Pseudorabies virus (PRV) is an important swine pathogen causing severe economic losses worldwide. Nasal mucosa serves as the initial entry site for PRV, highlighting the importance of nasal mucosal immunity in limiting infection. Here, we investigated the immune responses and protection against PRV for the intranasal vaccination of glycoprotein B (gB) subunit vaccine adjuvanted with poly(I:C). In piglets, immunohistochemistry showed rapid uptake of the gB by the nasal mucosa within 2 h, supporting subsequent immune activation. Upon challenge with the PRV variant ZJ01, intranasal gB vaccination provided complete clinical protection with the absence of clinical signs, substantially reduced virus load in tissues and viral shedding, and no pathological lesions. Relative to intramuscular gB or the live attenuated Bartha-K61 vaccination, intranasal gB vaccination elicited stronger mucosal antibody responses and greater infiltration of CD3⁺ T cells, CD19⁺ B cells, and IgA-secreting cells in the nasal cavity. Notably, intranasal immunization followed by challenge promoted the formation of tertiary lymphoid structure (TLS) in the turbinate, providing a local niche for adaptive immune responses. Consistent with histological observation, transcriptomic profiling of nasal mucosa revealed activation of the IL-17 and TNF signaling pathways, which are implicated in the formation and maintenance of TLS. These findings demonstrate that intranasal gB vaccination might represent a promising mucosal vaccination strategy for controlling PRV infection in swine.

Infectious bronchitis virus (IBV), a member of the gammacoronavirus family, can easily mutate, resulting in different subtypes of strains. QX and M41, two subtypes of IBV, exhibit distinct characteristics in terms of their pathogenicity and infection site. Immunohistochemistry and qRT–PCR revealed differences in the replication of QX and M41 in different tissues. Interestingly, we found that the viral load of QX steadily increased in “PBMCs-Mφ” chicken macrophages, whereas the opposite phenomenon occurred in the M41 group. We also observed synergistic antiviral effects of interferon and RNA interference (RNAi) on PBMCs-Mφ. Moreover, high-throughput sequencing and vsiRNA analysis revealed only partially detectable vsiRNA (NSP10) in the IBV M41-stimulated group, which explains the above phenomenon. We constructed a PS-vsiRNA plasmid capable of efficiently expressing small RNAs, and the antiviral test results revealed that transfection with PS-M10 significantly reduced the M41 viral load. Moreover, PC-QX-NSP10 and PC-QX-NSP16 overexpression in PBMCs-Mφ showed that the complex of NSP10/NSP16 could effectively reduce the mRNA level of Dicer (an RNAi pathway core enzyme) and increase the viral load, which is a potential viral suppressor of the RNAi (VSR) protein. In summary, for the first time, this study explains the differences between QX and M41 on PBMCs-Mφ from the perspective of vsiRNA in RNAi, which is highly important for the subsequent development of therapeutic IBV vaccines.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that causes lethal diarrhea in neonatal piglets, yet the viral pathogen-associated molecular patterns (PAMPs) that initiate mucosal antiviral immunity remain poorly defined. Here, integrating intestinal single-cell transcriptomics with in vivo and in vitro infection models, we delineate how PEDV is sensed by host innate immunity. We found that RIG-I, rather than MDA5, functions as the dominant cytosolic sensor that detects PEDV and initiates interferon-mediated antiviral defense in the intestinal mucosa. RIP-seq and RNA structural analyses identified nucleotides 375-760 within the 5' ORF1a region of the PEDV genome as a core RNA PAMP directly engaged by RIG-I. Molecular docking and mutational analyses further revealed that specific nucleotide motifs and cooperative stem-loop assemblies are required for optimal RIG-I activation, establishing higher-order RNA architecture as a key determinant of PAMP potency. Functionally, this PEDV-derived RNA PAMP robustly activated RIG-I signaling, amplified interferon and ISG responses, and restricted replication of PEDV and multiple heterologous RNA viruses, demonstrating broad-spectrum activity against several enteric and respiratory RNA viruses. Moreover, co-administration of this viral RNA with an inactivated influenza vaccine significantly enhanced antigen-specific immunity and protection. Together, these findings define the molecular basis of RIG-I-mediated recognition of PEDV, identify a structurally encoded viral PAMP with both antiviral and adjuvant properties, and provide new insights into how coronaviruses are sensed and controlled at mucosal surfaces.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) causes devastating enteric disease in newborn piglets, yet how the intestinal mucosa detects this coronavirus and mounts antiviral immunity has remained unclear. Our study identifies RIG-I as the dominant epithelial sensor responsible for detecting PEDV and initiating interferon-driven antiviral defense. We further define a structurally encoded RNA element within the 5' ORF1a region of the PEDV genome that functions as a core viral PAMP directly activating RIG-I. This RNA element acts as a molecular alarm that triggers robust innate immune responses, restricts infection by PEDV and other RNA viruses, and enhances the protective efficacy of an inactivated influenza vaccine. These findings illuminate fundamental mechanisms of coronavirus sensing at mucosal surfaces and highlight viral RNA structures as promising natural immunostimulants for antiviral and vaccine strategies.

