T. gondii is a globally prevalent intracellular parasite that poses significant public health challenges. However, its virulence mechanisms remain poorly understood. Here, we identified cleft lip and palate transmembrane protein 1 (CLPTM1, TGME49_205240) as a critical virulence factor and systematically characterized its role. The CLPTM1 deletion strain (Δclptm1) grows normally in vitro but completely loses virulence in vivo, with 100% survival of infected mice and no brain cyst formation. Serum IL-6 levels and tissue pathology in major organs were significantly reduced in Δclptm1-infected mice, indicating attenuated systemic inflammation and tissue damage. Transcriptomic analysis revealed that Δclptm1 infection markedly downregulated key chemokine genes (CCL5, CCR7 and CCL22) in macrophages. This trend was further supported by the reduced expression of these chemokines and decreased F4/80⁺ macrophage infiltration in liver and lung tissues. Concomitantly, diminished phosphorylation of IκB-α, along with decreased levels of p65 and its activated form pp65, suggests that CLPTM1 promotes chemokine expression by facilitating the activation of the NF-κB signaling pathway. Consistently, pp65 expression in liver and lung tissues was markedly reduced in the Δclptm1-infected group. Here, we delineate a mechanistic axis whereby CLPTM1 influences T. gondii virulence through the activation of the host NF-κB/chemokine/macrophage pathway, thereby promoting inflammation and immune cell infiltration. This study provides new insight into T. gondii pathogenesis and lays a foundation for the future development of diagnostic, therapeutic, and vaccine strategies against toxoplasmosis.

Lumpy skin disease (LSD), a highly contagious viral infection caused by Lumpy Skin Disease Virus (LSDV), has caused severe economic losses. We analyzed three representative outbreaks in 2024, combining clinical, histopathological, serological, and molecular data to refine regional control strategies. According to our findings, affected cattle presented with generalized cutaneous nodules, fever and peripheral edema; one-third of in-contact animals remained clinically normal yet seroconverted within ten days. Antibody titers peaked at approximately 20 d post-onset and were accompanied by persistent viral DNA in saliva, ocular secretions and skin scabs. Histology revealed alveolar septal edema, hepatocellular ballooning, and glomerular necrosis, whereas biochemical profiling revealed hypalbuminemia, hyperglobulinemia, and elevated alanine aminotransferase in one patient. Immunohistochemistry revealed intense LSDV antigen in the spleen, alveolar septa of the lung, and throughout the dermis of the skin nodules, identifying these tissues as the most reliable diagnostic targets. The combined findings confirm active viral replication in cutaneous, respiratory and lymphoid tissues, highlight the occurrence of subclinical infection and underscore the value of paired serology and PCR for herd screening. Prompt isolation, vector control and vaccination with homologous capripox vaccines are recommended to curtail transmission and economic impact.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating disease of domestic pigs with mortality rates approaching 100%, leading to severe global economic losses. No effective vaccines or antivirals are available, highlighting the urgent need for novel therapeutic strategies and efficient screening tools. We developed a recombinant ASFV-expressing dual reporter (mCherry and NanoLuc), rASFV_mChNluc, and established a high-content screening (HCS) platform optimized for cost-effective, low-labor analysis via the mCherry reporter. The assay demonstrated excellent robustness (Z′-factor = 0.669±0.064) and successfully verified the activity of the known ASFV inhibitor AraC, confirming inhibition at the postinfection stage. Screening of an in-house fungal extract library (493 extracts) identified 25 hits (5.07%) that reduced viral infectivity to < 5%. Extracts from the insect fungi Beauveria neobassiana and Samsoniella aurantia showed potent activity, with an SI > 62.81 (EC₅₀ = 1.99±0.71 µg/mL) and an SI > 42.92 (EC₅₀ = 11.65±2.99 µg/mL), respectively. Time-of-addition assays indicated that B. neobassiana acts at multiple replication stages, whereas S. aurantia targets the postinfection stage. This study establishes a robust ASFV HCS platform for efficient high-throughput antiviral discovery and highlights fungi as a promising source of novel ASFV inhibitors.

