remain largely unexplored. Tunneling nanotubes (TNTs) are actin-rich intercellular membrane extensions that have been found to mediate intercellular communication and facilitate the cell-to-cell transmisssion of viruses. Methods Immunofluorescence analysis, confocal imaging, time-lapse photography, in situ sectioning transmission electron microscopy, and scanning electron microscopy were employed to characterize the structure and function of PRV-induced TNTs in SK-N-SH cells widely utilized in neuroscience research. A cell compartment co-culture model was further constructed in this study, and the morphological differences in TNTs, as well as the corresponding viral transmission efficiency under immune pressure, were statistically analyzed and compared between different PRV strains. Additionally, we investigated the role of viral US3 in TNTs formation via proteins overexpression, gene-deleted virus construction and kinase-inactivated US3 protein expression. Results We demonstrate that PRV infection induces the extensive formation of TNTs in SK-N-SH cells. These structures act as protected conduits that facilitate efficient cell-to-cell viral transmission, enabling the virus to evade neutralizing antibodies. Critically, a human-isolate variant strain (hSD-1/2019) induced the formation of significantly more, longer and thicker TNTs than a classical PRV strain (Ea). In addition, hSD-1/2019 also exhibited more efficient viral transmission under immune pressure, providing a novel mechanism to explain the enhanced virulence of hSD-1/2019. Further investigation identified that the viral protein US3 and its kinase activity are essential for TNTs formation. However, TNTs induced by viral infection were morphologically distinct from those induced by US3 overexpression alone, suggesting that additional viral factors are required to regulate TNTs maturation and morphology. Conclusions Our findings provide novel insights into PRV neuropathogenesis from the perspective of viral transport dynamics, and identify virus-induced intercellular conduits as a potential therapeutic target against infections of PRV and similar neuroinvasive viruses.

Streptococcus suis serotype 2 (SS2), a significant zoonotic pathogen, initiates systemic infection by breaching the respiratory epithelial barrier. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, is increasingly implicated in the pathogenesis of various infectious diseases, yet its role in SS2-induced epithelial barrier dysfunction remains unknown. Here, we demonstrate SS2 infection sensitizes airway epithelial cells to ferroptosis, leading to the accumulation of lipid peroxides, upregulation of the transcriptional repressor Snail1, and subsequent downregulation of intercellular junction proteins. This cascade compromises epithelial integrity and promotes bacterial translocation. Mechanistically, we found SS2 overwhelms the cellular redox defense system and identified bacterial eukaryotic-like serine/threonine kinase 1 (Stk1) as the key mediator of this process. Stk1 directly interacts with host protein Keap1, which stabilizes the Keap1-Nrf2 complex. This stabilization enhances the ubiquitination and subsequent proteasomal degradation of Nrf2, the master regulator of antioxidant response, thereby crippling cell’s ability to neutralize lipid peroxides. In summary, this study unveils a novel virulence mechanism wherein SS2 effector Stk1 promotes Nrf2 degradation to trigger ferroptosis, ultimately leading to the disruption of respiratory epithelial barrier. These findings suggest that inhibiting ferroptosis could represent a promising therapeutic strategy for clinical prevention and treatment of SS2 infections.