Strangles, caused by Streptococcus equi subsp. equi (S. equi), is a highly contagious respiratory disease that causes substantial economic losses to the horse industry worldwide. Serological diagnosis currently relies mainly on the imported IDvet® dual-antigen sandwich ELISA kit, which shows good diagnostic performance but is expensive and lacks a domestic alternative in China, thereby limiting its routine use in primary veterinary settings. The aim of this study was to develop a domestic indirect ELISA based on novel antigen targets and an untagged antigen purification strategy. Using comparative bioinformatics analyses, two conserved fragments with species-level specificity for S. equi, designated EQ8 (150 aa) and SclF (44 aa), were identified and fused for recombinant expression. The fusion protein was purified by GST affinity chromatography, cleaved with PreScission protease, and subjected to secondary purification to obtain an untagged EQ8–SclF protein. Western blotting and ELISA demonstrated that the purified protein retained good immunoreactivity and that the GST tag did not cause detectable cross-reactivity, with all OD450 values obtained using GST-coated plates remaining below 0.09, well under the cut-off value of 0.115. Checkerboard titration established the optimal assay conditions as an antigen coating concentration of 1 μg/mL and a serum dilution of 1:100. Using the IDvet kit as the reference method, 157 clinical serum samples (67 positive and 90 negative) were tested in parallel. The newly developed assay showed a relative sensitivity of 89.55% (60/67), a relative specificity of 94.44% (85/90), an overall agreement rate of 92.36% (145/157), and a kappa value of 0.84, indicating excellent agreement between the two methods. Good inter-assay reproducibility was observed, with coefficients of variation all below 9.29%. In conclusion, an indirect ELISA based on an untagged EQ8–SclF fusion protein was preliminarily established for the serological detection of strangles. This assay showed diagnostic performance highly consistent with that of the imported commercial kit while offering potential advantages in terms of lower cost, simpler operation, and elimination of tag-associated interference. It therefore shows potential as a domestic alternative for the serological diagnosis of strangles.

RNA interference (RNAi) is a potent antiviral approach, outperforming traditional pesticides and broad-spectrum drugs. Its use in animal disease control faces two challenges: inefficient target design relying on computer-predicted small-interfering RNAs (siRNAs) rather than virus-derived siRNAs (vsiRNAs), and the lack of cost-effective siRNA delivery systems. In this study, we address both limitations by engineering a probiotic Bacillus subtilis 168 strain, called the recombinant B. subtilis AAD (Anti-AIV-DsRNA, targeted AIV), that constitutively expresses vsiRNA-enriched dsRNA targeting the H9N2 avian influenza virus. Oral administration of AAD leads to the release of double-stranded RNA (dsRNA)-loaded extracellular vesicles (EVs), which efficiently reduce H9N2 viral loads and mitigate pathological lesions. Mechanistically, virus-derived dsRNA is processed by the enzyme Dicer into siRNAs, which then activate RNAi and interferon signaling, resulting in approximately a 70% reduction in viral burden. Overall, these findings demonstrate that integrating the probiotic properties of B. subtilis with EV-mediated dsRNA delivery constitutes a sustainable, effective, and residue-free antiviral strategy for animal disease.

