Acidifiers are functional feed additives that may improve egg quality by modulating intestinal microbiota and host metabolism. This study investigated the effects of dietary acidifier supplementation on laying performance, yolk fatty acid composition, eggshell ultrastructure, intestinal function, cecal microbiota, serum metabolome, and antioxidant status in laying hens. Sixty 52-week-old Jingfen No. 6 laying hens were randomly assigned to either a control group or an acidifier-supplemented group for 6 weeks. Acidifier supplementation increased laying rate and egg mass while reducing the feed-to-egg ratio. It also increased the concentrations of seven yolk fatty acids, including tetradecanoic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, cis-vaccenic acid, and arachidic acid. In addition, acidifier supplementation resulted in a denser eggshell microstructure, improved cecal mucosal morphology, and enhanced the activities of lipase, chymotrypsin, amylase, and trypsin. Although cecal microbial alpha diversity remained unchanged, acidifier supplementation altered the overall microbial community structure and enriched several genera, including Slackia_A, Megasphaera_A, Veillonella_A, Merdimonas, and Caccocola. Serum metabolomic analysis identified 395 differential metabolites, primarily associated with lipid, amino acid, energy, and bile acid metabolism. Representative altered metabolites included taurodeoxycholic acid 3-glucuronide, LysoPE (0:0/20:0), 15-HETE, cholic acid, malic acid, and succinic acid. Acidifier supplementation also favorably affected serum HDL-C, LDL-C, and calcium concentrations and enhanced antioxidant capacity. Collectively, these findings indicate that dietary acidifier supplementation improves laying performance, yolk fatty acid deposition, eggshell structure, intestinal function, and antioxidant status, accompanied by alterations in cecal microbiota and serum metabolism. These coordinated changes may contribute to the observed improvements in egg quality.

Adaptor protein complex 2 (AP2), a central regulator of clathrin-mediated endocytosis and intracellular cargo trafficking, is hijacked by numerous viruses to complete their infectious cycles. This review systematically synthesizes the multifaceted roles of AP2 across the entire viral life cycle, from entry and replication to assembly and release, as well as in immune evasion. By delineating how diverse viruses exploit this key host machinery, we further consolidate the rationale and current progress in developing broad-spectrum antiviral strategies that target AP2 and its regulatory pathways. This work aims to provide a unified perspective on AP2 as a critical host-pathogen interface, offering new insights into viral pathogenesis and antiviral drug discovery.

A comparative experiment was conducted in a large-scale pig farm in Jiangsu Province, China. Emission characteristics of ammonia (NH3), methane (CH4), and nitrous oxide (N2O) were compared between a pig house with robotic daily manure cleaning (RC) and shallow manure pit storage (MS). The experiment was initiated in the spring of 2024 (April 15th) and concluded on June 14th, with a total duration of 61 days. The results showed no difference in ventilation or indoor temperature during the monitoring period, while pig weights ranged from 30 to 81 kg. Under the RC mode, the average daily concentrations of NH3 and CH4 in-barn concentration were 27% and 46% lower than under the MS mode. The corresponding average daily emission factors were 3.4 and 29.9 g d(-1) pig(-1) for NH3 and CH4, representing reductions of 22% and 46% compared to the MS mode. There was no difference in the emission of N2O (p > 0.05). The RC system significantly reduced in-barn concentrations and emission rates of NH3 and CH4, likely by shortening the manure retention time inside the barn. The study showed that robotic manure removal technology shortens manure storage time by increasing the frequency of manure cleaning, enabling the simultaneous reduction of ammonia and methane emissions. It offers a promising technical approach for mitigating gaseous pollution in large-scale pig production.

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.

