Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-origin porcine enteric alphacoronavirus first identified in Guangdong, China in 2017, which causes severe watery diarrhea and high mortality in neonatal piglets, leading to in substantial economic losses to the global swine industry. Here, we report that SADS-CoV nsp10 functions as an IFN-λ1 antagonist. Specifically, nsp10 suppressed IFN-λ1 production independently of its zinc finger domains. Furthermore, nsp10 inhibits MAVS- and IRF1-driven IFN-λ1 expression within the RLR signaling pathway. Nsp10 blocks poly (I:C)-induced nuclear translocation of IRF1, although no direct physical interaction is detected between the two proteins. Pull-down assays combined with mass spectrometry identified hnRNPU as a nsp10-interacting protein and their direct interaction was verified by co-immunoprecipitation (Co-IP). hnRNPU alone inhibits IFN-λ1 production and synergizes with nsp10 to enhance this suppression, consequently facilitating SADS-CoV replication. Collectively, our findings uncover a previously unrecognized immune evasion mechanism employed by SADS-CoV and provide a potential therapeutic target for controlling coronavirus infections.
Duck enteritis virus (DEV), a member of the Herpesviridae family, causes an acute and highly contagious disease in waterfowl characterized by vascular injury and severe gastrointestinal lesions. Although DEV infection is known to disrupt cellular homeostasis, the interplay between DEV-induced endoplasmic reticulum (ER) stress and host inflammatory responses in intestinal epithelial cells (IECs) remains poorly understood. We developed an in vitro model using DEV infection in primary duck IECs to study the activation of the unfolded protein response (UPR) and its association with downstream immunomodulatory effects. Our results demonstrated that DEV infection significantly altered ER ultrastructure, characterized by severe rough ER dilation, and triggered ER stress as evidenced by the early upregulation of the chaperone GRP78. Mechanistically, DEV infection was associated with the activation of the PERK-eIF2α-ATF4-CHOP and IRE1α-XBP1 axes of the UPR, while the ATF6 branch showed no significant cleavage under our experimental conditions. Furthermore, activation of the PERK branch was associated with sustained upregulation of the NLRP3 inflammasome and the NF-κB signaling pathway, which correlated with the accumulation of pro-inflammatory cytokines IL-1β and IL-6. Concurrently, the IRE1α branch was associated with the phosphorylation of JNK and the expression of TRAF2, further corresponding to the secretion of IL-1 and TNF-α. Notably, Tunicamycin (Tm)-induced ER stress partially mimicked, but did not fully recapitulate, the robust and sustained inflammatory profile induced by viral infection. These findings suggest a strong correlation between DEV-induced UPR activation and the inflammatory milieu of IECs, providing novel insights into the pathogenesis of DEV-induced enteritis and identifying potential targets for antiviral intervention.
Bovine mastitis is one of the most economically burdensome diseases in the global livestock industry. With the growing threat of antimicrobial resistance and the implementation of policies aimed at reducing or prohibiting antibiotic use, the development of novel antimicrobial strategies is urgently needed. Phage therapy, as a biological approach capable of precisely lysing pathogenic bacteria, holds promise as an alternative to antibiotics. This review systematically examines the scientific foundations of phage therapy, covering the phage life cycle, host recognition mechanisms, and the ability to degrade biofilms via depolymerases. It highlights the development strategies of phage cocktails, phage-based combination therapies, and preliminary evidence from preclinical models (e.g., mice) and limited bovine trials demonstrating the potential of phages in reducing bacterial load and alleviating inflammation. Key challenges are also discussed, including insufficient understanding of phage-host interactions within the complex mammary microenvironment, bottlenecks in phage preparation and formulation, the emergence of bacterial resistance, and the absence of a regulatory framework. Finally, this review outlines future research directions, emphasizing the potential of interdisciplinary strategies that integrate artificial intelligence-driven phage screening and rational design, nanomaterials, CRISPR-based gene editing, and novel delivery systems. By systematically summarizing concrete case studies and lessons learned from the clinical translation of phage therapy for bovine mastitis, with a particular focus on formulation engineering and delivery strategies applicable to livestock settings, this review provides a practical roadmap for advancing phage therapy from the laboratory to the farm.
Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is an important disease affecting livestock and public health. To achieve the goal of ending bTB, more effective vaccines for cattle are urgently needed. Recently, subunit vaccines have attracted widespread attention. An effective bTB subunit vaccine requires the identification of key components in the pathogen that can induce protective immune responses and relies on adjuvants to induce robust immunity against the target pathogen. In this study, we developed subunit vaccines employing BCG cell wall polysaccharides (CW) or Ag85A as antigens, formulated with either chitosan-based nanoparticles (CSNPs) or manganese jelly (MnJ) as adjuvants. The protective efficacy of these vaccines was evaluated following intranasal administration in a mouse model challenged with M. bovis. Our results demonstrated that vaccination with CW-MnJβ elicited a marked IL-17 response. Following M. bovis challenge, the data revealed that CW‑based vaccines (CW‑CSNPs, CW‑MnJα, and CW‑MnJβ) provided significant protection, with CW‑MnJα and CW‑MnJβ showing efficacy comparable to BCG, as evidenced by significant reductions in bacterial burden and obvious alleviation of histopathological damage in the lung and spleen compared to the PBS control. In contrast, Ag85A-based vaccines failed to provide comparable protection, although Ag85A-MnJα and Ag85A-MnJβ stimulated a substantial IgG response. These findings provide evidence that intranasal administration of a subunit vaccine incorporating BCG cell wall polysaccharide as an antigen, adjuvanted with either CSNPs or MnJ, holds substantial promise as an effective bTB vaccine.
Coronaviruses (CoVs) can cause severe respiratory and enteric diseases, posing major threats to human health and the livestock industry. However, the host metabolic mechanisms underlying viral replication remain unclear. Using porcine epidemic diarrhea virus (PEDV) as the primary model, this study investigated the role of iron metabolism in CoV replication and its mechanisms. Our results showed that PEDV infection markedly induced intracellular iron accumulation, while exogenous iron supplementation enhanced viral replication and iron chelation suppressed it. Mechanistically, iron enhances viral replication by promoting the formation of iron-sulfur (Fe-S) clusters, essential cofactors for the viral RNA-dependent RNA polymerase (RdRp). Based on these findings, we further evaluated the antiviral activity of TEMPOL, a small molecule targeting viral Fe-S clusters. TEMPOL reduced RdRp activity and effectively inhibited PEDV replication. Moreover, TEMPOL exhibited broad-spectrum antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infectious bronchitis virus (IBV), and porcine deltacoronavirus (PDCoV). Importantly, TEMPOL effectively reversed FAC-promoted viral pathogenesis, alleviating tissue lesions and clinical symptoms across multiple CoV animal models. Collectively, this study reveals a key role of iron metabolism in CoV replication and suggests that targeting viral Fe-S clusters may provide a novel strategy for anti-CoV therapy against diverse CoVs.
Bovine viral diarrhea virus (BVDV) has marked tropism for lymphoid tissues and is associated with thymic atrophy and immune dysfunction in calves. However, the transcriptional programs associated with thymic injury and longer-term recovery remain incompletely defined. To characterize these responses, thymus transcriptomes were profiled by RNA sequencing following experimental BVDV-2 infection, with sampling during the post-acute phase at 13 days post-inoculation (pi) and at a later time (day 42 pi). This transcriptomic analysis was performed in the same calf cohort previously shown to develop variable long-term thymic atrophy and impaired IDV-specific cell-mediated responses after prior BVDV exposure. Post-acute BVDV exposure was associated with a strong thymic immune and antiviral transcriptional signature, with 295 differentially expressed transcripts relative to controls, including prominent chemokine, complement, and antigen-presentation-associated signals. At day 42 pi, relatively few transcripts differed in the broad comparison group, indicating attenuation of the early transcriptional response. In contrast, marked heterogeneity was evident among BVDV-exposed calves, with persistent thymic atrophy versus apparent recovery. Persistent thymic atrophy was associated with enrichment of cell-cycle, chromosome-organization, DNA metabolic, and extracellular-matrix-related programs. Targeted RT-qPCR analysis supported selected extracellular-matrix, spindle/cell-cycle, and lymphoid-development-associated expression differences between calves with persistent thymic atrophy and calves with apparent recovery. The day-42 thymic outcome analysis was exploratory because it involved a small subgroup of calves with persistent atrophy versus apparent recovery. Together, these findings provide a molecular context for the previously observed association between persistent thymic atrophy and weaker T-cell responses to subsequent IDV challenge.
