Group A rotaviruses (RVs) continue to be one of the most important pathogens causing severe acute gastroenteritis in infants and young animals worldwide. Recently, the prevalence of porcine RV (PoRV) from pig farms has strikingly increased, adversely affecting the swine industry, particularly with the G9 genotype of PoRV VP7 emerging as the predominant genotype spreading in China. Current vaccines against PoRV fail to provide sufficient protective immunity, necessitating urgent development of effective vaccines and antiviral drugs against PoRV. Here, we successfully established and improved the entirely plasmid-based reverse genetics (RG) system to rescue a G9 genotype of recombinant PoRV AHFY2022 strain (G9P[23]). Using the improved RG system, we rescued recombinant AHFY2022 harboring the fluorescent UnaG protein or nano-luciferase (NLuc) reporter within gene segment 7 that encodes non-structural protein 3 (NSP3). Furthermore, we adopted the UnaG reporter virus to screen anti-PoRV drugs and identified two promising antiviral drugs, C8 and C9. Moreover, we generated the recombinant PoRV (rAHFY2022-G5-VP7) containing G5 genotype of VP7 from PoRV-positive samples in the backbone of AHFY2022 strain. The reassortant strain exhibited efficient replication and genetic stability. Mouse models were utilized to evaluate the immune responses elicited by rAHFY2022-G5-VP7 strain in vivo, revealing similar neutralizing antibodies and cellular immune response compared to the parental AHFY2022 strain in mice. Together, this study provides an important tool for screening potential anti-PoRV drugs and developing novel vaccines against prevalent PoRV strains.

Background Exosomes (EXOs) are promising biomarker sources for liquid biopsy. Due to their low abundance in biofluids and the presence of non-target vesicle proteins during the isolation process, traditional methods such as ultracentrifugation (UC) are time-consuming, yield low purity, and can cause damage to EXOs. Achieving rapid, high-purity isolation remains a significant challenge. The magnetic beads (MBs) method is the most effective way to enhance EXO purity, but existing MBs face bottlenecks, including poor suspension stability, limited surface functionalization, slow magnetic response, and increased adsorption of interfering contaminants, ultimately leading to the loss of critical proteomic information. Results In this study, a novel strategy for EXO separation was proposed based on the specific interaction between the tetraspanin protein CD63 on EXOs and CD63-targeted aptamers functionalized on immunomagnetic hydrogel nanofibrils (IMHNFs). The IMHNFs showed streamlined magnetic responsivity with a responsivity rate 8-fold faster than that of conventional magnetic beads and exhibited good suspension stability. The IMHNFs enabled rapid EXO isolation from biofluids within 30 min, faster than UC. Furthermore, IMHNFs offer universality for multiple biofluids, higher purity (3.28-8.05-fold compared with UC), lower pollution protein, richer proteomic information, and higher signal intensity (the signal intensity of the specific protein CD63 in urine-derived EXOs was over 10-fold that achieved with UC). Notably, 332 upregulated proteins were identified in urine-derived EXOs from prostate cancer (PCa) patients compared with normal controls, covering 61 PCa biomarkers confirmed and published in the literature, demonstrating the excellent potential of this method for future clinical research and applications. Significance The proposed IMHNFs method exhibited significant advantages in magnetic separation performance (rapid magnetic response and good suspension stability), high-purity EXO enrichment, and proteomic analysis—especially its universality in various biofluids. It not only provides new insights into immunomagnetic separation technology but also contributes to advancing EXO development in liquid biopsy.