IntroductionPorcine reproductive and respiratory syndrome virus (PRRSV) was first reported in pigs in China in 1996. At present, multiple PRRSV lineages and sub-lineages are circulating in Chinese swine herds.MethodsIn this study, PRRSV ORF5 sequences collected from 2019 to 2024 were analyzed to investigate their spatiotemporal distribution, molecular evolution, and major amino acid mutations.ResultsA total of 674 sequences were obtained from 25 provinces between 2019 and 2024, and their geographical distribution was generally consistent with the major pig-producing regions in China. Phylogenetic analysis revealed that these PRRSV ORF5 sequences mainly belonged to lineage 1 (L1A, L1C), L3, L5 (L5A), and L8 (L8E). From 2019 to 2024, the proportion of L1C initially increased, followed by a decrease and a subsequent rebound, while L1C remained the predominant sub lineage. The proportion of L1A showed a relative increase, whereas L3 was detected at very low levels. Meanwhile, L5A and L8E exhibited a downward trend. Amino acid mutations were observed in the signal peptide, neutralizing epitopes, and T-cell epitopes among different sub-lineages. Some mutations were variable, while others were sub-lineage-specific. Notably, partial L1C strains exhibited an amino acid deletion at position 34, which may affect the viral immune evasion mechanisms. Nucleotide and amino acid identities initially decreased and then increased, with the most pronounced divergence observed in 2021.DiscussionContinuous monitoring of the molecular epidemiology of PRRSV is essential and will provide a scientific basis for the further prevention and control of PRRSV in China.

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder, often accompanied by low-grade inflammation, visceral hypersensitivity and gut microbiota dysbiosis. In this study, the therapeutic potential of Lactiplantibacillus plantarum LPPerfectus001 (L. plantarum 001) was investigated to alleviate IBS symptoms. Using an Lipopolysaccharides (LPS)-induced RAW264.7 macrophage model, L. plantarum 001 demonstrated significant anti-inflammatory properties by inhibiting Nitric Oxide production and downregulating pro-inflammatory cytokines. Furthermore, in a mouse model of IBS induced by Citrobacter rodentium infection and water avoidance stress, L. plantarum 001 intervention reduced fecal moisture, improved intestinal barrier integrity via up-regulating of ZO-1 and MUC2, and attenuated visceral hypersensitivity. Transcriptomic analysis combining with RT-qPCR revealed that L. plantarum 001 modulated the NF-κB signaling pathway and Th1/Th2 cell differentiation, reducing expression of key inflammatory genes. Additionally, 16S rRNA sequencing showed that L. plantarum 001 restored gut microbiota diversity, enriched beneficial butyrate-producing Odoribacter, and suppressed pro-inflammatory Pseudomonadota. These findings suggested that L. plantarum 001 alleviates IBS through multi-targeted mechanisms involving barrier repair, microbiota modulation, and anti-inflammatory signaling, highlighting its potential as a probiotic therapy for IBS.

High salt stress can hinder plant growth and development, thus reducing crop yield and quality. As an important signaling molecule, NO is involved in regulating various physiological and biochemical processes in plants. In this study, tobacco seedlings treated with 200 mmol/L NaCl exhibited leaf etiolation, sharp increases in superoxide anion (O2−) and peroxynitrite anion (ONOO−) contents, reduced activities of photosystem I (PSI) and II (PSII) reaction centers, and obstructed electron transfer. However, overexpression of the gene encoding the NO regulator nitrosoglutathione reductase (GSNOR) reduced the excessive accumulation of nitric oxide (NO) and S-nitrosothiol (GSNO) during NaCl stress. It also enhanced antioxidant enzyme activity and the contents of antioxidant substances, thereby maintaining the homeostasis of reactive oxygen species and reactive nitrogen species. Moreover, GSNOR also promoted photosynthesis in tobacco leaves at the transcriptional level. Through enrichment analysis of differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) based on physiological and RNA-seq data, it was found that photosynthesis-related pathways, including “Porphyrin and chlorophyll metabolism”, “Carbon fixation in photosynthetic organisms,” and “Photosynthesis,” were significantly enriched. In GSNOR-overexpression plants, the expression of genes encoding proteins involved in chlorophyll synthesis, the Calvin cycle, PSII, and PSI were upregulated. Meanwhile, leaf chlorophyll increased, and the net photosynthetic rate, stomatal conductance, and activity of the reaction centers were enhanced. In contrast, GSNOR-inhibited plants exhibited the opposite trends. These results indicate that NO enhances the salt tolerance of tobacco seedlings by promoting photosynthesis and enhancing antioxidant capacity.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a major viral disease that poses a serious threat to the global swine industry. Although progress has been made in understanding its life cycle, the molecular mechanisms underlying PRRSV entry and replication remain incompletely understood. Multiple RNA viruses hijack the endocytic sorting complex required for transport (ESCRT) machinery to orchestrate various stages during infection. In the current study, we identified ESCRT-II subunit ELL-associated protein 20 (EAP20) as an important host factor involved in PRRSV entry and replication. Mechanistically, EAP20 participated in the transport of internalized PRRSV particles to early endosomes via the clathrin-mediated endocytosis pathway. During replication, EAP20 interacted with PRRSV nonstructural protein (Nsp) 2, Nsp5, and Nsp9. Specifically, EAP20 anchored the core replicase Nsp9 on the perinuclear endoplasmic reticulum (ER) and coordinated with the transmembrane proteins Nsp2/Nsp5 to form ER-derived double-membrane vesicles. Collectively, our findings demonstrate that PRRSV exploits EAP20 for viral entry and replication, highlighting EAP20 as a novel proviral factor and a potential antiviral target. IMPORTANCE:PRRSV remains one of the most economically significant pathogens in the global swine industry. Current control strategies are largely hindered because PRRSV pathogenesis has not been fully elucidated. In this study, we identified EAP20, a core subunit of ESCRT-II, as a multifaceted proviral factor that participated in PRRSV entry and replication. These findings provide new insights into the interplay between PRRSV and the host ESCRT machinery, laying a foundation for the development of more effective strategies for PRRS control.