The distinction between feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) has long provided a practical framework for understanding how common feline coronavirus (FCoV) infection can give rise to feline infectious peritonitis (FIP). The internal mutation hypothesis remains central to explaining many cases. However, the two biotype labels are often used to imply genetic, anatomical, cellular, clinical, and epidemiological properties simultaneously, although these properties are neither synonymous nor invariably linked. Longitudinal observations made possible by effective antiviral treatment and the emergence of the recombinant lineage FCoV-23 have made these interpretive limitations particularly apparent. Clinical remission after treatment does not necessarily coincide with clearance of viral RNA, cessation of faecal shedding, or normalisation of all host-response measures, and post-treatment RNA detection does not by itself establish persistence of replication-competent virus or treatment failure. FCoV-23 combines between-cat transmission of a viral lineage with heterogeneous, cat-specific spike domain 0 deletions consistent with within-host emergence or selection. In this review, we reassess the FECV/FIPV dichotomy by separating five levels of description: genetic background, anatomical compartment, cell tropism, host clinical state, and transmissibility. These levels are analytically distinct but biologically interconnected, and experimentally measurable viral properties can provide mechanistic links between genetic variation and downstream biological outcomes. We examine how molecular markers, PCR findings, faecal shedding, experimental phenotypes, and post-antiviral observations should be interpreted in relation to the biological processes they directly reflect. We argue that FECV and FIPV remain useful terms, but should not be treated as fixed viral entities or used as substitutes for direct evidence. Matching the level of inference to the level of observation will improve interpretation of FCoV studies, diagnostic reporting, and the design of future work on within-host evolution, transmission, and treatment response.
Pseudorabies virus (PRV) remains a major threat to the swine industry, particularly due to the emergence of highly virulent PRV variants that have caused severe outbreaks in Asia in recent years. These emerging strains exhibit enhanced pathogenicity and immune evasion, resulting in insufficient protection by traditional inactivated or attenuated vaccines derived from classical strains such as Bartha. Therefore, novel antiviral vaccine strategies with improved immunogenicity are urgently needed. In this study, an attenuated recombinant PRV (rPRV) was constructed from the emerging HNX strain by removing the TK and gE loci and introducing a CCL3L1 expression cassette (HNX-ΔTK/ΔgE-CCL3L1). The immunogenicity and protective efficacy of this recombinant virus were systematically evaluated. HNX-ΔTK/ΔgE-CCL3L1 significantly promoted the activation of bone marrow-derived dendritic cells (DCs) in vitro and enhanced DC activation in lymph nodes (LNs) in vivo. Vaccination with HNX-ΔTK/ΔgE-CCL3L1 induced robust humoral immune responses, including increased virus-neutralizing and glycoprotein B-specific antibody levels observed from the second week post-immunization onward. Furthermore, rPRV-expressed CCL3L1 enhanced T cell-dependent germinal center (GC) responses, resulting in improved protection against lethal PRV challenge. Our results demonstrate that CCL3L1 acts as an effective molecular adjuvant by potentiating humoral immunity through activation of the conventional DC-T follicular helper cell-GC B cell axis, supporting HNX-ΔTK/ΔgE-CCL3L1 as a potential vaccine for the control of emerging PRV variants.
Goose astrovirus (GAstV) is a significant pathogen affecting goslings by inducing visceral gout, yet no commercial vaccine is currently available. This study involved the serial passaging of the GAstV-GXNN strain in LMH cells to investigate alterations in viral replication, genomic stability, and pathogenicity, as well as to assess the potential of a vaccine candidate. The findings indicated that the viral titer increased progressively with each passage, reaching 107.35 TCID50/mL by the 120th passage (GAstV-GXNNP120). Whole-genome sequencing revealed the presence of 6, 19, 26, and 28 nucleotide mutations at the 30th, 60th, 90th, and 120th passages, respectively. Pathogenicity assays demonstrated a reduction in virulence with successive passages, culminating in the complete attenuation of GAstV-GXNNP120, which did not induce clinical signs or lesions in one-day-old goslings. Following five successive passages in goslings, the attenuated strain exhibited stable genetic characteristics without any reversion to virulence. Goslings aged one day, inoculated with GAstV-GXNNP120 at dosages ranging from 102.0 to 105.0 TCID50, developed neutralizing antibodies by the third day post-vaccination. Antibody levels increased in a dose-dependent manner, peaking at day 21 and remaining elevated through day 42. Challenge experiments utilizing the virulent GAstV-GXNN strain revealed that groups vaccinated with doses of 103.0 TCID50 and above achieved complete protection. These groups exhibited no clinical symptoms or pathological damage post-challenge, and both tissue viral loads and virus shedding levels were significantly reduced compared to the control group. Consequently, the minimum effective vaccination dose was established at 103.0 TCID50. These results provide a crucial foundation for the development of a live attenuated GAstV vaccine.