The immunopotentiation efficacy of polydimethyl diallyl ammonium chloride (pDMDAAC) nanoparticles (NPs) as an adjuvant for the spike (S) protein subunit vaccine of porcine deltacoronavirus (PDCoV) was investigated, with particular emphasis on the combination of the NPs with the STING agonist cGAMP. Following dorsal subcutaneous immunization in mice, ELISA and virus neutralization assays revealed that the combination of pDMDAAC NPs and cGAMP (S-pDMDAAC-cGAMP) significantly enhanced PDCoV-specific IgG antibody titers and neutralizing antibody (nAb) potency in serum compared with the S protein alone. Further analysis via CCK-8 assay indicated there was markedly augmented splenic lymphocyte proliferation capacity in the S-pDMDAAC-cGAMP group, indicating robust cellular immune activation. Cytokine profiling showed elevated secretion levels of IFN-gamma, IL-4, and TNF-alpha in splenocytes, while flow cytometric analysis revealed increased proportions of CD3+ CD4+ and CD3+ CD8+ T lymphocytes, accompanied by elevated B-cell populations in inguinal lymph nodes and spleen. These results collectively indicated that pDMDAAC NPs substantially potentiate both humoral and cellular immunity toward the PDCOVID-19S protein and cGAMP amplified this adjuvant effect.

Porcine epidemic diarrhea virus (PEDV) represents a highly contagious enteropathogenic coronavirus affecting swine. This virus elicits acute diarrhea in neonatal nursing piglets, with mortality approaching 100%, and, thus, imposes enormous economic burdens on the worldwide pork industry. Therefore, developing safe and effective vaccines remains a top priority for controlling PEDV. Here, we constructed a lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccine. This vaccine encodes the four major structural proteins of PEDV: spike (S), membrane (M), envelope (E), and nucleocapsid (N), which self-assemble into virus-like particle (VLP). Compared with a PEDV S-only mRNA vaccine and a commercial PEDV/transmissible gastroenteritis virus (TGEV) bivalent inactivated vaccine in a mouse model, this VLP mRNA vaccine induced significantly higher levels of IgG, mucosal IgA, and neutralizing antibodies. It also enhanced the lymphocyte proliferation index and expanded the populations of T and B cells. Subsequent evaluations in sows showed that the VLP mRNA vaccine elicited robust PEDV-specific immune responses in vivo. Furthermore, piglets that suckled colostrum from these vaccinated sows gained protection against challenge with a virulent PEDV strain. These protected piglets exhibited lower diarrhea scores, reduced viral shedding, less severe intestinal lesions, and decreased mortality. Collectively, these findings demonstrate that the developed PEDV VLP mRNA platform constitutes a highly promising strategy for preventing PEDV infections.IMPORTANCEPEDV-induced watery diarrhea in neonatal piglets remains a major threat to the global swine industry, while current commercial vaccines are predominantly inactivated formulations or S-only subunit vaccines, whose efficacy and broad-spectrum protection continue to be limited. We, therefore, incorporated the four PEDV structural proteins (S-M-E-N) into a LNP mRNA vaccine that self-assembles into VLPs in vivo. In mice, the mRNA-VLP platform elicited significantly higher systemic IgG, mucosal IgA, and neutralizing antibody titers than an S-only mRNA control or a commercial PEDV/TGEV inactivated vaccine, while expanding both T- and B-cell populations. Maternal immunization of pregnant sows converted these responses into potent systemic immunity that protected suckling piglets against virulent challenge, as evidenced by markedly reduced diarrhea scores, intestinal lesions, viral shedding, and mortality. Collectively, the developed PEDV mRNA-VLP platform offers a highly promising strategy for preventing PEDV infection.