Nipah virus disease, caused by Nipah virus (NiV), is a zoonotic infectious disease with an extremely high fatality rate. Owing to the absence of effective countermeasures, Nipah virus disease has caused substantial economic losses in the swine farming industry. Vaccines represent the most cost-effective approach for prevention and control. Herein, we describe the development of a recombinant pseudorabies virus (PRV)-vectored vaccine (designated PRV-HNX-ΔTK/gE-NiV-G), which expresses the codon-optimized full-length glycoprotein of Nipah virus (NiV-G). The biological characteristics of this strain, including plaque morphology, growth kinetics, and genetic stability, were evaluated in vitro. Furthermore, the recombinant virus exhibited favorable safety profiles in mice. Intramuscular administration of PRV-HNX-ΔTK/gE-NiV-G elicited high-titer neutralizing antibodies against NiV-G and a robust cellular immune response in BALB/c mice. Taken together, these findings indicate that PRV-HNX-ΔTK/gE-NiV-G has potential as a promising candidate for the development of bivalent vaccines that target both NiV and PRV infections.

Feline panleukopenia virus (FPV) is a highly contagious parvovirus that causes acute gastroenteritis, leukopenia, and high mortality in felids. Although domestic cats are commonly vaccinated, FPV continues to threaten captive and wild felids because of its environmental stability, rapid progression, and potential antigenic drift. In June 2022, a one-year-old captive female cougar (Puma concolor) at Hongshan Forest Zoo, Nanjing, China, was found dead following a peracute course without prodromal signs. The animal had previously received a trivalent feline vaccine containing feline panleukopenia, herpesvirus, and calicivirus antigens. Postmortem examination revealed perianal fecal staining, oral discharge, pulmonary congestion with peripheral emphysema, a darkened liver, and intestinal mucosal sloughing with petechiae. Histopathology revealed crypt epithelial necrosis, mucosal sloughing, and lymphoid depletion, which was consistent with parvoviral enteritis. Fecal and intestinal samples tested positive for FPV according to the lateral-flow assay and PCR. Viral replication was confirmed in CRFK cells via indirect immunofluorescence, and FPV antigen was shown to be localized to the intestinal crypt epithelium via immunohistochemistry. VP2 gene sequencing (1,753 bp) revealed that the isolate clustered with field strains from domestic cats in Jiangsu and Shanghai, which share near-complete nucleotide identity but differ from the Felocell vaccine strain in three amino acid substitutions (A91S, I232V, and L562V), two of which lie in antigenic loops and may affect antigenicity. No further FPV cases were detected during or after the 14-day observation period, reflecting successful containment through disinfection, relocation, feral cat control, and movement restrictions. This represents the first confirmed fatal FPV infection in a captive cougar in China and highlights the potential for local spillover from domestic reservoirs. The case underscores the need for continuous molecular surveillance, vaccination evaluation, and One Health–based biosecurity to protect susceptible wildlife populations at the human–domestic–wildlife interface.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) has emerged as a critical pathogen that threatens swine herds across China. In this study, a novel PRRSV-1 strain, designated XJEU2308, was isolated from a PRRSV outbreak in a previously confirmed PRRSV-negative (both RNA and antibody negative) swine herd in Xinjiang, China. During the outbreak surveillance period, production records revealed a mean stillbirth rate of 12.19% and a suckling piglet mortality rate of 56.07%. Phylogenetic analysis on the basis of the ORF5 gene classified XJEU2308 as a BJEU06-1-like strain, whereas whole-genome analysis clustered it within the newly identified "New subgroup 1" of Chinese PRRSV-1. Notably, this strain carried a unique 3-amino acid deletion (at positions 693–695) in nonstructural protein 2 (NSP2). In challenge experiments, XJEU2308 induced typical clinical symptoms and exhibited moderate pathogenicity. Importantly, the implementation of the load-close-exposure (LCE) strategy combined with field virus (FLV) exposure successfully restored the herd to a provisional PRRSV-negative status. Overall, this study isolated a new subgroup 1-like PRRSV-1 strain from a swine farm that experienced a reproductive failure outbreak; the strain is characterized by a unique 3-amino-acid deletion in the NSP2 gene and moderate pathogenicity. Additionally, this study validated the effectiveness of the LCE-FLV strategy for containing PRRSV-1.

