Varicella-Zoster Virus (VZV) infection remains a globally prevalent disease, posing serious health risks, particularly to the elderly and immunocompromised populations. Traditional serological detection methods are often time-consuming and operationally complex, making them unsuitable for the need for rapid screening in primary healthcare settings. Quantum dot-based fluorescent immunochromatographic technology, with its high sensitivity, strong photostability, and operational simplicity, provides a promising approach for the rapid detection of VZV antibodies. In this study, we expressed and purified the recombinant VZV gL protein, screened high-affinity monoclonal antibodies, and constructed a quantum dot-labeled immunochromatographic test strip. Validation using 22 clinical samples demonstrated that the method exhibits high specificity and sensitivity, with a detection limit of 1:10⁶. The concordance rate with a commercial ELISA kit was 95.5%, offering a reliable technical tool for the rapid diagnosis of VZV infection and assessment of immune status.

The high genetic diversity of infectious bronchitis virus (IBV) poses a significant challenge for broad‐spectrum detection. To address this, we developed a rapid, specific, and visual detection platform by integrating recombinase‐aided amplification (RAA) with CRISPR/Cas13a mediated collateral cleavage, targeting the conserved viral nucleocapsid (N) gene. Following optimization of key reaction parameters, the assay achieved a limit of detection (LOD) of 1 copy/μL. It demonstrated broad‐spectrum detection capability against major circulating IBV lineages in China, including QX (GI‐19), TW (GI‐7), 4/91 (GI‐13), LDT3 (GI‐28), and Mass (GI‐1), while maintaining high specificity with no cross‐reactivity against other common avian pathogens such as infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), and avian influenza virus (AIV). Clinical validation using 38 field samples showed 100% concordance with conventional RT‐PCR. The entire detection process is completed within 50 minutes, with results detectable via real‐time fluorescence or directly visualized under blue light. This study provides a sensitive, specific, and field‐deployable tool for point‐of‐care testing (POCT) of IBV, offering a practical solution for timely disease management.

In recent years, food safety concerns associated with veterinary drug residues have aroused significant public attention, highlighting the critical need for continuous monitoring in both animal-derived foods and the environment to safeguard public health. Medroxyprogesterone acetate (MPA), a major anabolic steroid widely used in animal husbandry, poses potential human health risks according to recent studies. Although conventional analytical techniques based on chromatography and immunological assays are effective, they often suffer from complex operations, the need for skilled personnel, high cost, and time consumption. To overcome these limitations, electrochemical immunosensors modified with advanced nanomaterials offer a promising alternative. Herein, a highly sensitive electrochemical immunosensor was developed for trace MPA detection utilizing core–shell SiO2-PEI@Ag nanoparticles as the sensing matrix. In this nanocomposite, the structural synergy between the SiO2 core and outer Ag NPs shell not only provides a high-density platform for specific antibody immobilization, but also drastically expands the electroactive surface area and accelerates electron transfer to maximize signal amplification. Benefiting from these synergistic advantages, the constructed immunosensor exhibited a limit of detection (LOD) of 0.014 ng/mL and a wide linear range of 0.03–100 ng/mL. Furthermore, the recovery rates in pork and milk samples were 91.32–97.29% and 95.14–102.54%, respectively. In addition, negligible cross-reaction was observed toward structurally related steroid hormones related to MPA and common antibiotics. The immunosensor shows good stability, reproducibility, and accuracy, demonstrating great potential as a promising candidate to complement conventional tools for future MPA residue screening.