Summary Influenza neuraminidase (NA) is a promising target for universal flu vaccines, yet eliciting potent B-cell responses against its conserved epitopes remains challenging. Here, we developed a membrane-anchored, folding-domain-free NA (mNA) that elicited superior head-specific germinal center B cell and antibody responses compared to soluble tetrameric NA. In non-human primates, mNA immunization induced cross-reactive memory B cell (MBC) responses, expanding clones with the conserved “DR” motif in HCDR3, a hallmark of human broadly reactive NA antibodies. These MBCs conferred cross-inhibitory activity against diverse NA variants and in vivo cross-protection. Cryo-EM analysis revealed that the 554-C2 clone targets the conserved enzymatic pocket via the “DR” motif, while the 554-C1 clone recognizes previously uncharacterized epitopes at the interface between two adjacent N2 monomers, effectively reducing plaque formation by contemporary H3N2 strains. Our findings highlight the immunological advantages of membrane-anchoring, providing a robust strategy for designing next-generation vaccines against influenza and other pathogens.

Paneth cells, a type of intestinal epithelial cell, are primarily located at the base of the intestinal crypts and adjacent to intestinal stem cells. They play a crucial role in maintaining the intestinal mucosal barrier by regulating the gut microbiota and supporting intestinal stem cells. Although some studies reported the morphology and distribution of Paneth cells in humans and mice, controversy persists regarding their distribution in farm animals, particularly in the small intestine of pigs. This study optimized the immunohistochemical staining method for Paneth cells by testing different fixatives and using lysozyme and Sox9 as markers to identify Paneth cells in the pig small intestine. The study found that Paneth cells are mainly distributed in the duodenal segment of the pig small intestine, and their population increases as piglets grow. Paneth cells are more widely distributed in black pigs, which have strong disease resistance. The number of Paneth cells is dramatically reduced after enterovirus infection. Furthermore, pretreatment with Bacillus subtilis effectively rescues the reduction in Paneth cell number caused by porcine epidemic diarrhea virus infection in piglets. L-arginine treatment can rescue the reduced Paneth cell population caused by PDCoV infection. This study comprehensively analyzed the distribution and function of Paneth cells in the pig small intestine, providing a new theoretical basis for improving the intestinal mucosal barrier and enhancing immune defense.

Plant growth regulators (PGRs) play a critical role in modulating plant development and stress responses. This study investigates the mechanism by which 2-amino-3-methylhexanoic acid (AMHA), a novel natural small-molecule PGR, stimulates cucumber growth. Results show that AMHA increased trehalose-6-phosphate and indole-3-acetic acid levels, promoting root development. The improved root architecture enhanced the uptake and utilization efficiency of key nutrients, including N, P, K, and Fe. In shoots, AMHA upregulated photosynthetic genes (CHL27, MgCh, LHCB3, LHCB4, and PsbY), leading to an increased stomatal conductance and net photosynthetic rate. Additionally, elevated sucrose production in source leaves, coupled with its SWEET-transporter-mediated allocation to sink tissues, further fueled growth. By integrating these pathways, AMHA targets core physiological and molecular processes, demonstrating strong potential to improve crop productivity. These findings establish AMHA as a promising PGR that coordinates phytohormone signaling, nutrient uptake, and sucrose metabolism to enhance plant vigor, offering valuable insights for modern, eco-friendly agricultural practices.

Porcine epidemic diarrhea virus (PEDV) is an enteric alphacoronavirus that causes severe diarrhea and high mortality in neonatal piglets, posing a persistent threat to the global swine industry. Despite extensive vaccination, PEDV continues to circulate in China, underscoring the need for updated molecular epidemiological surveillance. Here, we investigated the prevalence, genetic diversity, and pathogenicity of PEDV strains circulating in China during 2023-2024. A total of 714 clinical samples were collected from diarrheic pigs in seven major swine-producing regions. PEDV RNA was detected in 36.27% of samples, with substantial positivity observed in both anal and nasal swabs. Phylogenetic analysis based on the full-length S gene revealed that circulating strains clustered into multiple subgroups, including GIa, GIIa, GIIb, and GIIc, with GIIc being predominant, indicating pronounced genetic diversity and co-circulation of distinct lineages. Two PEDV strains were isolated from PEDV-positive intestinal tissues and designated PEDV-HeiHo-2024 (GIIb) and PEDV-NJZU-2024 (GIa). Recombination analysis identified PEDV-HeiHo-2024 as a putative recombinant derived from distinct GII lineage strains. Comparative analyses of key neutralizing epitopes and N-linked glycosylation sites in the S protein revealed substantial divergence from classical vaccine strains. Experimental infection of neonatal piglets demonstrated that PEDV-HeiHo-2024 induced severe diarrhea and characteristic intestinal lesions, confirming its high pathogenicity, whereas PEDV-NJZU-2024 showed no apparent pathogenic effects. Collectively, these findings highlight the continued circulation of genetically diverse and pathogenic PEDV strains in China and emphasize the importance of sustained molecular surveillance and vaccine updates.