The broiler industry faces an urgent demand for the development of sustainable and antibiotic-free methods to combat widespread environmental and metabolic stressors, resulting in decreased performance of birds, compromised welfare, and substantial financial losses. This complete review focuses on the use of plant flavonoids (PFs) as a multicomponent antistress approach to address the common outcomes of all these stressors: systemic oxidative stress and gut barrier dysfunction. Through the detailed evaluation of over 300 scientific references, this review has demonstrated that PFs have dual-signaling pathway actions to protect animals against these stresses. The first action involves powerful antioxidant activity via the activation of the Keap1-Nrf2 signaling pathway, leading to the upregulation of phase II detoxification enzymes and cytoprotective enzymes (GPX and GST) to restore the cellular redox state. These enhanced defense systems are critical for protecting cells against chronic heat stress and for providing clearance mechanisms against highly toxic mycotoxins (Aflatoxin B1; Ochratoxin A). The second action of PFs is strong anti-inflammatory protection via the inhibition of the antagonistic NF-κB signaling pathway, which attenuates proinflammatory cytokine storms. This attenuation directly supports intestinal barrier function by promoting the expression of tight junction proteins in response to both heat stress and enterotoxic mycotoxins (T-2 toxin), thus preventing leaky gut syndrome. Additionally, PFs can be used to mitigate acute physiological challenges. By modulating the HPA axis, they can reduce the circulating levels of stress hormones (corticosterone/cortisol) in birds subjected to handling and transportation stress. Finally, the antioxidant properties of PFs penetrate muscle tissue, thereby preventing oxidation and stabilizing the myofibrillar protein structure postmortem. This action results in higher-quality poultry meat with lower drip loss. Although specific flavonoid compounds such as those found in Silphium perfoliatum L. may hold promise, the real value of PFs lies in their ability to act via these common downstream pathways (Nrf2/NF-kB) to position them as emerging, sustainable alternatives. Therefore, future research should prioritize controlled feeding trials and cost‒benefit analyses to quickly develop PF-based feed additives that improve global growth performance, decrease mortality, and increase the quality of poultry meat.

Porcine enteric viruses are major etiological agents of viral diarrhea in piglets, posing a serious threat to the sustainable development of the swine industry. To systematically characterize the epidemiological status and evolutionary dynamics of porcine enteric viruses in China, we collected 736 diarrheic pig samples between 2022 and 2025 from Hubei, Jiangxi, Shanxi, Guizhou and Tibet Provinces. Molecular epidemiological surveillance and virus isolation were performed for porcine epidemic diarrhea virus (PEDV), group A porcine rotavirus (PoRVA), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV). PEDV emerged as the predominant pathogen, with positivity rates increasing annually and reaching 70.75% in 2025. PoRVA ranked second, with a stable prevalence, whereas TGEV and PDCoV presented lower detection rates with regional variation. A total of six viral strains were successfully isolated, including two PEDV strains (HuB2023 and HuB2025), three PoRVA strains (HuB2023, JX2024 and SX2024) and one TGEV strain (JX2024). Phylogenetic analysis revealed that circulating PEDV evolved from G2a to G2c, with the detection of an S‑INDEL–like cluster. Notably, the PEDV HuB2025 represents a G2c recombinant strain harboring critical amino acid substitutions within the neutralizing epitopes of the spike protein. All PoRVA isolates belonged to the G9P[23] genotype, and whole-genome sequencing of PoRVA SX2024 confirmed strong homology with reported strains and defined its genotype constellation as G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1, highlighting intragenotypic diversity. The TGEV JX2024 isolate clustered within the Purdue subgroup with limited variation. Collectively, these findings delineate the evolutionary landscape of porcine enteric viruses in China and provide valuable reference strains for vaccine development and the optimization of control strategies.