African swine fever (ASF) is a highly contagious and virulent infectious disease caused by infection of pigs with the double-stranded DNA virus African swine fever virus (ASFV). The p72 protein is the major component of the ASFV icosahedral capsid, comprising approximately 32% of the viral particle, and is essential for viral adsorption and cellular entry. In this research, we expressed the p72 protein using a eukaryotic system, and it exhibited strong reactivity with ASFV-positive porcine serum. We developed eight monoclonal antibodies using hybridoma technology, six of which recognized the native conformation of the p72 protein. Using truncated protein mapping, we identified two linear B-cell epitopes, designated 138PRNGYDWDNQTPLEGA153 and 291GKQDITPITDATYLD305, and defined residues W144, D145, Q147, T148, and Q293, D294, I295 as critical for antibody binding via alanine-scanning mutagenesis. Notably, the epitope 138PRNGYDWDNQTPLEGA153 was identified for the first time using truncated protein mapping. These results expand the known epitope repertoire of the ASFV p72 protein, providing a foundation for advancing ASF diagnostic tools and studies of immune recognition.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a porcine enteropathogenic coronavirus, causes acute diarrhea, vomiting, and death in newborn piglets, leading to economic losses in the pig industry. Previous studies have shown that SADS-CoV can infect a variety of mammalian cell lines, including human-derived cells, indicating its potential public health risk of cross-species transmission. In this study, we successfully expressed the SADS-CoV S1 protein using a eukaryotic expression system and screened monoclonal antibodies 1B8 and 3C9. Antibody 1B8 was used as a detection antibody coupled with quantum dots to synthesize a fluorescent probe, while 3C9 was used as a capture antibody. The assembled fluorescent immunochromatographic strip reacted well with SADS-CoV. The detection limit of this test strip for the S1 protein is 0.78 ng mL-1, and the detection limit for SADS-CoV is 102 TCID50 per mL. No cross-reactions were observed with three common porcine enteric coronaviruses, namely porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Moreover, in the detection of newborn piglets' fecal clinical samples, the results of the test strip were highly consistent with those of quantitative fluorescent PCR. The results indicate that the detection method established in this study can be used for the rapid screening and diagnosis of SADS-CoV infection in pig herds.

Apramycin (APR), a widely used broad-spectrum aminoglycoside antibiotic, poses potential risks to human health due to residue accumulation in animal-derived foods. Therefore, high-throughput rapid detection methods with high sensitivity and low cost advantages are urgently needed to implemented to safeguard public health. In this study, high-affinity and target-selective monoclonal antibody (mAb) against APR was developed, and a highsensitivity quantum dot-based direct competitive fluorescence-linked immunosorbent assay (dc-FLISA) was established for rapid APR detection. The dc-FLISA demonstrated a 50 % inhibition concentration (IC50) of 3.91 ng/mL, a limit of detection (LOD) of 0.38 ng/mL, and a linear range of 0.68-22.39 ng/mL. Recoveries from spiked food samples ranged from 94.3 % to 108.3 %, with coefficients of variation (CV) less than 10 %. This established method combines high sensitivity, cost-effectiveness, and rapid detection, offering a reliable technical solution for large-scale detection and regulatory compliance monitoring of APR livestock-derived food products, ultimately safeguarding consumer health.

The high incidence of swine enteric coronavirus co-infections necessitates highly specific diagnostic tools targeting key viral antigens, such as the porcine epidemic diarrhea virus (PEDV) nucleocapsid (N) protein. In this study, we generated monoclonal antibodies (mAbs) against the recombinant PEDV N protein and mapped a novel, highly conserved linear B-cell epitope, 272-ERDLKDIPEWR-282. To define the molecular basis of this interaction, alanine scanning mutagenesis identified residues Leu275, Lys276, Asp277, Ile278, Glu280, Trp281, and Arg282 as the core motif essential for mAb recognition. Furthermore, structural modeling predicted that this epitope adopts an exposed α-helix and flexible loop conformation. Crucially, sequence alignments revealed poor conservation of this epitope across other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV). Subsequent in vitro validation via indirect immunofluorescence assays (IFA) and ELISA confirmed that the targeting mAb, 1D1, exclusively recognizes PEDV antigens, exhibiting no detectable cross-reactivity with TGEV or PDCoV. These findings elucidate the fine antigenic profile of the PEDV N protein and highlight mAb 1D1 and its corresponding epitope as promising candidates for the differential diagnosis of major swine enteric coronaviruses.