Zhi Bai Heye Fang (AR-PCC-NF) exerts a positive effect on glycolipid metabolic disorders in the clinical setting; however, its efficacy components and mechanisms of action remain unclear. Glycolipid metabolic disorders in mice were used to evaluate the therapeutic effects of AR-PCC-NF and its individual components, and the chemical components of AR-PCC-NF were detected by HPLC. An insulin-resistant cell model was then treated with 12 biological components in vitro, and seven candidate active components were administered to mice with glycolipid metabolic disorders to investigate the efficacy and mechanism of AR-PCC-NF. AR-PCC-NF improved glucolipid metabolism more effectively than did the individual components. The protein expression of INSR and GLUT4 was elevated, and FOXO1 expression and impaired mitochondrial debris in the liver were reduced by AR-PCC-NF. Furthermore, neomangiferin, chlorogenic acid, isomangiferin, 2-hydroxy-1-methoxyaporphine, hyperoside, nuciferine, and berberine improved glucose consumption or T-CHO in vitro. Interestingly, in vivo, neomangiferin, chlorogenic acid, isomangiferin, 2-hydroxy-1-methoxyaporphine, hyperoside, nuciferine, and berberine partially improved abnormal glucolipid metabolism in mice when used separately, but the effects were equivalent to those of AR-PCC-NF when the seven active components were used in combination. Moreover, AR-PCC-NF and its efficacy components upregulated the protein expression of p-AMPK/AMPK and PGC-1α, decreased the levels PPARα, and reduced mitochondrial debris in the liver. In conclusion, neomangiferin, chlorogenic acid, isomangiferin, 2-hydroxy-1-methoxyaporphine, hyperoside, nuciferine, and berberine are the main active components of AR-PCC-NF in the treatment of glycolipid metabolic diseases, and the mechanism is related to the regulation of the AMPK/PGC-1α.

Low temperature triggers Ca2+ signalling and reprogramming of gene expression and metabolism in plants. However, how the Ca2+ signal is transduced to the downstream metabolic pathways remains unknown. The involvement of a cold-induced calmodulin-like protein, MfCML50, from Medicago falcata in regulation of cold tolerance was examined in the present study. The interaction of MfCML50 with isopiperitenol/carveol dehydrogenase (MfCDH) was identified, which was dependent upon Ca2+ and led to activated activity of MfCDH. MfCDH catalysed the production of carvone from carveol, with Km value of 10.25 μM. Overexpression of MfCML50 or MfCDH in Medicago truncatula led to enhanced cold tolerance with increased accumulation of carvone under cold conditions. The knockout mutation of the ortholog MtCML50 and MtCDH in M. truncatula led to reduced cold tolerance with decreased accumulation of carvone in the mtcml50, mtcdh, or mtcml50 mtcdh mutants under cold conditions, which could be recovered by expressing MfCML50 or MfCDH or exogenous application of carvone. The diphenyl-2-picrylhydrazyl (DPPH) assay showed that carvone exhibited strong antioxidant activity. Less ROS were accumulated in MfCML50 and MfCDH overexpressing lines, but more ROS were accumulated in mtcml50 and mtcdh mutants under cold conditions. The results suggested that the Ca2+ signal activated the MfCML50-MfCDH module regulates cold tolerance through promoted production of carvone to maintain ROS homeostasis.