Enterovirus G (EV-G) is an important enteric pathogen widely circulating in swine populations and is characterized by considerable genetic diversity and recombination potential. In recent years, recombinant EV-G strains carrying exogenous papain-like cysteine protease (PLCP) gene insertions have been increasingly reported; however, their genotype distribution and molecular characteristics in major pig-producing regions remain poorly understood. In this study, 356 clinical samples collected from Guangxi, southern China, between 2020 and 2025 were screened for EV-G, and 13 representative strains were subjected to whole-genome sequencing and sequence analysis. The overall EV-G positivity rate in Guangxi was 20.51% (73/356). Phylogenetic analysis showed that the 13 Guangxi EV-G strains were mainly classified into three genotypes, G1, G2, and G8, with G1 being the predominant genotype. Notably, PLCP gene insertions of 573-642 nt were identified at the 2C/3A junction in seven strains belonging to three distinct genotypes, G1, G2, and G8, demonstrating the cross-genotype distribution of PLCP insertions within a single geographic region. Phylogenetic analysis of the PLCP sequences demonstrated that all Guangxi-derived PLCP sequences clustered within the EV-G-PLCP clade and were clearly separated from the torovirus PLCP clade. Recombination analysis retained three potential recombination events with clearer combined support from RDP4 and SimPlot analyses, involving Guangxi strains GX3008, GX3022, and GX4292. Selection pressure analysis showed that the VP1 gene was overall under negative selection. Collectively, these findings demonstrate the co-circulation of multiple EV-G genotypes, the cross-genotype distribution of PLCP insertions, and the presence of potential recombination events in Guangxi. This study provides new evidence for understanding the genetic diversity, genomic plasticity, and regional molecular characteristics of EV-G, and also provides an important basis for future PLCP-related functional studies and continued EV-G surveillance.

Background:Asymptomatic hosts can shed pathogens without showing clinical symptoms, making them invisible to routine screenings and potent drivers of pathogen dissemination and epidemic outbreaks. The lack of reliable, cost-effective tools for large-scale identification of asymptomatic infections hampers early intervention and control strategies. Porcine deltacoronavirus (PDCoV), a zoonotic pathogen with potential for cross-species transmission, presents a critical case for improving such detection methodologies. Methods:We improved the IgG serodynamics-based epitope discovery method by integrating clustering and high-level analysis, which helped us identify linear epitopes with immunogenicity from a large number of candidate epitopes. Epitopes were filtered using negative sera identified by virus neutralization tests (VNT) to eliminate highly antigenic probes. These remaining low antigenicity probes were used to construct a protein-peptide hybrid microarray (PPHMPDCoV). The platform was applied to detect PDCoV-specific transiently produced IgGs (TPIs) in serum samples collected from pigs aged 28-174 days. Results:The PPHMPDCoV successfully detected asymptomatic PDCoV infections in pigs, particularly showing a peak infection rate of 15% at 45 days of age. The platform enabled not only the detection of asymptomatic carriers but also the characterization of infection stages. Conclusion:The study provides a novel, specific, and practical platform for detecting asymptomatic PDCoV infections based on serological TPI signatures. It offers early warning and disease prevention strategies in livestock and establishes a framework for future monitoring of potential interspecies transmission.

Oral vaccines have attracted considerable attention due to their advantages of convenient administration and ability to induce mucosal immunity. However, unfavorable conditions such as the gastrointestinal barrier and acidic environment constrain their immunogenic efficacy. To address these issues, a novel oral vaccine delivery platform has been developed, in which mannose-decorated liposomes are complexed with antigen and retinoic acid, then enveloped by a thiolated alginate gel microsphere (MLip@Gel). Mannose-modified liposomes adsorb porcine epidemic diarrhea virus (PEDV) through electrostatic interactions, targeting intestinal macrophages and enhancing uptake. Thiol-modified sodium alginate is used as a gel shell, preventing PEDV from being destroyed in the stomach and promoting retention in the intestines. Retinoic acid (RA) facilitates the differentiation of cells that promote secretory IgA production. The delivery system maintain stability under acidic conditions, while decomposing at pH ≥ 6.8 to release the antigen. Experiments on mice demonstrate that PR-MLip@Gel can induce a high level of α4β7+CCR9+ cell activation and IgA level, compared to PEDV(IM) group. Systemic responses, such as IgG and CD4+/CD8+ T, B cells, are also significantly increased. The challenge experiments demonstrate that piglets immunized with PR-MLip@Gel exhibit better protection against PEDV infection, as PR-MLip@Gel can simultaneously induce robust mucosal immunity and humoral immunity. These findings suggest that the oral vaccine effectively induces strong mucosal and systemic immune responses, offering a promising strategy for developing oral vaccines against intestinal infectious diseases.