Bluetongue virus (BTV) is a segmented double-stranded RNA virus of ruminants transmitted by Culicoides spp. biting midges. Although the genome consists of ten segments, classification into serotypes is primarily based on genome segment 2. However, reassortment among genomic segments is a major driver of BTV evolution and diversity. This study used longitudinal whole-genome sequencing to characterize BTV genomes collected from 2021 to 2023 within a single sheep flock in Colorado, where multiple serotypes co-circulate. Whole-genome sequences were generated from fourteen blood samples representing four serotypes: BTV-6, -11, -13, and -17. Longitudinal sampling identified multiple BTV serotypes within individual sheep across consecutive years. Tanglegram analysis comparing segment phylogenies to the segment 2 tree demonstrated incongruent topologies across all genomic segments, suggestive of reassortment or the circulation of distinct genomic constellations. Nucleotide-level comparisons revealed high sequence homology among same-serotype samples from the same year, while the greatest genetic divergence was observed among BTV-17 genomes collected in different years. Additionally, all BTV-13 genomes contained a previously undescribed nonsynonymous substitution in segment 10 predicted to extend the encoded protein by three amino acids. Together, these findings demonstrate that highly conserved BTV genomes and distinct genomic constellations can be detected at the flock level across multiple years. This longitudinal whole-genome approach reveals the genetic complexity of endemic BTV populations, including novel variants and genomic patterns consistent with reassortment that are lost with conventional serotyped-based approaches, highlighting the need to integrate whole-genome characterization into endemic BTV monitoring programs.
Viral infection triggers innate immune responses that are essential for restricting early pathogen replication. Lipid droplets (LDs), traditionally recognized as neutral lipid storage organelles, have recently emerged as regulators of immune signaling. In this study, we investigated the modulatory role of LDs in antiviral immunity using porcine reproductive and respiratory syndrome virus (PRRSV) and pseudorabies virus (PRV) as models. We demonstrated that oleic acid (OA)-induced LD accumulation significantly enhanced type I interferon (IFN) responses and suppressed viral replication. Mechanistically, increased LD content potentiated the activation of both the RIG-I/MDA5 and cGAS-STING signaling pathways. Moreover, LD accumulation led to upregulation of viperin, a key interferon-inducible antiviral protein. Pharmacological inhibition of LD formation or DGAT2 activity reduced viperin expression and impaired antiviral efficacy, indicating that LD dynamics are functionally linked to the antiviral state. Collectively, our findings uncover an important role of LDs in coordinating innate immune signaling and viperin-mediated antiviral defense, positioning LDs as potential therapeutic targets for enhancing host immunity against viral infections.
Japanese encephalitis virus (JEV), a typical mosquito-borne neurotropic pathogen, triggers neuronal cell death and robust inflammatory responses within the central nervous system, posing severe threats to public health and animal husbandry worldwide. The host protein cylindromatosis (CYLD) acts as a vital deubiquitinating enzyme and negatively modulates a spectrum of inflammatory signaling cascades via its deubiquitinase activity; nevertheless, its functional role and underlying mechanism during JEV infection remain poorly defined. The present study was designed to explore the regulatory effects and molecular mechanism of CYLD in JEV replication as well as virus-elicited neuronal pyroptosis. Our results revealed that JEV infection markedly downregulated endogenous CYLD expression in a time-dependent fashion. Functional validation experiments demonstrated that CYLD overexpression remarkably restrained JEV replication, while CYLD knockdown exerted the opposite pro-viral effect, and such regulatory functions were independent of the interferon signaling pathway. Additionally, ectopic CYLD expression effectively mitigated JEV-triggered neuronal pyroptosis, whereas CYLD deficiency significantly exacerbated this pyroptotic process. Notably, the inhibitory effects of CYLD on viral replication and neuronal pyroptosis were completely abrogated by the application of deubiquitinase activity-deficient mutants and truncated CYLD constructs, confirming that the biological functions of CYLD are strictly dependent on its deubiquitinase active domain. Consistently, in vivo mouse infection models further validated the critical involvement of CYLD in JEV pathogenicity. Mechanistically, combined in vivo and in vitro assays illustrated that CYLD alleviates JEV-induced neuronal pyroptosis through targeted inhibition of the JNK/p38 MAPK signaling pathway and subsequent suppression of downstream NLRP3 inflammasome activation. Collectively, this study for the first time identifies CYLD as a pivotal negative regulator of neuronal pyroptosis upon JEV infection. CYLD exerts prominent neuroprotective effects and restricts JEV replication via suppressing NLRP3 inflammasome-dependent pyroptosis, which sheds new light on the molecular pathogenesis of Japanese encephalitis and provides a promising novel therapeutic target for the prevention and clinical treatment of JEV encephalitis.