Porcine Circovirus 3 (PCV3) has been detected in pigs worldwide, including Thailand. It is associated with a range of clinical problems, including reproductive disorders, but its pathogenic role remains unclear. To date, PCV3 detection in Thailand has been reported only in pigs with respiratory signs. An outbreak of stillbirths and mummified fetuses occurred at a farrow-to-nursery farm in 2023. Clinical assessment, farm management review, and molecular testing for African swine fever virus (ASFV), porcine parvovirus (PPV1, PPV2, PPV3, PPV4, PPV5, PPV6, and PPV7), porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2), PCV2, PCV3, and PCV4 were performed. PCV3 DNA was detected in all the samples, whereas other major reproductive pathogens were not detected. A positive PCV3 sample was subjected to full-genome sequencing and phylogenetic analysis. The complete genome (2,000 nt) of this strain shares 99.35%-99.70% identity with other PCV3 strains in the GenBank database and is classified into the genotypes PCV3a1 or PCV3b, similar to previously reported strains causing reproductive disorders in other countries. No significant environmental or nutritional stressors were identified. The findings of this study provide the first molecular evidence in Thailand, suggesting that PCV3 may be the cause of reproductive failure and possible vertical transmission. PCV3 should be considered in the differential diagnosis of reproductive disorders in swine production.

Hydatid disease, a neglected zoonotic infection caused by Echinococcus granulosus, remains hyperendemic in pastoral regions with limited resources underresourced veterinary surveillance systems. Consequently, deploying point-of-care diagnostic tools for the field surveillance of E. granulosus infection is critical for interrupting transmission chains and curbing echinococcosis dissemination. Here, we developed a rapid fluorescent immunochromatographic test strip using quantum dot (QD) nanobeads as signal reporters and antigen B as the capture probe. Compared with conventional techniques, this novel assay detects E. granulosus antibodies within 15 min, significantly reducing the time needed. Furthermore, it exhibits a sensitivity at least an order of magnitude greater than that of conventional colloidal gold-based lateral flow strips. More importantly, the results of our fluorescent immunochromatography test strips were strongly consistent with those of commercial ELISA kits. Owing to its rapid, sensitive, and user-friendly format, this test strip provides a practical point-of-care tool for field-based hydatid disease surveillance and control.

Salmonella strains carrying the bla_CTX-M-55 gene encoding the β-lactamase CTX-M-55 present markedly greater resistance to ceftazidime (CAZ) than those carrying the bla_CTX-M-14 gene encoding CTX-M-14, but the structural basis remains unclear. In this study, we combined susceptibility testing, binding affinity measurement, molecular dynamics simulation, and site-directed point mutation to examine how CTX-M-55 enhances CAZ hydrolysis. Compared with CTX-M-14, CTX-M-55 conferred an eightfold higher minimum inhibitory concentration for CAZ and exhibited more than tenfold stronger CAZ binding affinity. Structural and simulation analyses revealed that CTX-M-55 possesses a larger and more hydrophobic active pocket that supports more rapid interactions with CAZ and forms a more stable binding environment, driven mainly by favorable van der Waals and solvation energies. Mutation analysis further revealed two functionally distinct classes of residues contributing to CAZ resistance. Asp-131 and Asn-132 represent functional determinants of CAZ hydrolysis in CTX-M-55, as their substitution disrupts the structure of the binding pocket and hinders the effective binding of the substrate. In contrast, Ser-272 acts as an optimized residue that strengthens the hydrolytic capacity of CTX-M-55 compared with that of CTX-M-14 by modulating CAZ accommodation within a stable pocket. Together, these results indicate that the stronger CAZ resistance of CTX-M-55 results from both a favorable pocket structure and specific residue effects, with Asp-131 and Asn-132 providing the basic hydrolysis capacity and Ser-272 playing an optimized role for CAZ. This work helps clarify the differences in CAZ resistance among the CTX-M family and points to pocket features that may be useful targets for inhibitor design.

H9N2 avian influenza virus poses a persistent threat to poultry and public health because of its widespread circulation and role in generating novel viruses. To investigate the genetic determinants of HA antigenic diversity and adaptation, we constructed a hotspot library targeting 179 substitution hotspots identified through large-scale sequence analysis of 13,984 HA sequences distributed across the HA protein. The highly variable residues 145 and 168 represent prominent substitution hotspots that correlate with host-specific lineages and highlight poultry as a key adaptation reservoir. The library was subjected to replication in multiple host-cell systems, as well as immune and thermal selection. Functional screening revealed that N115H enhances viral replication in mammalian cells, residue 164 is critical for antigenic escape, and I134L, L230S, and V306L collectively increase HA thermostability. These results link natural sequence variation to functional phenotypes, demonstrating that H9N2 HA antigenic diversity and cross-species potential are shaped by mutation hotspots. Our integrated computational and experimental framework provides a platform for probing viral fitness landscapes, with implications for risk assessment and the rational design of next-generation vaccines.