Porcine rotavirus (PoRV) is a significant pathogen that causes diarrhea in piglets, with the G9, G5, and G4 genotypes being the most prevalent in China. Although vaccination is the most effective strategy to prevent PoRV infections, the currently available G5 genotype-based vaccine offers limited cross-protection against other circulating PoRV genotypes in pig farms. In this study, we developed an entirely plasmid-based reverse genetics (RG) system for the PoRV strain NJ2012 (G9P[7]) and generated two recombinant reporter viruses expressing the fluorescent UnaG and NLuc proteins, respectively. Furthermore, we successfully constructed multivalent recombinant PoRV strains by inserting the VP7 gene of G4 genotype into the gene segment 7 (NSP3) and/or the VP7 gene of G5 genotype into the gene segment 5 (NSP1) within the backbone of rNJ2012-WT strain. These multivalent recombinant viruses efficiently expressed G4 and/or G5 genotype of the VP7 protein in infected cells and elicited robust immune responses in mice. The adult mice immunized with the trivalent recombinant PoRV (rNJ2012-fG5-VP7/haG4-VP7), which simultaneously expressed VP7 proteins from G4, G5, and G9 genotypes, conferred passive protection to suckling mice against infections caused by multiple G genotypes of PoRV. In summary, these findings established a platform for efficient generation of multivalent recombinant PoRV, offering a scalable methodology to facilitate the development of next-generation PoRV vaccines.IMPORTANCEPorcine rotavirus (PoRV) is a primary etiological agent of diarrhea in swine, posing significant challenges due to the diversity of circulating genotypes and the limited cross-protection offered by existing PoRV vaccines. To address this, we developed multivalent recombinant vaccine candidates capable of eliciting robust immunity against multiple genotypes of PoRV strains. Using the established entirely plasmid-based reverse genetics (RG) system based on the G9 genotype of PoRV strain NJ2012 (G9P[7]), we further engineered the multivalent recombinant virus simultaneously expressing VP7 proteins from G4, G5, and G9 genotypes. Immunization of adult mice with this trivalent recombinant virus conferred broad-spectrum passive protection to their suckling mice against infections by multiple PoRV genotypes. Our findings established a novel platform for efficiently developing multivalent PoRV vaccines, offering a promising strategy for the prevention and control of PoRV outbreaks.

Inflammation serves as a crucial feedback mechanism in response to external stimuli, with severe inflammatory reactions leading to significant damage in the body. Evidence indicates that Lactobacillus plantarum can suppress inflammatory responses, effectively maintaining intestinal balance and stability, and are widely used in pig farming. Prebiotics, when compared to live bacteria, demonstrate a more pronounced probiotic function. In this study, lipopolysaccharides (LPS) was utilized as a stimulus for an inflammation model. Through in vitro cell experiments and in vivo pig trials, it was observed that oral administration of Lactobacillus plantarum prebiotics effectively inhibited inflammation. Moreover, the anti-inflammatory effect was improved with higher doses of prebiotics, without any observed intestinal damage. Additionally, flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) and Peyer's patches (PPs) revealed alterations in various immune cell populations, including T cells, B cells, dendritic cells (DCs), and natural killer (NK) cells. Overall, the results showed an increase in T cell proportion during inflammation and a decrease upon resolution. B cells and DCs were suppressed during both inflammation and recovery periods. NK cells were unaffected by inflammation but their proportion decreased during the recovery phase. This study, for the first time, highlights that while Lactobacillus plantarum prebiotics alleviate clinical symptoms of inflammation, immune responses involving B cells and DCs are also suppressed, in addition to T cell immune responses. This finding not only enhances our understanding of the mechanisms of action of Lactobacillus plantarum prebiotics but also provides fundamental data for future therapeutic interventions and immunological applications.