STING serves as a central adaptor in innate immunity, with its trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus determining its signaling outcomes. Retention of STING within the ER is associated with enhanced inflammatory responses, whereas its localization to the Golgi apparatus facilitates the production of type I interferon. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV), an economically significant swine pathogen characterized by causing interstitial pneumonia, promotes the retention of STING within the ER, thereby enhancing inflammatory responses. Mechanistically, high mobility group box 1 (HMGB1) mediates PRRSV-induced impairment of STING trafficking. Conversely, the knockout of HMGB1 restores STING localization to the Golgi apparatus and attenuates proinflammatory cytokine expression. Furthermore, we identify PRRSV nonstructural protein 2 (Nsp2) as a viral determinant that amplifies inflammatory responses through the HMGB1-STING axis. Nsp2 facilitates HMGB1-dependent impairment of STING trafficking, thereby reinforcing inflammatory responses. Collectively, our study identifies the spatial regulation of STING as a novel mechanism exploited by PRRSV to bias immune signaling toward inflammation, providing new insights into viral pathogenesis.
Recently, Brazil has experienced a zoonotic emergence of sporotrichosis. The associated cutaneous lesions are often extensive and slow to heal, thereby providing a gateway for opportunistic bacteria belonging to the normal skin microbiota. Among these, Staphylococcus spp. are of particular concern due to their high prevalence and notable levels of antimicrobial resistance. The objective of this study was to identify and characterize Staphylococcus spp. isolated from the cutaneous wounds of domestic cats undergoing treatment for sporotrichosis and exhibiting clinical signs of secondary bacterial infection. A total of 233 samples from 203 cats were analyzed. Staphylococcus spp. was isolated from 156 samples (67%), with S. aureus (42.3%) and S. felis (25.6%) being the most prevalent. Antimicrobial susceptibility testing revealed high levels of resistance to penicillin (51.9%), erythromycin (28.8%), and clindamycin (19.2%). In contrast, most isolates were susceptible to chloramphenicol (98%), ciprofloxacin (96.7%), and nitrofurantoin (93%). Multidrug-resistant strains were identified in 24% (38/156) of the isolates. Overall, 12 isolates (7.7%) were classified as methicillin-resistant staphylococci, including four methicillin-resistant S. pseudintermedius (MRSP) and one methicillin-resistant S. aureus (MRSA). To investigate the genetic profiles and epidemiological relationships of these isolates, all the MRSP and MRSA strains were subjected to whole-genome sequencing. Among the MRSP isolates, four sequence types (STs) were identified, including ST551, the founder of clonal complex (CC)551, which is commonly associated with infection in dogs. The MRSA isolate belonged to ST1176, a member of CC5, which is a globally prevalent lineage and is frequently associated with nosocomial infections in humans. This study demonstrates that Staphylococcus species, including methicillin-resistant isolates, are frequently present in the wounds of sporotrichosis-infected cats exhibiting clinical signs of secondary bacterial infection. The detection of MRSA and MRSP in a cat highlights an additional public health concern associated with feline sporotrichosis and further reinforces the growing concern regarding antimicrobial resistance in companion animals.
In recent years, Swine Acute Diarrhea Syndrome Coronavirus (SADS-CoV) has attracted our attention as a new outbreak of porcine coronavirus. Reports on SADS-CoV indicate that this virus is widely involved in innate immunity, but its mechanism of involvement in antagonizing interferon signaling is still unclear. In this article, we found that the nucleocapsid protein (N) of SADS-CoV can inhibit the JAK-STAT signaling pathway mediated by IFN. Specifically, we found that N protein can degrade STAT1 through the ubiquitin proteasome pathway, and further discovered that N protein can promote K48 ubiquitination modification of STAT1. In addition, through mass spectrometry, we identified that the recruitment of E3 ligase TRIM28 by N protein promotes the degradation of STAT1. In addition, SADS-CoV infection of cells downregulates the protein expression level of STAT1, while overexpression of STAT1 inhibits the replication of SADS-CoV. The above results indicate that SADS-CoV N protein antagonizes the JAK-STAT signaling pathway by recruiting E3 ligase TRIM28 to mediate STAT1 degradation. These findings reveal a new mechanism by which SADS-CoV N protein antagonizes host innate immunity and provide us with novel strategies for antagonizing SADS-CoV.