Florfenicol (FFC) has become a significant antibiotic in the global aquaculture industry, particularly in the treatment of bacterial infections in finfish and shellfish. FFC is valued for its broad-spectrum bacteriostatic activity and effectiveness. This comprehensive review consolidates and expands upon existing research, offering in-depth information on FFC application across various aquaculture practices. This review addresses the key pharmacokinetic aspects of FFC, such as its absorption, distribution, metabolism, and excretion, in aquatic species. Significant attention is given to the safety profile of FFC, with discussions on residue accumulation, elimination dynamics, and the established withdrawal periods necessary to ensure consumer safety. Furthermore, this review highlights emerging issues related to FFC in aquaculture, particularly with respect to plasmid-mediated resistance, the dispersal of resistant bacteria and resistance genes, and their impact on the aquatic environment and nontarget organisms, laying a foundation for future research in this critical area. This evaluative assessment offers a novel contribution to the field, serving as an essential reference for ongoing and future studies on the appropriate use and prevention of FFC resistance in aquatic animal medicine.

Necrotic enteritis (NE), primarily caused by Clostridium perfringens, remains a major challenge to poultry production worldwide, resulting in substantial economic losses and compromised animal welfare. Historically, antibiotics have been widely used to control NE; however, increasing concerns regarding antimicrobial resistance and regulatory restrictions have accelerated the search for effective nonantibiotic alternatives. This review synthesizes current knowledge on nutritional interventions aimed at mitigating NE in poultry production systems. The multifactorial nature of NE is discussed, highlighting the interactions among diet composition, the gut microbiota, intestinal integrity, and host immune responses. The key dietary strategies evaluated included probiotics, prebiotics, synbiotics, phytogenic compounds, enzymes, organic acids, functional lipids, immunomodulators, bacteriophages, antioxidants, dietary fibers, and optimized protein and amino acid nutrition. These interventions influence gut health through multiple mechanisms, including modulation of microbial communities, enhancement of epithelial barrier function, reduction in pathogenic bacterial proliferation, and regulation of host immune responses. Evidence from experimental and field studies indicates that many of these strategies can reduce C. perfringens colonization, improve intestinal morphology, and enhance growth performance in broilers. However, the effectiveness of individual interventions may vary depending on diet composition, management practices, and production conditions. Therefore, integrated nutritional strategies combined with effective management and biosecurity practices are likely to provide the most reliable approach for NE control in antibiotic-reduced poultry production systems. This review highlights key nutritional approaches and emerging research directions that may support sustainable NE control and improved gut health in modern poultry production.

Goats are important livestock in China, and their close contact with humans raises concerns about zoonotic enteric parasite transmission. However, data on the prevalence and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Hubei goat populations remain limited. Five hundred fecal samples were collected from goats across five regions of Hubei Province (2024–2025). Nested PCR and sequencing were used to detect and characterize Cryptosporidium spp., G. duodenalis, and E. bieneusi. Risk factors were also evaluated. The prevalence rates were 3.2% for Cryptosporidium spp., 29.4% for G. duodenalis, and 16.2% for E. bieneusi. The dominant species/assemblage/genotypes included C. bovis, C. xiaoi, C. parvum, and C. ubiquitum; assemblage E (85.7%) for G. duodenalis; zoonotic assemblages A and B; and CHG3 (25.9%), BEB6 (23.5%), and CHG1 (21.0%) for E. bieneusi. Significant regional variation was detected for G. duodenalis and E. bieneusi infections, which were significantly greater in the winter than in the spring. Conclusion: This first comprehensive molecular survey in Hubei goats revealed a high prevalence and genetic diversity of these parasites, with clear evidence of zoonotic strains. The findings support a “One Health” approach to control zoonotic transmission in the region.