Coronavirus infections can trigger multiple modes of cell death, leading to severe infectious diseases. The process is modulated by host factors with mechanisms yet to be fully elucidated. Here, we first demonstrated that the host factor A20 regulated PANoptosis during porcine deltacoronavirus (PDCoV) infection, thereby contributing to the antiviral defense response. We found that PDCoV could induce PANoptosis in intestinal epithelial cells, which facilitates the extracellular release of viral particles through this form of programmed cell death. A20 restricted the PANoptosome assembly and downstream death signaling by targeting RIPK3 ubiquitin chains for degradation. Consequently, loss of A20 exacerbated cell lysis and enhanced the release of viral particles, although this effect does not alter viral entry or replication. We further established that PDCoV-induced PANoptosis-dependent release was driven by osmotic imbalance resulting from membrane pore formation mediated by GSDMD and MLKL, rather than by direct transmembrane egress of viral particles. Transwell models showed that pharmacological inhibition of the pore-forming activities of GSDMD and MLKL reduced viral dissemination and preserved epithelial barrier integrity. These findings advance our understanding of enteric coronavirus pathogenesis and suggest that the A20-PANoptosis axis represents a potential target for antiviral intervention.IMPORTANCECoronaviruses have repeatedly posed significant threats to both human and animal health. Here, we used porcine deltacoronavirus (PDCoV), a highly enterotropic zoonotic pathogen, to uncover a novel mechanism by which coronaviruses exploit PANoptosis to facilitate viral egress. We demonstrate that PDCoV infection triggers PANoptosis in intestinal epithelial cells, leading to plasma membrane rupture and subsequent viral release. Importantly, we identified the host ubiquitin-editing enzyme A20 as a critical negative regulator of this process. A20 restricts PANoptosome assembly by specifically deubiquitinating RIPK3, thereby limiting cell lysis and suppressing viral dissemination without affecting viral replication. Our findings offer fundamental insights into coronavirus-host interactions and highlight the therapeutic potential of targeting lytic cell death to combat viral dissemination.

The persistent mutation and global dissemination of epidemic viruses pose major threats to public health, driving demand for safer vaccine strategies. While subunit vaccines offer enhanced safety by using defined antigens, their weak immunogenicity necessitates the use of adjuvants. Traditional adjuvants such as aluminum salts, however, often raise safety concerns. In this study, a nanoparticle-engineered subunit vaccine was presented using quaternized aminated glucan (QAG) as adjuvant and Omicron S protein as antigen. QAG nanoparticles exhibited strong protein-loading capacity and promoted efficient antigen uptake by macrophages through electrostatic interactions, significantly upregulating CD80, CD86, and MHC II expression. In vivo, Omicron/QAG immunization induced robust humoral and cellular responses, including a 2.71-fold increase in IgG levels compared to Omicron alone, along with enhanced activation of T cells, B cells, and NK cells. Neutralization assays confirmed improved protection against the Omicron B.1.1.529 strain. Furthermore, QAG demonstrated excellent safety in cytotoxicity and hemolysis assays. These findings highlight QAG as a safe and effective nanoparticle adjuvant that enhances the immunogenicity of subunit vaccines, providing a promising platform for next-generation vaccine development.

Porcine rotavirus (PoRV) is a major cause of severe diarrhea in piglets, highlighting the need for efficient serological tools to support surveillance and vaccine evaluation. While virus neutralization tests (NTs) remain the gold standard for detecting functional antibodies, they are labor-intensive and low-throughput. To address this limitation, we developed a competitive enzyme-linked immunosorbent assay (cELISA) based on a high-affinity monoclonal antibody (mAb16) that targets the VP4 spike protein of PoRV. When validated against a panel of porcine serum samples previously characterized by NT, the cELISA demonstrated excellent diagnostic performance, with an area under the receiver operating characteristic curve (AUC) of 0.995, 96.00% sensitivity, and 98.28% specificity. Notably, the percent inhibition values from the cELISA exhibited a strong positive correlation with neutralizing antibody titers (Pearson r = 0.846). This correlation was further confirmed across multiple PoRV genotypes—including G4P[23], G5P[23], and G9P[7]—in a controlled vaccine trial. The assay also showed high analytical sensitivity, no cross-reactivity with other common porcine pathogens, and excellent intra- and inter-assay reproducibility. Epitope mapping localized the mAb16 binding site to the VP5 subunit of VP4, providing a structural basis for its broad genotype across-reactivity. Collectively, this cELISA provides a sensitive, specific, and high-throughput alternative to conventional NT for PoRV serosurveillance and vaccine immunogenicity assessment.