Rabies virus (RABV), a single-stranded RNA virus, invades the central nervous system and causes acute, zoonotic diseases worldwide. The glycoprotein (G) of RABV is the only surface antigen responsible for inducing potent neutralizing antibodies, making it a key target for medical countermeasures. In this study, two anti-G protein monoclonal antibodies (mAbs) were prepared using hybridoma cell technology, with their high specificity and robust reactivity validated by indirect enzyme-linked immunosorbent assay (i-ELISA), indirect immunofluorescence assay (IFA) and Western blot. Through systematic epitope mapping using a series of truncated G protein fragments, two mAbs (1G2 and 4E5) were found to recognize a minimal linear epitope, AEDFVEVHLP (amino acids 412–421) through Dot-ELISA and i-ELISA. Alanine-scanning mutagenesis further defined the core binding residues within this epitope as 412Ala-Glu-Asp-Phe-Val416, 418Val, 420Leu, and 421Pro. Bioinformatic analysis with MEGA software revealed highly conserved with conservative substitutions of this epitope across diverse RABV of genotype 1. Homology modeling with SWISS-MODEL localized the epitope “412AEDFVEVHLP421” to a solvent-exposed α-helical conformation on the G protein surface. And this epitope is located in the extracellular C-terminal region, Prior studies have demonstrated that this region plays a role in stabilizing the trimerization of the rabies virus glycoprotein (RVG). In conclusion, our study identifies a novel, highly conserved with conservative substitutions linear B-cell epitope on the RABV glycoprotein, providing a precise molecular target for the rational design of next-generation epitope-based vaccines and specific diagnostic tools.

The endoplasmic reticulum (ER) maintains protein-folding homeostasis and initiates antiviral responses via the unfolded protein response (UPR). Porcine epidemic diarrhea virus (PEDV) has evolved to exploit this process through its nonstructural protein 3 (NSP3). Here, we identify NSP3 as a multifunctional deubiquitinase that remodels ER stress signaling and redox homeostasis to facilitate viral replication and immune evasion. NSP3 suppresses GRP78 expression while selectively activating PERK-eIF2α-ATF4 and IRE1α-XBP1s pathways but inhibits ATF6 nuclear translocation. Mechanistically, NSP3's PLP2 domain removes K48- and K63-linked ubiquitin chains from KEAP1, IRE1α, and ATF6, thereby fine-tuning their activity. Deubiquitination of KEAP1 promotes KEAP1-CUL3 complex stability and enhances NRF2 degradation, disrupting the antioxidant axis (NRF2-NQO1-HMOX1) while activating the ATF4-ERO1A-NOX4 pathway to elevate reactive oxygen species (ROS) levels without cytotoxicity. Functionally, activation of the PERK-NRF2 axis suppresses PEDV replication, whereas inhibition of either component enhances viral growth, highlighting a cooperative antiviral role. In contrast, activation of the IRE1α-XBP1s branch promotes replication but can be antagonized by NRF2 activation, indicating crosstalk between these pathways. Although ATF6 alone has minimal impact, its modulation by NRF2 shapes viral replication outcomes. Collectively, our findings reveal that PEDV NSP3 orchestrates differential deubiquitination of ER stress sensors to manipulate UPR-ROS signaling and redox balance, thereby subverting host defenses and promoting viral survival, providing mechanistic insight into coronavirus-host interactions and potential antiviral targets.

African swine fever in both domestic and wild pig populations is caused by the extremely infectious African swine fever virus (ASFV). It seriously endangers biodiversity and results in large financial losses for the worldwide pork sector. The major capsid protein p72 is molecularly chaperoned by the ASFV pB602L protein, which is essential to viral assembly. Furthermore, as a nonstructural protein expressed at late stages of infection, pB602L induces a distinct antibody response that may complement existing serological assays based on structural proteins. Given its strong immunogenicity, pB602L represents a promising antigen for developing supplementary diagnostic tools for African swine fever (ASF). In this study, we successfully generated and separated the ASFV pB602L protein, and we verified its responsiveness using serum from pigs infected with ASFV. Additionally, we produced four monoclonal antibody-specific hybridoma cell lines that targeted the pB602L protein exclusively. These cell lines demonstrated high immunoreactivity and responsiveness toward ASFV pB602L. These results highlight the potential enhancement of diagnostic skills. We have detected two previously unknown linear B-cell epitopes (138TIDSFL143 and 164TNVDTC169) using overlapping peptide and truncated protein fragment analysis. Due to their high degree of conservation across various ASFV strains, these epitopes offer trustworthy candidates for the creation of particular diagnostic instruments. This study expands the known ASFV antigenic repertoire by systematically mapping immunodominant epitopes of pB602L. The identified epitopes provide potential molecular targets for the rational design of multi-epitope subunit vaccines.