RNA interference (RNAi) is a key antiviral immune mechanism in eukaryotes. However, antiviral RNAi in vertebrates has only been observed in cells with poor interferon systems or in viral suppressors of RNAi (VSR) deficiency virus infections. Our research discovered that infecting macrophages with wild-type coronavirus (Infectious bronchitis virus, IBV) and influenza viruses (Avian influenza virus, AIV) can trigger RNAi antiviral immunity and produce a certain amount of virus-derived siRNA (vsiRNA). These vsiRNAs have an inhibitory effect on the virus and carry out targeted silencing along the Dicer-Ago2-vsiRNA axis. Notably, these vsiRNAs are distributed throughout the virus's entire genome, with a predilection for A/U at the 5' and 3' termini of vsiRNA. In addition, Dicer cleavage produces vsiRNA based on the RWM motif, where R represents A/G, W represents A/C, and M represents A/U. We also discovered that avian LGP2 and MDA5 proteins positively impact the expression of the Dicer protein and the Dicer subtype "DicerM." Most importantly, the PS-vsiRNA plasmid combined with nanomaterial polyetherimide (PEI) showed excellent anti-virus activity in specific-pathogen-free (SPF) chickens. These findings show that RNA viruses trigger the production of the vsiRNA in avian somatic cells, which is of great significance for the application of therapeutic vaccines.

Coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had given rise to a massive epidemic. Owing to the high morbidity and mortality of COVID-19 and the lack of effective therapies, safe and effective vaccination is the optimum choice for controlling this epidemic and preventing infection. The protein subunit vaccine ZF2001, which targets the receptor-binding domain (RBD) protein of SARS-CoV-2, has a significant protective effect against COVID-19. At the beginning of the COVID-19 epidemic, to promote the early approval of ZF2001 for clinical trials by the National Medical Products Administration of China (NMPA), a comprehensive evaluation of its toxicity in vivo was warranted. In the present study, a major part of the above series of studies, we evaluated the safety, immunogenicity and efficacy of the ZF2001 vaccine for the first time in adult Sprague Dawley (SD) rats. The male and female rats were administered three doses of the ZF2001 vaccine (25 μg or 50 μg NCP-RBD protein/dose, containing the aluminum-based adjuvant). The safety profile of ZF2001 was assessed by observing the general health status, local toxicity at the site of administration, immunotoxicity, immunogenicity, blood chemistry and hematology parameters in SD rats. In general, our results indicated that the ZF2001 vaccine did not induce significant systemic toxicity in rats, with a no-observed adverse effect level (NOAEL) of 50 μg NCP-RBD protein/rat. Moreover, the ZF2001 vaccine showed good immunogenicity by inducing the production of specific IgG antibodies in rats after three consecutive immunizations. In addition, histological examination revealed recoverable inflammatory changes in quadricep muscles and adjacent lymph nodes at the vaccine injection site. In summary, our systematic toxicology study proves the safety, tolerability and immunogenicity of the ZF2001 vaccine, which further supports the results of clinical trials of ZF2001.

Outbreaks and the widespread prevalence of porcine respiratory infectious diseases have led to substantial economic losses worldwide. In this study, epidemiological surveillance revealed lower viral detection in association with an increased abundance of Bacillaceae in pigs with outdoor access; thus, Bacillus subtilis NS12, a strain with enhanced mucosal colonization and superior broad-spectrum antiviral activity, was isolated from the nasal mucosa of these pigs for further investigation. This mechanistic study revealed that the antiviral efficacy of B. subtilis NS12 is primarily attributed to bioactive metabolites, including a novel surfactin with high safety and antiviral activity and piceatannol, a potent antioxidant molecule. These metabolites modify the structure and fluidity of phospholipids within the viral envelope, thereby inhibiting viral entry by impeding membrane fusion. Therefore, with its broad-spectrum antiviral activity, B. subtilis NS12 offers a probiotic-based, environmentally sustainable, and noninvasive antiviral strategy for preventing and controlling respiratory viral infections.