ABSTRACT Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder marked by chronic low‐grade inflammation, heightened visceral sensitivity, and disruptions in gut microbiota composition. In this study, the alleviating effects and underlying mechanism of Lacticaseibacillus rhamnosus LRPerfectus158 was investigated using an IBS mouse model established through Citrobacter rodentium infection followed by water‐avoidance stress. LRPerfectus158 administration significantly alleviated IBS‐like symptoms, including reduced fecal moisture content, restored colon length, and attenuated visceral hypersensitivity. Behavioral assessments further showed that LRPerfectus158 alleviated anxiety‐like behaviors. Moreover, LRPerfectus158 administration decreased serum corticosterone levels, accompanied by reduced hippocampal injury, suggesting a potential association with altered hypothalamic–pituitary–adrenal (HPA) axis activity. Colonic histology and molecular analyses revealed that LRPerfectus158 mitigated inflammation, increased goblet cell abundance, and enhanced epithelial barrier integrity by upregulating ZO‐1 and MUC2 at both mRNA and protein levels. Transcriptomic profiling further confirmed the dual effects of LRPerfectus158 in IBS, demonstrating its ability to alleviate intestinal neural hypersensitivity while concurrently suppressing mucosal inflammation and strengthening epithelial barrier integrity. Metagenomic sequencing showed that LRPerfectus158 was associated with alterations in gut microbiota composition, including increased relative abundances of taxa such as Muribaculum, Duncaniella, Bacteroides uniformis, and Ligilactobacillus murinus. Collectively, these findings suggest that LRPerfectus158 is associated with alleviation of IBS‐like symptoms, correlating with modulation of the gut microbiota, reinforcement of the intestinal barrier, and reduced HPA axis hyperactivation, which highlights its potential as a promising probiotic candidate for IBS intervention.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen that poses a considerable threat to the global swine industry, particularly with emerging variants that complicate control efforts. However, the host factors involved in PRRSV entry are not well understood. In the present study, we identified members of the numb-associated kinase (NAK) family, specifically adaptor-associated kinase 1 (AAK1), G-associated kinase (GAK), and BMP-2-inducible kinase (BMP2K), as essential regulators of PRRSV entry utilizing both genetic and pharmacological approaches. Mechanistically, NAKs facilitate PRRSV entry by phosphorylating the adaptor protein complex 2 subunit mu-1 (AP2M1) at threonine 156, enhancing AP2M1 activation and thereby promoting its interaction with the YxxØ motif in PRRSV glycoprotein (GP) 5 and the receptor cluster of differentiation 163 (CD163). This interaction is critical for efficient trafficking of the virions to early endosomes (EEs). Disruption of AP2M1 phosphorylation or blockade of the AP2M1-YxxØ interaction significantly impaired PRRSV internalization, indicating the potential for targeting this pathway to inhibit infection. Notably, inhibition of the NAKs-AP2M1 axis effectively reduced infection across multiple PRRSV strains, highlighting its capacity as a broad-spectrum antiviral target. Collectively, our findings provide novel insights into PRRSV entry mechanisms and offer a promising therapeutic strategy to control emerging variants of this economically significant disease.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to the global swine industry due to its extensive genetic diversity and limited vaccine cross-protection. Antiviral strategies that target both viral infection and host pathological responses are urgently needed. Here, we evaluated the antiviral efficacy and underlying mechanisms of sodium copper chlorophyllin (SCC) against PRRSV infection. In vitro analyses demonstrated that SCC exhibited potent and dose-dependent inhibitory activity against both classical and highly pathogenic PRRSV-2 strains in Marc-145 cells and immortalized porcine alveolar macrophages (iPAMs). Cotreatment produced the strongest antiviral effect, indicating that SCC primarily targets early stages of viral infection. Mechanistically, SCC directly disrupted viral particle integrity, resulting in viral RNA release and loss of infectivity. In addition, SCC significantly inhibited viral adsorption, internalization, and intracellular genome replication, while exerting no detectable effect on progeny virion release. SCC also reduced the expression of the key PRRSV entry receptor CD163 in porcine macrophages, thereby impairing virus-host interactions. Furthermore, SCC markedly alleviated PRRSV-induced oxidative stress and inflammatory responses by reducing intracellular reactive oxygen species (ROS) and enhancing antioxidant gene expression (heme oxygenase-1 [HO-1], NAD(P)H quinone oxidoreductase 1 [NQO1], and glutamate-cysteine ligase modifier subunit [GCLM]), while suppressing proinflammatory cytokines (TNF-α, IL-6, and IL-8). Network pharmacology analysis supported the involvement of oxidative stress- and metabolism-related pathways. Importantly, in a PRRSV challenge model, oral administration of SCC significantly reduced viral loads, alleviated fever, and attenuated lung pathology. Collectively, SCC displays broad antiviral activity through direct virucidal effects, inhibition of early viral entry, and modulation of host responses, highlighting its potential as a therapeutic candidate for PRRSV control.