[Background]Porcine Rotavirus(PoRV)is one of the key pathogens causing viral diarrhea in newborn and young piglets,and its infection can lead to severe gastrointestinal dysfunction in the host,with clinical manifestations,including dehydration,diarrhea,and even death,resulting in huge economic losses to the global pig industry.The VP4 protein is one of the key structural proteins on the surface of PoRV virions.The VP4 protein is cleaved by trypsin to generate two functional subunits,such as VP8 and VP5,which play a core role in the initial stage of viral infection of host cells,mediating the adsorption of the virus to host cell receptors and the subsequent membrane penetration process.Meanwhile,the VP4 protein is also an important target antigen that elicits the host immune response.However,current research on specific monoclonal antibodies(mAbs)against the PoRV VP4 protein is relatively scarce,which limits the development of related diagnostic methods and new vaccines.[Objective]This study aimed to prepare specific monoclonal antibodies(mAbs)against the VP4 protein of Porcine Rotavirus(PoRV),and on this basis,comprehensive biological characterization of these mAbs were analyzed,including reactivity,antigenic epitope types(conformational or linear),and subtype identification,and key neutralizing activities were evaluated too.The aim was to precisely identify the antigenic epitope regions with important functional significance on the VP4 protein,thereby providing support for the accurate diagnosis of PoRV infection and the design of novel vaccines.[Method]BALB/c mice were immunized with purified VP4*P23 recombinant protein,and hybridoma cell lines were screened using splenocyte fusion technology.The reactivity of monoclonal antibodies was identified by Western blot,indirect immunofluorescence assay(IFA),and immunoperoxidase monolayer assay(IPMA).The conformational sensitivity of monoclonal antibodies was evaluated by indirect ELISA.VP5 truncated proteins were constructed to determine the antigenic epitope regions,and the neutralizing ability of VP5 monoclonal antibodies was assessed by in vitro neutralization assay.[Result]A total of 26 hybridoma cell lines stably secreting antibodies were successfully obtained.The antibody subtypes included multiple types such as IgG1,IgG2a,IgG2b and IgM,among which the light chain type was mainly κ chain.Among the obtained 26 mAbs,15 of them were confirmed to specifically react with natural PoRV virions(detected by IFA and IPMA).Indirect ELISA detection showed that mAb11,14,15 and 23 were conformation-insensitive mAbs,while mAb16,17,18,19,21,24,25 and 26 were conformation-sensitive mAbs.It was worth noting that the reactivity of three mAbs(mAb1,2 and 22)was enhanced after antigen denaturation.Western blot analysis further focused on mAbs recognizing linear epitopes(mAb11,14,15),and the results showed that they could all specifically recognize the linear epitope within the amino acid region of about 300-360 on the VP5 protein,possibly targeting the same antigenic epitope.However,in vitro neutralization assay evaluation showed that these three mAbs had no neutralizing effect on PoRV strains.[Conclusion]Multiple mAbs targeting the PoRV VP4 protein were successfully prepared,among which 15 mAbs exhibited the ability to bind to natural viruses.Through systematic characterization,not only the subtype distribution and light chain type of mAbs were clarified,but more importantly,the antigen recognition characteristics of mAbs were deeply analyzed:conformation-sensitive and conformation-insensitive mAbs were successfully distinguished,and the linear antigenic epitope regions recognized by mAb11,14,and 15 were precisely located.This study provided key antibody resources for the optimization of PoRV diagnostic reagents and the development of subunit vaccines,and laid a foundation for in-depth research on the immunological functions and antiviral mechanisms of the VP4 protein.