Bovine nebovirus (BoNeV), a single-stranded positive-sense RNA virus belonging to the Caliciviridae family, genus Nebovirus, has a genome length ranging from 7453 to 7460 nt. Initially, identified in 1978 within fecal samples from calves experiencing diarrhea in the United Kingdom, BoNeV was subsequently detected in beef cattle, cows, and yaks across 13 countries spanning four continents, thereby demonstrating its extensive global distribution. Although a stable cell culture system for BoNeV is not yet available, its pathogenicity in calves has been confirmed through experimental infection studies. With the increasingly severe losses caused by viral diarrhea to calves, research on this virus has attracted widespread attention. This article reviews the latest research on the molecular epidemiology, genome structure, genetic evolution, recombination, detection methods, and pathogenesis of BoNeV. In summary, BoNeV has a high prevalence and coinfection rate, and its gene sequence is characterized by genetic diversity and a high recombination frequency. Different evolutionary subtypes of strains exhibit lower amino acid homology and structural differences. This review provides important insights for advancing future genetic evolution research and prevention strategies related to BoNeV.

The giant panda (Ailuropoda melanoleuca) is a flagship species for biodiversity conservation, yet the viral communities inhabiting its upper respiratory tract (URT) remain poorly characterized. In this study, we performed viral metagenomic sequencing on 13 pooled libraries derived from 130 nasopharyngeal swabs collected from giant pandas between 2018 and 2021. The assembly yielded 16 complete or near-complete viral genomes, predominantly of DNA viruses belonging to the families Papillomaviridae, Genomoviridae, and Parvoviridae. Notably, we identified the complete genome of a novel Parvoviridae species within the subfamily Densovirinae. Phylogenetic analysis revealed that this virus clusters with invertebrate-infecting viruses, suggesting that it likely represents an arthropod-associated viral element derived from respiratory parasites rather than a direct vertebrate pathogen. Although the alpha diversity remained stable, beta diversity analysis revealed significant temporal shifts in viral community composition (P = 0.02). This study provides the first systematic characterization of the giant panda URT virome, establishing a critical baseline for disease surveillance and highlighting the complex interplay between the host and its environment-associated viral elements.

Host genetic background plays a decisive role in susceptibility to pathogens, and hybrid offspring often exhibit heterosis that surpasses parental phenotypes. However, the underlying regulatory mechanisms remain poorly understood. In this study, we generated an F1 hybrid mouse model by crossing highly susceptible BALB/c mice (maternal strain) with resistant C57BL/6 mice (paternal strain) to systematically investigate the immune regulatory strategies underlying heterosis during Mycobacterium tuberculosis infection. Our results demonstrate that F1 hybrid mice display pronounced heterosis following infection, characterized by a significantly reduced pulmonary bacterial burden, attenuated pathological damage, and enhanced infiltration of innate immune cells, particularly natural killer cells. Transcriptome sequencing (RNA-seq) revealed that F1 hybrids integrate parental genetic information via parent-of-origin effects: maternally expressed genes were predominantly enriched in signal transduction and immune dysregulation pathways, whereas paternally expressed genes were significantly associated with metabolic processes and homeostatic maintenance. Furthermore, by integrating padlock probe based single-nucleotide polymorphism (SNP) in situ hybridization, we demonstrate that this allelic expression bias is highly cell type specific. Notably, in infection-recruited macrophages and neutrophils, key genes such as Plin2 (lipid metabolism) and Tap2 (antigen presentation) exhibited a pronounced paternal allele biased expression pattern. Collectively, our findings indicate that hybrid offspring achieve coordinated optimization of immune defense and metabolic homeostasis by preferentially utilizing paternally derived genetic information within specific innate immune cell populations. This study provides new insights into the genetic basis of infectious disease susceptibility and the molecular mechanisms underlying heterosis.