Effective brucellosis control necessitates rapid serological screening assays suitable for on-site deployment. B. neotomae, a smooth Brucella species handled under lower-containment conditions, is a potential alternative source of lipopolysaccharide (LPS) antigen. This study aimed to develop and evaluate a competitive colloidal gold immunochromatographic strip based on B. neotomae LPS for detecting antibodies against B. melitensis and B. abortus in cattle and goats. A screened LPS-reactive monoclonal antibody, mAb 1B4, was incorporated into a dual-label format with an independent control line. The strip detected Brucella Positive National Standard Antiserum at 5.0 IU/mL, whereas the detection limit of the Rose Bengal test (RBT) exceeded 20 IU/mL. No cross-reactivity was observed with the limited panel of sera positive for Escherichia coli O157, Salmonella Dublin, Yersinia enterocolitica O:9, or rough Brucella. The strip also showed good repeatability and stability. Using commercial competitive ELISA (cELISA) as the confirmatory method, overall agreement was 97.37% ((kappa = 0.94) for goat sera and 98.75% ((kappa = 0.98) for cattle sera, both higher than the corresponding values for RBT. These findings indicate that the B. neotomae LPS-based strip has the potential to serve as a rapid preliminary screening assay for detecting antibodies against B. melitensis and B. abortus in cattle and goats. Further validation using larger, geographically diverse serum panels with well-defined infection and vaccination status is warranted before broader field application.

Abstract Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

The complex epidemic profile of swine infectious diseases hinders effective disease control, as separate multiple-vaccine inoculation causes excessive body stress, leading to insufficient immune responses, vaccine failure, reduced production performance and increased breeding costs. Based on our pre-established FMDV/SVA nanoparticle vaccine platform, this study screened and optimized nanoparticle antigens FMDV T3D-LS-LOOP and SVA VP1(B)-β-VP2, developed a bivalent FMDV/SVA nanoparticle vaccine via Escherichia coli prokaryotic expression system, and systematically evaluated its immune protective efficacy and biosafety in pigs. Results showed that this bivalent vaccine induced high-level neutralizing antibodies and specific IgG responses equivalent to monovalent inactivated vaccines, with strong cross-neutralizing activity against type O FMDV Mya98/Cathay lineages. It triggered Th2-type cellular immune responses against both pathogens, building a dual immune barrier via humoral and cellular immunity synergy. The vaccine showed excellent safety with no notable adverse reactions; challenge tests confirmed it completely inhibited viral replication, dissemination and shedding, reduced tissue viral loads, blocked horizontal transmission, eliminated vesicular lesions, and provided full immune protection against both viruses. In conclusion, the prepared FMDV/SVA divalent nanoparticle vaccine elicits potent specific humoral and cellular immunity against both viruses and achieves complete immune protection. As a promising candidate for FMDV and SVA coinfection control, it offers novel strategies and technical support for integrated multi-pathogen disease prevention in pig herds.

Seneca Valley virus (SVV), a member of the Picornaviridae family，which is closely associated with porcine idiopathic vesicular disease (PIVD), spread quickly and has posed a potential threat to the swine industry in several countries. Currently, SVA demonstrate persistent genetic evolution, yet no licensed vaccines or effective therapeutics are commercially available for disease containment，which underscores the significance of strategies for preventing and controlling SVA infection. A Venezuelan equine encephalitis virus (VEEV) replicon system expressing Vesicular stomatitis virus glycoprotein (VSVG) was constructed in the preliminary research. In this study, we developed a recombinant virus-like vesicles (rVLVs) vaccine expressed SVA VP1 and VP2 protein based on the VEEV-VSVG system and evaluated the characterization and stability. Subsequently, immunization with rVLVs vaccine elicited robust humoral and cellular immune responses in both mice and swine. In addition, the swine challenge experiment manifested that immunization with rVLVs-SVA-VP2 conferred complete protection, comparable to the inactivated vaccine, whereas rVLVs-SVA-VP1 vaccination demonstrated a 60% protective efficacy.