ABSTRACT RNA viruses have evolved diverse strategies to evade host interferon (IFN)-stimulated gene (ISG) defenses; however, how they exploit host epitranscriptomic regulation remains poorly understood. Here, we identify an immune-evasion mechanism in which porcine reproductive and respiratory syndrome virus (PRRSV) targets the m6A demethylase fat mass and obesity-associated protein (FTO) to suppress antiviral signaling. Mechanistically, the viral endoribonuclease nsp11 inhibits STAT5-dependent transcription through the key residues Q96 and S104, thereby reducing FTO expression. Loss of FTO increases m6A modification of STAT2 and STAT3 transcripts, impairing their translation and phosphorylation, thereby attenuating ISG responses. Reduced STAT3 activity further dampens STAT5 signaling, establishing a feed-forward circuit that amplifies suppression of antiviral immunity. Functionally, disruption of this regulatory region (Q96A and S104A) attenuates viral pathogenicity in vivo and restores ISG induction. These mutations also reduce infection-associated inflammatory responses and the accumulation of reactive oxygen species. Together, these findings define a nsp11-STAT5-FTO-STAT2/3 axis that enables PRRSV to reprogram host epitranscriptomic control of innate immunity. Our work reveals a mechanism of epitranscriptomic hijacking and identifies FTO as a key host factor exploited by RNA viruses, highlighting m6A regulation as a potential target for antiviral intervention. IMPORTANCE Viruses must overcome host innate immune defenses to establish infection; however, the mechanisms by which they manipulate host RNA regulation remain incompletely understood. In this study, we show that porcine reproductive and respiratory syndrome virus (PRRSV) suppresses interferon responses by targeting the host m6A demethylase FTO through its endoribonuclease nsp11. This process involves the inhibition of STAT5 phosphorylation, which reduces FTO expression and increases m6A modification of key immune regulators, including STAT2 and STAT3, thereby impairing their activation. Disruption of this pathway attenuates viral pathogenicity in vivo and restores antiviral signaling. These results demonstrate that PRRSV can reprogram host epitranscriptomic regulation to modulate innate immunity and suggest that m6A -related pathways may be potential targets for antiviral intervention.

Since 2010, millions of piglets have died from porcine epidemic diarrhea virus (PEDV) variant strains. Compared with classical strains, variants exhibit enhanced virulence and immune evasion capacity, rendering classical strain-based vaccines poorly effective. However, the critical mutants responsible for increased pathogenicity and immune evasion remain unclear. This study aims to identify the key mutations that drive humoral immune evasion in PEDV variants and to elucidate further the underlying molecular mechanisms. Cross-neutralization assays and recombinant virus screening identified the 894-993 amino acid (aa) mutation region of the S2 subunit as a key determinant of humoral immune evasion in variants. The challenge experiments in piglets demonstrated that the 894-993 aa mutation region plays a critical role in determining variants' virulence. Subsequent vaccination-challenge experiments further clarified the pivotal contribution of the 894-993 aa mutation region in the humoral immune evasion in vivo. In addition, the 894-993 aa region determines the membrane fusion characteristics, enabling variants to resist neutralizing sera through cell-to-cell transmission. Moreover, structural analyses revealed that mutations in this region altered the surface electrostatic potential and increased hydrophobicity and rigidity of the S protein. The findings of this study are the first to demonstrate that the 894-993 aa mutation region of the S2 subunit drives humoral immune evasion, modulates the virulence and membrane fusion of PEDV variant strains, and provides a theoretical foundation for the formulation of new prevention and control strategies.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) variant strains pose a serious threat to piglet health worldwide. Despite the availability of commercial vaccines developed based on classical PEDV strains, they have shown limited efficacy against newly emerging variants. Moreover, variant strains exhibit varying degrees of genomic mutations compared to classical strains, and the regulatory effects of these mutations on viral biology have not been systematically studied. In this study, we identify the 894-993 amino acid (aa) region within the S2 subunit as a key determinant of humoral immune evasion in variants. Mutations in this region were shown to reduce neutralization sensitivity, alter membrane fusion activity, and increase the structural rigidity of the S protein. These findings greatly enhance our understanding of the biological characteristics of PEDV variants and provide a new potential strategy and important theoretical support for viral control and vaccine development.