Through structure-based virtual screening, this study identified the botanical compound fangchinoline (FAN) as an inhibitor of the porcine aminopeptidase N (pAPN) receptor and evaluated its anti-transmissible gastroenteritis virus (TGEV) activity, mechanism, and safety. FAN exhibited potent antiviral efficacy against TGEV in PK-15 and IPEC-J2 cells (EC₅₀ = 2.862 μM and 3.670 μM, respectively), significantly reducing viral RNA copies and N protein expression while suppressing the upregulation of the inflammatory cytokines IL-6 and TNF-α. It also showed broad-spectrum potential by inhibiting PEDV (EC₅₀ = 2.243 μM). Mechanistic studies using a cellular thermal shift assay (CETSA) and drug affinity-responsive target stability (DARTS) confirmed that FAN can bind to pAPN, affecting its thermal and enzyme stability. Time-of-addition assays indicated that FAN interferes with both the early (attachment and internalization) and late replication phases of TGEV. In acute toxicity tests, FAN showed low toxicity (LD₅₀ in mice: 2060.23 mg/kg for males, 1481.93 mg/kg for females) and no mutagenic potential in the Ames test. Favorable pharmacokinetic parameters were also observed in rats. In conclusion, FAN exerts its anti-TGEV effect by targeting the critical pAPN receptor. Its plant origin, potent and broad-spectrum antiviral activity, elucidated mechanism, and promising safety profile support its further development as a lead compound for anti-coronavirus strategies.

Coccidiosis caused by Eimeria spp. remains a major constraint to poultry production worldwide, and the discovery of new anticoccidial agents is hindered by the lack of scalable in vitro screening systems. Here, we developed a phenotypic high-throughput screening (HTS) platform based on a transgenic E. tenella line coexpressing enhanced yellow fluorescent protein (EYFP) and nanoluciferase (Nluc), enabling sensitive and quantitative assessment of parasite growth in vitro. Using Madin-Darby bovine kidney (MDBK) cells as host cells, parasite replication was reliably monitored within a 45 h infection window via a luminescence readout. The assay exhibited strong linearity with the parasite inoculum and was validated using known anticoccidials, monensin and salinomycin, whose expected dose–response profiles were obtained. The platform was applied to screen 1,770 compounds, identifying 28 primary hits (> 50% inhibition). After the samples were filtered for their cytotoxicity and drug-like properties, three compounds (alendronate, pemetrexed, and torsemide) were selected for further evaluation. These compounds exhibited micromolar in vitro activity (EC₅₀ ~ 8–10 μM) with favorable selectivity indices, particularly for pemetrexed and torsemide (> 100). In a chicken infection model, compared with the vehicle control, all three compounds significantly reduced oocyst shedding. Using the anticoccidial index (ACI), a composite metric incorporating oocyst shedding, mortality, weight gain, and lesion severity, torsemide was most effective (ACI = 163.3), followed by pemetrexed (ACI = 153.5). Alendronate showed only marginal activity (ACI = 110.8). Overall, this study establishes a robust, scalable in vitro HTS platform for anticoccidial drug discovery and highlights several approved drugs as potential leads for further optimization.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major immunosuppressive pathogen involved in the porcine respiratory disease complex. Licorice extract (LE) has anti-inflammatory, immune-regulating, and growth-promoting effects. In this study, the use of LE as a novel feed additive in PRRSV-infected nursery pigs was evaluated, and the effects of dietary supplementation and drinking water were compared. A total of 264 weaned piglets were allocated into 8 groups: three dietary supplementation groups (low, medium, and high doses: BLL, BLM, and BLH), three drinking water supplementation groups (low, medium, and high doses: WLL, WLM, and WLH), a farm control group (Farm), and a negative control group (CON). Compared with the CON group, dietary LE supplementation in the BLL and BLM groups significantly increased the average daily gain (ADG) by 1.02-fold and 1.18-fold (P < 0.001) and reduced the feed-to-gain ratio (F/G) by 3.58% and 5.35% (P < 0.05), respectively, compared with their drinking water counterparts, with the effects in the BLM group being the most pronounced. Additionally, compared with CON, LE administration via either route alleviated coughing, with significant downregulated (P < 0.001) of cough scores in the BLH, WLL, and WLH groups during weeks 3–4. Furthermore, compared with the CON group, the BLM group exhibited increased tracheal cilia integrity (P < 0.1) and significantly decreased (P < 0.05) lung injury scores. While LE did not alter PRRSV-specific antigen or antibody levels (P=0.065), it significantly modulated immune responses by downregulating IL-1β and IL-6 (P < 0.05), upregulating IL-10 (P < 0.05), and increasing IgA, IgG, and IgM levels (P < 0.05). Gut metagenomic analysis revealed that LE notably increased the abundance of multiple beneficial bacteria and reshaped the structure of the gut microbiota. Specifically, LE upregulated Anaeromassilibacillus sp. An172 and reversed the antibiotic-induced decrease in the abundance of Bacteroides bouchesdurhonensis and Bacteroides sp. OF04‑15BH. These findings demonstrate that dietary LE, at an optimal dose of 2.6 g/kg, is a promising feed additive for PRRSV-infected pigs, with benefits mediated through immune modulation and gut microbiota remodeling.