Abstract Background Glaesserella parasuis ( G. parasuis ) is a prevalent opportunistic pathogen of the porcine upper respiratory tract, causing substantial economic losses to the global swine industry. Current commercial vaccines exhibit suboptimal heterologous cross-protection and inherent biosafety concerns, highlighting the need for a safe, broadly effective multi-serotype vaccine. Results This study targeted outer membrane proteins (OMPs) with robust cross-serotype immunogenicity from virulent G. parasuis strains. Comparative proteomic analysis of five strains identified highly abundant, co-expressed OMPs, which were screened based on antigenicity and physicochemical properties. Epitope prediction for helper T lymphocytes (HTL), cytotoxic T lymphocytes (CTL), and B-cells was performed on selected OMPs. Immunodominant epitopes were concatenated using flexible linkers and fused with the TLR2 agonist phenol-soluble modulin α4 (PSMα4) to engineer a multi-epitope vaccine, designated ZZ1. Screening yielded eight conserved OMPs with high antigenicity, hydrophilicity, and thermostability, from which 6 HTL, 7 CTL, and 11 B-cell epitopes were predicted. Immunoinformatic evaluations revealed that ZZ1 possesses a high antigenicity score of 1.018 (threshold: 0.4) and is non-allergenic and non-toxic. Following successful expression, in vivo trials demonstrated that recombinant ZZ1 elicited robust, specific IgG antibody responses. Challenge tests in piglets revealed 100% protective efficacy against G. parasuis serotype 4, and 80% against serotypes 5 and 13. Conclusions These findings indicate that ZZ1 is a promising candidate capable of conferring broad cross-protection against multiple prevalent G. parasuis serotypes, offering an innovative strategy for next-generation subunit vaccine development.

Ferroptosis is a distinct modality of programmed cell death driven by iron-dependent lipid peroxidation, exerting a pivotal regulatory influence on the infection dynamics and immune responses associated with zoonoses. This article systematically elucidates the mechanistic interplay between significant zoonotic pathogens and the core regulatory networks of host ferroptosis, encompassing iron metabolism, the antioxidant defense systems typified by GPX4, and lipid peroxidation. Furthermore, it synthesizes current advancements and challenges regarding small molecules, natural products, and bioactive components of traditional Chinese medicine that target key ferroptotic checkpoints for anti-infective therapy. This review aims to establish a theoretical foundation for the development of novel, precision prevention and control strategies for zoonoses, grounded in ferroptosis modulation within the “One Health” perspective.

Streptococcus suis is an important zoonotic pathogen that is widely distributed in the global swine industry. Our previous studies demonstrated that the bacterial elongation factor IF-2 protein provides protection against S. suis infection in mice. On this basis, the T-cell and B-cell epitopes of IF-2 were predicted and concatenated to generate the IF2-1 and IF2-2 polypeptides. We successfully constructed IF2-1-T4 and IF2-2-T4 nanoparticles on the basis of the IF-2 protein via the T4 in vivo assembly platform. Both IF2-1-T4 and IF2-2-T4 nanoparticles exhibited superior immunoprotective efficacy compared with IF-2 in mouse models, regardless of the presence of adjuvants. Among them, IF2-2-T4 showed the strongest protection and demonstrated the potential to induce cross-protection against multiple S. suis serotypes, providing a promising strategy for the development of broad-spectrum S. suis vaccines. Furthermore, this study systematically evaluated the immunoprotective efficacy of a recombinant T4 phage-based vaccine in a swine model and compared the effects of intramuscular and needle-free immunization routes. Collectively, these results establish a foundation for the application of T4 nanoparticle-based vaccines in swine and offer new insights into the design of cross-protective vaccines against S. suis and other bacterial pathogens.