Irpex lacteus is a metabolically versatile white-rot fungus capable of producing a wide range of structurally diverse secondary metabolites, including terpenoids, phenolics, steroids, peptides, and polysaccharides. Many of these compounds exhibit notable biological activities, such as antioxidant, antimicrobial, anti-inflammatory, and cytoprotective effects, highlighting their potential relevance to food chemistry and agricultural applications. Owing to its highly efficient ligninolytic enzyme system and flexible secondary metabolic network, I. lacteus has emerged as a promising biological platform for lignocellulose valorization, microbial biotransformation, and the discovery of functional food ingredients and natural preservatives. In recent years, significant progress has been made in elucidating the chemical diversity and biosynthetic logic of its characteristic metabolites, particularly tremulane-type sesquiterpenoids with unusual skeletal rearrangements. This review systematically summarizes 226 secondary metabolites reported from I. lacteus, covering their chemical classification, biosynthetic features, biotransformation capabilities, and biological activities. Special emphasis is placed on advances enabled by genome mining, heterologous expression, and co-culture strategies that activate cryptic biosynthetic pathways. Finally, the potential applications of I. lacteus metabolites in agriculture, food chemistry, and sustainable bioprocessing are discussed, and future perspectives based on multi-omics integration and metabolic engineering are proposed.

Functional constipation (FC) significantly impacts children’s health. This study investigates the prevalence and microbiota characteristics of FC in children aged 0–4 years in Zunyi area. From October to December 2023, 2039 children aged 0–4 years in Zunyi were selected using stratified sampling and cross-sectional survey methods. A questionnaire based on Rome IV diagnostic criteria was used. Twenty-nine children with FC were randomly selected as the functional constipation group (FCG), and 26 healthy children, matched for age, sex, and area, were selected as the control group (CG). A total of 2051 questionnaires were collected, with 2039 valid responses. Among them, 151 children had FC, resulting in a prevalence rate of 7.4

Dissolved oxygen (DO) plays a vital role in the emission of carbon-nitrogen gaseous (CNs: NH3, CH4 and N2O) in livestock wastewater, but the effect of different DO concentration on the emission of CNs is yet unclear. In this study, the continuous emission of CNs from swine manure biogas digestate (BD) storage under different DO concentrations was monitored using the dynamic emission vessel method for 60 consecutive days, and the emission mechanism was explored combined with the microbial analysis. Results showed that the cumulative emission fluxes of GHGs in BD with DO concentrations of 1.0, 2.0, and 4.0 mg L- 1 were reduced by 58.66 %, 70.46 %, and 66.81 %, respectively, compared with the static storage BD. Verticia might inhibits the activity of acetoclastic genera (Methanosaeta and Methanosarcina). DO concentrations was positively correlated with the Verticia. Increasing the concentration of dissolved oxygen reduce CH4 emissions. N2O and NH3 emissions were positively correlated with DO concentration, but the total N loss showed the opposite changes. The results of this study provide a deeper insight into the effect of DO concentration on the CNs emission, and an appropriate concentration of 1.0-2.0 mg L- 1 is recommended for the reduction of CNs in swine manure BD.