Parainfluenza virus type 5 (PIV5) can infect a variety of animals and can lead mainly to respiratory symptoms. In recent years, PIV5 has been frequently detected in swine fecal samples with diarrhea symptoms, but its role in pig diseases remains unclear. In this study, a swine PIV5 strain, CHN-SCMY2025, was isolated from diarrheal piglets in Sichuan Province, Southwest China. The cytopathic effect of the isolate was systematically characterized by electron microscopy and immunofluorescence. The results demonstrated that CHN-SCMY2025 is approximately 150–250 nm in diameter and has a morphology typical of parainfluenza virus. In addition to Vero cells, CHN-SCMY2025 exhibits broad cellular tropism and can replicate in BHK-21, LLC-PK, IPEC-J2, ST, and 293 T cells. Sequence alignment based on the NP (PX583268.1), HN (PX583267.1), F (PX583266.1), M (PX963773.2), and L (PZ011079.1) of CHN-SCMY2025 was determined, which shares 95.83–99.82%, 96.53–99.88%, 96.67–100%, 95.51–99.21%, and 97.89–99.78% nucleotide identity with other PIV5 reference strains in GenBank, respectively. Phylogenetic analysis revealed that CHN-SCMY2025 is closely related to the swine PIV5 isolate (KC237064.1). In vivo infection demonstrated that CHN-SCMY2025 could induce mild to moderate respiratory symptoms and mild diarrhea in suckling piglets. The virus was shed daily in the feces of infected suckling piglets and upon necropsy. The virus was distributed in multiple organs, but relatively high viral loads were detected mainly in the lung, duodenum, and jejunum. CHN-SCMY2025 is the first systematically characterized swine PIV5 strain from southwestern China. Our results provide information on epidemiology, pathogenicity, and interspecies transmission associated with swine PIV5.

Bovine brucellosis remains endemic in Bangladesh and poses significant public health and economic risks. Traditional risk factor analyses predominantly employ binary outcome classifications, failing to capture the spectrum of serological states from healthy through latent infection to active disease. In this study, a Bayesian mixed-effects multinomial regression was applied to identify risk factors associated with latent and disease states, and a linear mixed-effects model was used to quantify milk yield loss. A cross-sectional study was conducted across 17 large-scale dairy herds in Bangladesh (n = 2,696). Milk samples from 2,696 lactating cows were analyzed using indirect ELISA (iELISA). On the basis of Bayesian mixture model thresholds, the animals were classified as healthy (S/P% < 10.64), latent (10.64 ≤ S/P% ≤ 82.0), or diseased (S/P% > 82.0). Bayesian mixed-effects multinomial logistic regression with herd-level random intercepts was implemented in Stan to identify risk factors. Milk yield was further analyzed using a linear mixed-effects model to quantify the magnitude of production loss associated with brucellosis status. Retained placenta was the strongest predictor for both latent (RRR = 5.82) and disease (RRR = 11.81) states. High parity (3rd–5th calf) was significantly associated with active disease. Anestrous increased latent risk but not disease status. Higher milk yield was inversely associated with both states. The model showed excellent discrimination for disease classification (AUC = 0.90) and moderate discrimination for latent status (AUC = 0.74). Compared with healthy cows, latent and diseased cows produced 0.53 L/day and 1.01 L/day less milk, respectively. These findings demonstrate distinct epidemiological risk patterns and measurable production losses associated with different brucellosis states, informing targeted surveillance and culling strategies.