ABSTRACT African swine fever (ASF) is a highly contagious and fatal hemorrhagic disease of swine. While vaccines have been licensed in Vietnam and the Philippines, their global application remains constrained by insufficient large-scale validation of safety and efficacy, rendering rapid and reliable diagnostic assays crucial for effective prevention and control. In this study, a multi-epitope antigen (MEA) was rationally designed by utilizing informatics to predict and select 15 conserved B-cell epitopes from four major African swine fever virus (ASFV) structural proteins: p30, p54, p72, and pA104R. The recombinant MEA was expressed in Escherichia coli, and its immunogenicity was validated by Western blot and indirect immunofluorescence assays. This antigen was subsequently utilized as the basis for an indirect enzyme-linked immunosorbent assay (iELISA) whose reaction conditions were systematically optimized. The optimal cutoff value for the iELISA was determined to be 0.315 from the analysis of 100 known ASFV-negative serum samples. The assay demonstrated excellent reproducibility, with intra- and inter-assay coefficients of variation below 10%, and exhibited high specificity, with no cross-reactivity against sera positive for other common swine viruses. Notably, the developed assay exhibited high sensitivity, capable of detecting ASFV antibodies in serum samples at dilutions up to 1:6,400. Furthermore, it demonstrated superior sensitivity compared to two commercial ELISA kits in validating clinical samples from regions with circulating Genotype II strains. These findings highlight the successful development of a sensitive and specific iELISA for ASFV antibody detection, representing a valuable new tool for large-scale epidemiological surveillance and the implementation of robust control strategies for ASF.IMPORTANCEAfrican swine fever (ASF) is a devastating viral disease of swine causing substantial economic losses globally. Although vaccines have recently been licensed in countries such as Vietnam and the Philippines, the global availability of a universally standardized and safe vaccine remains a significant challenge. Control efforts continue to rely heavily on rapid and accurate diagnosis. A significant limitation of current serological assays is their variable sensitivity, which can lead to missed detections and compromise surveillance programs. This study addresses this deficiency through a rational, epitope-based engineering approach. We designed a single, chimeric antigen by genetically fusing 15 conserved, immunodominant B-cell epitopes from four major African swine fever virus proteins. The resulting indirect enzyme-linked immunosorbent assay demonstrated high specificity, excellent reproducibility, and, critically, superior diagnostic sensitivity compared to two commercial kits. This work reports the development of a significantly improved serodiagnostic tool, offering enhanced reliability for large-scale epidemiological surveillance and supporting more robust control strategies against ASF.

Classical swine fever virus (CSFV) and pseudorabies virus (PRV) remain significant threats to the swine industry. Although recombinant PRV vectors represent promising platforms for bivalent vaccine development, their application is frequently limited by insufficient expression of heterologous antigens, which may compromise protective efficacy. To address this limitation, we employed a multi-copy expression strategy to enhance CSFV E2 protein levels in a PRV-based vector. Using CRISPR/Cas9-mediated gene editing, we constructed recombinant PRVs expressing one, two, or three copies of the E2 gene by sequential insertion into the gE/gI, tk, and gG loci. In vitro validation demonstrated increased detectable E2 expression in the multi-copy recombinant viruses. Meanwhile, the recombinant viruses maintained virion morphology and replication kinetics comparable to those of the parental strain PRV-GX. Immunogenicity studies in rabbits showed that PRV-3CE2 elicited stronger E2-specific humoral responses and E2-associated cytokine recall responses than PRV-2CE2, while both recombinant viruses induced detectable neutralizing activity. In challenge experiments, although vaccination did not completely prevent febrile responses following CSFV challenge, both PRV-2CE2 and PRV-3CE2 reduced CSFV RNA loads in blood compared with the mock group, suggesting partial protective efficacy. Additionally, both PRV-2CE2 and PRV-3CE2 provided complete protection against lethal PRV challenge in rabbits. These findings suggest that multi-copy E2 expression enhances E2-associated immunogenicity and supports further optimization and evaluation of PRV-3CE2 as a PRV-CSFV bivalent vaccine candidate.