For viruses that replicate in the proximity of or bud at the endoplasmic reticulum (ER) associated membranes, proper processing of their glycoproteins is critical for successful infection. Rotavirus outer capsid protein VP7 is an ER-resident protein. However, its N-terminal signal peptide is removed by an unknown proteolytic mechanism. In this study, we leveraged tandem affinity purification followed by high-resolution mass spectrometry to profile host proteins that interact with VP7. We identified members of the signal peptidase complex (SPC) family as important host factors that facilitate rotavirus infection. CRISPR knockout or siRNA knockdown of distinct SPC subunits resulted in significant decrease in infectious rotavirus titers in a viral strain- and cell type-independent manner. While viral transcription, translation, and replication were not altered in the absence of SPC, we observed formation of abnormal viral particles by transmission electron microscopy (TEM) in SPCS1 knockout cells. Mechanistically, loss of SPC proteins led to inefficient cleavage of VP7 signal peptide and severely impaired the final steps of virion maturation and assembly. Additionally, we identified residue E256 within VP7 as a key site for SPC binding. An E to R mutation abolished VP7 interaction with SPC and subsequently led to reduced viral infectivity. Taken together, these findings define SPC as a novel regulator of VP7 maturation and rotavirus assembly and highlight its role as a novel cellular target for potentially broad-spectrum antiviral therapeutic development.

Porcine epidemic diarrhea virus (PEDV) G2c subtype has emerged as an increasingly prevalent variant causing widespread epidemic, high mortality and severe economic losses in the Chinese swine industry. The lack of specific and efficient detection methods hinders its surveillance, early detection and control. In this study, a TaqMan-MGB probe-based duplex quantitative real-time reverse transcription PCR (RT-qPCR) assay was developed for simultaneous detection of pan-PEDV and differentiation of the G2c subtype. Universal primers/probe targeting the conserved N gene of PEDV and G2c-specific primers/probe targeting the unique mutation sites in the S gene were designed. The assay was validated for performance, including specificity, sensitivity, and repeatability, and further evaluated using artificial challenge models and clinical samples. The standard curves exhibited excellent linearity (R2 > 0.999) with amplification efficiencies of 99.1

Coronaviruses (CoVs) pose serious threats to both animal and human health, underscoring the urgent need for broad-spectrum antiviral strategies that can provide rapid and immune-independent protection beyond vaccination. In this study, we developed zeolitic imidazolate framework-8 (ZIF8) as an innovative zinc-based anti-coronavirus nanoagent and revealed that ZIF8 exhibits markedly enhanced antiviral activity relative to the commonly used zinc oxide (ZnO). Using porcine epidemic diarrhea virus (PEDV) as a model α-coronavirus, mechanistic studies demonstrated that ZIF8 alleviates excessive reactive oxygen species (ROS) accumulation and preserves mitochondrial homeostasis, while simultaneously promoting cytosolic mtDNA release through moderate opening of the mitochondrial permeability transition pore (mPTP) and coordinately regulates NLRP3 inflammasome activation and STING signaling, thereby enhancing host antiviral responses. Consistent with this mechanism, ZIF8 nanoparticles displayed broad-spectrum inhibitory effects against representative β-, γ-, and δ-coronaviruses, including bovine coronavirus (BCoV), infectious bronchitis virus (IBV), and porcine deltacoronavirus (PDCoV). Moreover, in a PDCoV-infected mouse model, ZIF8 provided effective in vivo protection by reducing viral replication and preventing intestinal damage under both preventive and therapeutic regimens. Collectively, these findings establish the ROS-mitochondria-NLRP3/STING axis as a key mechanistic basis for ZIF8-mediated antiviral activity and provide new insights for the rational design of nanomaterial-based broad-spectrum antiviral therapeutics.