Urbanization alters wildlife communities and creates epidemiological bridges between wild, domestic, and synanthropic hosts. Data on multiple pathogen taxa circulating simultaneously in urban mammals in Central Europe remain limited. Between July 2023 and July 2025, 240 wild mammals (native and invasive) were collected in the urban and peri-urban district of Brno, Czech Republic. Intestinal content, tongue, lymph nodes and other organs were examined using multiplex qPCRs for selected viral, bacterial and parasitic pathogens, including hepatitis E virus (HEV), Francisella tularensis, Echinococcus multilocularis, Toxoplasma gondii, Toxocara spp., Baylisascaris procyonis and others. Basic statistical analyses were used to explore risk factors and co-infections. Overall, 43.3% animals were positive for at least one pathogen. Multiple tissue positivity either with the same or more pathogens was observed. The work showed first molecular evidence of HEV and F. tularensis in free-ranging European raccoon dogs, and HEV in stray cats. Highest prevalence was observed for Toxocara spp. in cats and red foxes. E. multilocularis, Toxocara spp. and B. procyonis were detected across several hosts. Strong associations were found between Toxocara and B. procyonis in cats and between Giardia duodenalis and Cryptosporidium parvum in cats. Age was identified as a significant factor with young foxes being infected more frequently by Toxocara than adults. These findings indicate substantial pathogen circulation and environmental contamination within urban adapted mammals. The pilot study provides a multi-pathogen baseline for urban wildlife in Central Europe and underscores the need for integrated, long-term One Health surveillance.
American alligators (Alligator mississippiensis) in production systems within the United States experience occasional disease outbreaks and die-offs. However, outbreak management is limited by an incomplete understanding of common causes of mortality in commercially raised crocodilians. In this study, we examined the gut microbiota of healthy (n = 18) and sick alligators (n = 17) during an active outbreak on an alligator farm in Southeast, USA. Sick alligators were all exhibiting generalized signs of disease. The gut microbial composition of alligators with disease differed significantly from that of healthy alligators. One ASV in the genus Peptostreptococcus and one in the genus Wolinella were found at increased relative abundances in alligators with disease while one ASV, Clostridium sensu stricto 13, was found at increased relative abundances in healthy alligators. Notably, the Peptostreptococcus ASV was found in all but two of the 17 alligators with disease and was not detected in any healthy alligator. Chlamydia species were also found at increased abundance in alligators with disease; although, Chlamydia was widely present in healthy alligators too. Additional studies are required to assess the clinical significance of these taxa as primary / secondary pathogens or protective (probiotic) species.
Porcine reproductive and respiratory syndrome virus (PRRSV) remains a significant pathogen in swine, causing major economic losses globally, with few antiviral treatment options available. Ebselen, a synthetic organoselenium compound known for its antioxidant, was explored for its potential to inhibit PRRSV replication. Our findings suggest that Ebselen significantly reduced viral RNA and protein levels across multiple PRRSV strains in a dose-dependent manner. Time-of-addition assays revealed that Ebselen primarily interferes with viral biosynthesis phase, though it does not inhibit the viral 3C-like protease (nsp4) directly. Transcriptomic profiling and biochemical assays indicated that Ebselen activates the NRF2 antioxidant response pathway by upregulating TRIM16 and SQSTM1, which disrupt KEAP1-NRF2 interactions, thereby stabilizing NRF2. This leads to increased expression of NRF2-regulated antioxidant enzymes and mitigation of PRRSV-induced oxidative stress. Moreover, Ebselen exhibited anti-inflammatory effects by reducing proinflammatory cytokine production. In vivo challenge experiments further showed that Ebselen treatment significantly decreased pulmonary viral loads and alleviated lung histopathological damage. These findings support potential of Ebselen as a host-targeted antiviral strategy, highlighting its dual role in managing oxidative stress and inflammation, offering a promising approach to combat PRRSV.