The silo composting reactor is applicable for the in-situ treatment of organic waste. Ensuring an adequate supply of oxygen can enhance the compost maturity. This study investigated the effects of aeration modes and rates on composting effectiveness and nitrogen conversion. The 24 h uninterrupted aeration mode (K60T0) was conductive to achieving an elevated temperature of 78 degrees C. The germination index value in the lower layer consistently exceeded 80 % with different aeration modes. The content of total nitrogen (excluding K60T0) and ammonia nitrogen increased progressively with the increase of aeration time. Meanwhile, nitrate nitrogen content exhibited fluctuations, with the lowest content observed in the middle layer. An aeration cycle of 50 min followed by a 10-min pause within an hour resulted in the highest amide nitrogen content of 5.93 g/kg and organic nitrogen content of 2 % in the upper layer. The dominant phylum and genera were Firmicutes and Ureibacillus, respectively. The relative abundance of Firmicutes initially decreased and then increased in general with prolonged aeration time. Additionally, the extension of aeration time altered the nitrogen cycling function within the reactor.

The biosynthesis of anthocyanin in plants is regulated by temperature, light, jasmonic acid (JA), among others. JASMONATE ZIM-DOMAIN (JAZ) proteins are critical repressors of the jasmonic acid (JA) pathway, the role of JAZ proteins on anthocyanin biosynthesis under high-temperature or dark conditions remains unexplored. Here, we identified 16 SmJAZ genes in eggplant and characterized their evolutionary relationships, gene structures, and high-temperature (HT) and darkness stress-responsive expression patterns. Both the HT and darkness treatments significantly inhibit anthocyanin biosynthesis in eggplant peel. Correlation analysis revealed that the SmJAZ9 expression strongly correlated with anthocyanin biosynthesis-related genes under HT and darkness conditions. Transient expression assay in eggplant peels demonstrated that nuclear-localized SmJAZ9 is a positive regulator, enhancing anthocyanin biosynthesis via interaction with the transcription factor SmMYB113, thereby promoting SmCHS and SmDFR transcription. Additionally, SmJAZ9 interacted with SmMYC2 but not SmCOI1, suggesting a JA signaling-independent regulatory mechanism under HT and darkness conditions. This study revealed a novel JAZ-mediated regulatory module integrating JA signaling, environmental stress responses, and anthocyanin biosynthesis, providing potential targets for improving eggplant fruit quality.

Hyperuricemia (HUA), a metabolic disorder characterized by elevated serum uric acid resulting from imbalanced production and excretion, is associated with gout and other serious health issues. This study aimed to screen out the potential probiotics with HUA-alleviating properties among 20 Lactobacillus strains. The results showed that L. reuteri Urob-7 exhibited the highest degradation rates for inosine and guanosine (82.10% and 88.78%, respectively) and strong xanthine oxidase (XOD) inhibitory activity (62.86%). In a HUA mouse model induced by inosine, guanosine, and potassium oxonate, L. reuteri Urob-7 intervention significantly reduced serum uric acid levels by 46.54%, restoring them to levels similar to control groups, and improved kidney function indicators. Moreover, Urob-7 reduced hepatic XOD activity by 37.6% and downregulated XOD expression in the intestines, decreasing excessive uric acid synthesis. It also significantly inhibited the NF-κB/NLRP3 inflammatory pathway, reducing the expression levels of NF-κB and NLRP3 in the kidneys by 39.3% and 47.6%, respectively. Furthermore, L. reuteri Urob-7 increased the abundance of short-chain fatty acid-producing bacteria (e.g., Ruminococcus and Intestinimonas) while reducing the proportion of pathogenic bacteria (e.g., Bacteroides and Anaerovorax), thus ameliorating gut microbiota dysbiosis and intestinal barrier dysfunction. In summary, L. reuteri Urob-7 effectively relieved HUA by modulating uric acid metabolism, suppressing inflammation, and improving gut microbiota balance. These results highlighted its potential as a promising candidate for HUA.