Respiratory infections caused by pathogenic bacteria pose a rapidly growing public health threat. The nasal mucus layer serves as the first line of defense against pathogen invasion; however, in nasal mucus, the antimicrobial components and their underlying mechanisms remain unclear. Here, we collected nasal mucus from goat nasal mucosal explant models and identified significant antimicrobial activity in the total protein fraction. Subsequent fractionation indicated that proteins < 30 kDa exhibited the most potent bactericidal activity. Nano LC–ESI–MS/MS analysis identified galectin-3 as a key protein with potent activity against Gram-positive bacteria, particularly Streptococcus suis (S. suis). Galectin-3 targeted teichoic acids on the bacterial surface, disrupting membrane integrity. Additionally, it inhibited the synthesis of three critical bacterial proteins: enoyl-ACP reductase (FabK), carbamate kinase (CK), and small ribosomal subunit protein uS2 (rpsB), thereby destroying bacterial growth and metabolism. In the murine nasal infection model, galectin-3 accelerated the clearance of S. suis and alleviated pathological damage caused by the infection. Taken together, our findings provide the first evidence of the direct antimicrobial action of galectin-3 in nasal mucus and elucidate its mechanisms involving bacterial membrane disruption and inhibition of key metabolic proteins. These results highlight galectin-3 as a promising therapeutic target for S. suis infections.

Endometriosis (EMs) with anxiety/depression is a common comorbidity of EMs, yet effective treatments are still lacking. Leaves of Paeonia suffruticosa Andr. (PSL) have potential therapeutic effects on EMs and their associated psychiatric disorders. In this study, 57 chemical compounds were identified using Global Natural Products Social Molecular Networking (GNPS) and semi-preparative HPLC. Network pharmacology analysis revealed that these compounds shared 68 common targets with the disease, primarily enriched in the PI3K/AKT signaling pathway. Protein-protein interaction analysis identified TNF, IL-6, ESR1, AKT1, and TP53 as core targets. Molecular docking confirmed that apigenin, quercetin, kaempferol, luteolin, and isorhamnetin exhibited strong binding affinities with these core targets. In conclusion, PSL may exert beneficial effects in treating EMs with anxiety/depression by modulating the PI3K/AKT signaling pathway through key targets including TNF, IL-6, ESR1, AKT1, and TP53.

Transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are major enteric coronaviruses responsible for severe diarrhea in neonatal piglets. Retinoic acid‐inducible gene I (RIG‐I) is a key sensor against RNA viruses, yet its distribution in the porcine intestine and regulatory roles during TGEV and PDCoV infections remain insufficiently understood. In this study, we show that under normal conditions, RIG‐I predominantly localizes in lamina propria antigen‐presenting cells, with its expression increasing with age. Following viral infection in vivo and in vitro , both TGEV and PDCoV induce RIG‐I expression, although TGEV elicits a more robust activation of RIG‐I and downstream interferon pathways. Mechanistically, RIG‐I overexpression inhibits replication of both viruses, whereas RIG‐I knockdown significantly enhances TGEV proliferation only, implying that TGEV primarily depends on RIG‐I–mediated immune responses, while PDCoV may rely on other pattern recognition receptors (PRRs). These findings unveil distinct immune regulatory strategies of TGEV and PDCoV and highlight the central role of RIG‐I in controlling TGEV infection, offering a theoretical foundation for targeted preventive and therapeutic interventions.

Host determinants are critical for shaping the outcomes of viral mucosal infections and developing effective antiviral strategies. However, the regulatory roles of "nonsusceptible" mucosal cells remain unclear. Here, we show that while swine influenza and porcine epidemic diarrhea (PEDV) viruses infect piglet nasal and intestinal epithelia, each virus establishes efficient infection only in its preferred mucosal niche. Goblet cell activity significantly influences mucosal infection outcomes; increased mucus secretion effectively blocks viral entry, while its reduction facilitates viral dissemination. Notably, PEDV activates acetylcholine-cholinergic receptor muscarinic 3 signaling in submucosal enteric neurons to induce goblet cell-associated antigen passages. This mechanism enables the translocation of intestinal bacteria to the lamina propria in early infection, even when the epithelial barrier remains intact, triggering inflammation and exacerbating mucosal damage. Our findings emphasize the crucial role of goblet cells in controlling viral tropism and their potential as targets for developing effective broad-spectrum antiviral approaches.