The rapid assembly of alpha-herpesvirus imposes a substantial metabolic burden on the host secretory pathway, requiring efficient ER-to-Golgi transport of viral glycoproteins. However, the mechanisms by which viruses remodel ER exit sites (ERES) to accommodate this surge in cargo remain poorly understood. Here, we report that Pseudorabies virus (PRV) exploits the host autophagy regulator TECPR2 (Tectonin beta-propeller repeat containing 2) to orchestrate COPII (Coat protein complex II) component accumulation and facilitate viral egress. We demonstrate that the viral immediate-early protein EP0, interacts with the WD40 domain of TECPR2 in the cytoplasm during late infection. This interaction recruits the host kinase Casein Kinase 2 (CK2) to form a cascade that stabilizes both EP0 and the COPII outer coat protein SEC31A. Consequently, this axis enhances the secretory capacity of infected cells in a manner dependent on CK2 activity but independent of transcriptional upregulation. It is worth noting that under this pathway, 25-hydroxy cholesterol (25-HC) can inhibit viral transmission, which disrupts the EP0-TECPR2-CK2 axis and suppresses the accumulation of COPII components. In vivo, 25-HC treatment significantly reduced viral loads and attenuated neurovirulence in a murine infection model. These findings reveal how PRV utilizes autophagy factors to promote secretory pathways, and highlight the EP0-TECPR2-CK2 axis, which is cholesterol-dependent, as a potential therapeutic target.

Pseudorabies virus (PRV), the etiological agent of Aujeszky's disease, remains a major threat to the swine industry. Several human PRV infections have recently been reported, including cases with severe ocular involvement, but how human-derived PRV isolates injure retinal cells remains poorly understood. Here, we used the human-derived PRV isolate hSD-1/2019 and ARPE-19 cells to define an injury-associated Ca2+ signaling pathway in retinal epithelial cells. hSD-1/2019 infection induced pronounced extracellular Ca2+ influx, CaMKII and JNK1/2 activation, mitochondrial dysfunction, and membrane-compromising cellular injury. Pharmacological screening identified a prominent Cav2.2-sensitive component in the infection-associated Ca2+ influx. Blockade of this component attenuated JNK1/2 activation, mitochondrial injury, and loss of membrane integrity, linking extracellular Ca2+ entry to downstream epithelial injury. In addition, PRV glycoprotein K (gK) expression promoted Cav2.2-sensitive Ca2+ elevation and JNK1/2 activation in transfected cells, supporting gK as a candidate upstream viral contributor to this response. Together, these findings suggest that hSD-1/2019 converts infection into a Ca2+ influx-driven injury program in retinal epithelial cells and identify a Cav2.2-sensitive Ca2+/JNK axis as a focused mechanism of PRV-associated retinal epithelial injury.IMPORTANCEPseudorabies virus (PRV) has long been considered primarily an animal pathogen, but recent human infections with severe ocular disease have raised concerns about its ability to damage human retinal cells. This study shows that the human-derived PRV isolate hSD-1/2019 engages extracellular Ca2+ influx to drive a JNK1/2-linked mitochondrial injury response in ARPE-19 cells. A Cav2.2-sensitive Ca2+ entry component was functionally associated with this process, and PRV gK was identified as a candidate viral contributor capable of engaging the Ca2+/JNK response. These findings provide a mechanistic framework for PRV-associated retinal epithelial injury and support further investigation in primary retinal cells and in vivo models.

ABSTRACT Zoonotic respiratory pathogens commonly disrupt epithelial barrier integrity across animal species and humans, yet the host mechanisms coupling immune activation to structural barrier failure remain elusive. Here, we report that Pasteurella multocida infection is associated with activation of an NF-κB–microtubule–STING signaling axis, which contributes to epithelial barrier dysfunction. Infection activated NF-κB, induced microtubule depolymerization, and impaired microtubule-dependent trafficking of STING, consistent with defective delivery to degradative compartments, accompanied by intracellular accumulation, enhanced TBK1–IRF3 signaling, excessive interferon-β production, and loss of junction proteins. STING inhibition restored barrier integrity in vitro and in vivo , while alleviating lung injury and improving survival in infected mice. These findings highlight STING as a potential host-directed therapeutic target for pasteurellosis. IMPORTANCE Pasteurella multocida is a zoonotic pathogen that causes respiratory diseases in livestock, poultry, and even humans, resulting in substantial economic losses and posing threats to public health. The mechanism by which its infection impairs the airway epithelial barrier function remains unclear. This study identifies the NF-κB–microtubule–STING pathway as critical: P. multocida infection activates NF-κB, disrupts microtubule integrity, and impairs STING trafficking, which leads to excessive IFN-β production and the degradation/loss of junctional proteins. Inhibition of STING can restore airway epithelial barrier function, alleviate lung injury in mice, and improve survival rates. These findings provide novel therapeutic targets for host-directed therapy against P. multocida infections, with clinical and veterinary application value.