BACKGROUND:The gut microbiome and the host immune system work together to maintain intestinal health and protect against infections. Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and plays a crucial role in this network. However, the precise role of TLR5 in regulating gut microbiota and resistance to infection remains unclear. This study utilized a TLR5 intestine-specific overexpression mouse model to explore these interactions and their impact on Salmonella infection. METHODS:TLR5 intestine-specific overexpression mice (TLR5+/+) and wild-type (WT) mice were infected with Salmonella to assess TLR5's protective role. Survival time, fecal Salmonella load, and intestinal tissue integrity were evaluated. Subsequently, 16S rRNA sequencing and LC-MS-based metabolomics were performed to analyze gut microbiota composition and fecal metabolites. Fecal microbiota transplantation (FMT) and metabolite transplantation experiments were conducted to evaluate the functional impact of microbiota and metabolites on resistance to infection. RESULTS:TLR5 overexpression significantly improved survival time and reduced fecal Salmonella load, demonstrating its protective role against infection. 16S rRNA sequencing revealed enrichment of beneficial taxa, while metabolomic analysis identified altered metabolites in TLR5+/+ mice. Although fecal microbiota and metabolite transplantation did not fully replicate the protective effects, these experiments highlighted the important roles of microbiota and metabolites in infection outcomes, with limitations in transplantation likely affecting the results. These findings underscore the significance of microbiota and metabolites in TLR5-mediated gut immunity. CONCLUSION:TLR5 overexpression significantly alters gut microbiota and metabolite profiles, contributing to improved infection outcomes. These findings highlight the critical roles of microbiota and metabolites in TLR5-mediated immunity and provide a foundation for exploring targeted strategies to enhance resistance against enteric pathogens.

Transmissible Gastroenteritis Virus (TGEV) is a major pathogen causing swine enteric diseases, necessitating effective control strategies. Vaccination plays a key role, but assessing vaccine efficacy remains challenging due to variations in immune response and existing detection limitations. Current antibody detection methods, such as neutralization assays and ELISA, are often subjective, labor-intensive, and time-consuming, highlighting the need for a more efficient evaluation approach. The TGEV S gene was amplified and inserted into the eukaryotic vector PM2.G-ΔG-HA to construct the recombinant plasmid PM2.G-ΔG-TGEV-S-HA. Transfecting ST cells with this plasmid, followed by infection with G*VSV-GFP/LUC, successfully produced TGEV P0 pseudoviruses. Western blot and electron microscopy confirmed the presence of TGEV S and VSV N proteins and the distinct pseudovirus morphology. Optimization determined that 0.5 μg/well of plasmid, 24 h transfection, and 24 h post-infection harvest yielded a viral titer of 106-107 TCID50/mL. The pseudoviruses exhibited strong ST cell tropism and were effectively neutralized by TGEV-positive sera. A pseudovirus-based neutralization test (pNT) was established, showing 100% sensitivity, 96.6% specificity, no cross-reactivity with PEDV, PPV, PDCoV, or PRoV, and a 94% concordance with the live virus neutralization test. The method effectively tracked antibody level changes post-TGEV vaccination. This study successfully developed a novel pseudovirus-based detection method, overcoming traditional assay limitations. The pNT method provides a scalable, efficient, and reliable tool for TGEV antibody evaluation, with broad potential applications in pathogen detection and vaccine assessment.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral disease that causes substantial economic losses in the swine industry. This study aimed to develop a PRRS virus (PRRSV) detection assay using multienzyme isothermal rapid amplification (MIRA) and to analyze the genetic variation of PRRSV in Southwest China. A total of 13,863 samples, including blood and lung tissues from pigs suspected of PRRSV infection, were collected. The MIRA assay was designed with primers and probes targeting conserved regions of the PRRSV‐M gene, demonstrating high specificity with no cross‐reactivity to other swine pathogens and an estimated detection threshold sensitivity of 1.0 copy/μL. Prevalence analysis revealed that, although vaccinated pigs showed relatively high antibody levels, the virus continued to circulate, particularly in unvaccinated herds. Genetic analysis of the predominant PRRSV strains indicated an increasing prevalence of NADC30‐like strains and notable genetic variation in genes such as ORF5 and nonstructural protein 2 (NSP2), including amino acid deletions and alterations of glycosylation sites. Recombination events were also observed in some isolates. These findings provide essential insights into the epidemiology and genetic diversity of PRRSV in Southwest China, contributing critical data for the development of more effective control and prevention strategies.