Streptococcus suis (S. suis) is a significant pathogen responsible for swine respiratory infectious diseases, capable of causing severe clinical manifestations and posing a substantial threat to both the swine industry and public health. With the continuous increase in antibiotic-resistant strains, vaccination has emerged as an urgently needed strategy for controlling S. suis infection. Consequently, the development of subunit vaccines with cross-protective efficacy has become a major research focus. Suilysin (SLY) is a protein with favorable immunogenic properties; however, recombinantly expressed SLY exhibits pronounced hemolytic activity. Previous studies have reported that mutations at amino acid positions 353 and 461 individually could completely abolish the hemolytic activity of SLY. In our study, however, we found that this loss of activity was not complete. We therefore constructed a novel mutant protein, mSLYW461F, P353L (mS43), by simultaneously introducing mutations at both positions 353 and 461. Through a series of in vitro toxicity assays, in vivo toxicity tests, and immunization-challenge experiments, we successfully obtained a mutated SLY variant that is completely devoid of hemolytic activity and other toxic effects while retaining its immunogenicity. Furthermore, immunization-challenge experiments conducted in piglets identified an excellent candidate subunit vaccine antigen combination, mS43+rPepO+rMRP, against S. suis. This combination provided 100% protection against challenge with the S. suis serotype 2 strain.

Orthoflaviviruses are recognized as significant emerging mosquito-borne pathogens globally. Members of the Japanese encephalitis (JE) serogroup can be distinguished from other orthoflaviviruses by the presence of an additional non-structural protein (NS1’). However, the role of the NS1’ protein in the virus’s life cycle and transmission remains incompletely understood. This study demonstrates that the acquisition of the secretory NS1’ protein from JE serogroup viruses by mosquitoes from infected hosts significantly suppresses the antiviral effect of the RNA interference pathway, thereby facilitating viral infection in mosquitoes. Furthermore, immunization of mice with the NS1’ protein showed considerable efficacy in preventing the transmission of orthoflaviviruses from mice to mosquitoes. These findings indicate that targeting the NS1’ protein may represent a promising strategy for controlling the spread of JE serogroup viruses and other orthoflaviviruses between vertebrate hosts and mosquitoes.

Japanese encephalitis virus (JEV), a neurotropic flavivirus, poses a significant public health threat, yet the molecular mechanisms underlying its interaction with host immunity remain poorly understood. This study reveals that zinc finger protein ZNF33B promotes JEV replication by subverting the RLR-mediated innate immune response through orchestrating m⁶A RNA modification. ZNF33B directly binds to antiviral transcripts Ifih1 (encoding MDA5), Mavs, and Irf3, recruiting the m⁶A methyltransferase METTL14 to enhance their m⁶A methylation. Concurrently, ZNF33B interacts with the nuclear m⁶A reader YTHDC1 to facilitate the export of these methylated transcripts from the nucleus to the cytoplasm. In the cytoplasm, the m⁶A-modified transcripts are recognized by the cytoplasmic reader YTHDF2, leading to accelerated RNA decay. This process downregulates MDA5 and IRF3 protein levels, suppressing type I interferon production and downstream antiviral responses, thereby creating a permissive environment for JEV replication. Our findings establish a regulatory axis where ZNF33B integrates m⁶A modification and RNA metabolism to evade host immunity, highlighting the potential of targeting epitranscriptomic pathways for antiviral therapy.