Rabies is a fatal zoonotic disease caused by rabies virus (RABV), resulting in approximately 59,000 deaths annually worldwide and posing a serious threat to public health. The RABV phosphoprotein (P protein) plays crucial roles in viral replication, transcription, and immune antagonism; however, its immunogenic properties have not been fully characterized. In this study, the RABV P protein was expressed in an Escherichia coli expression system and used to immunize mice, resulting in the generation of six P protein-specific monoclonal antibodies (mAbs). Using an overlapping peptide-based truncation strategy, two linear B-cell epitopes were identified: 52DMKRLHLDDEKSSNL66 and 177VAPGPPALEWSATNE191. Alanine-scanning mutagenesis revealed that residues D52, M53, R55, L56, and L58 were critical for the recognition of epitope 52DMKRLHLDDEKSSNL66 by mAbs 2D2 and 18G7. Residues G180, P181, and W186 were essential for recognition of epitope 177VAPGPPALEWSATNE191 by mAbs 3D7, 15D8, 16D3, and 16C6. Notably, although some amino acid residues within epitopes P1-5 and P4-2 exhibited high variability among representative RABV strains, the critical residues recognized by these monoclonal antibodies were highly conserved. These findings provide new insights into the antigenic structure of the RABV P protein and may contribute to future studies on its functional characterization, as well as the development of P protein-based diagnostic reagents and subunit vaccines.

Infectious bursal disease (IBD) is a highly contagious avian disease caused by infectious bursal disease virus (IBDV), mainly affecting the bursa of Fabricius immune organ in young chickens. The virus has heat and acid resistance and can be spread through contaminated environments, feed and insects, leading to immunosuppression and increasing the risk of secondary infections. Affected chickens often show symptoms such as diarrhea, tremors and dehydration. Chickens aged 3-6 weeks are the most susceptible, with an incidence rate of up to 100% and a mortality rate of 5-30%. Therefore, from the perspective of infection control, effective monitoring and prevention of IBDV are of vital importance. The quantum dot fluorescence immunochromatographic strip established in this study can complete the specific detection of infectious bursal disease in chickens within 15 min, with a detection limit of 102.5 TCID50/mL for infectious bursal disease virus liquid. It does not cross-react with avian influenza viruses (H9N2, H10N8) and infectious bronchitis virus. This test strip can rapidly and accurately detect infectious bursal disease virus, providing a valuable tool for on-site detection.

The NF-κB pathway is an important signal for the body to respond to viral invasion and produce inflammatory responses. African swine fever virus (ASFV) can not only activate the host NF-κB signal when invading the host but also regulate this signal through its own encoded viral proteins. In this study, we find pH108R, an inner envelope protein of ASFV, can significantly downregulate NF-κB activation induced by IL-1β or TNFα. The overexpression of pH108R suppressed transcription of several proinflammatory cytokines such as IL-6, IL-8 induced by IL-1β or TNFα. Consistently, the levels of phosphorylated p65 and IκBα were decreased in pH108R overexpression cells after IL-1β and TNFα stimulation. In addition, transcriptome sequencing showed that it could significantly downregulate the expression of TNFα-induced immune and inflammatory genes in PK-15 cells. Collectively, our study indicates that ASFV pH108R markedly suppresses NF-κB activation stimulated by IL-1β or TNFα, clarifies the immunosuppressive activity of pH108R, and provides a new perspective for analyzing the immune escape of ASFV.

Pseudorabies virus (PRV), a zoonotic alphaherpesvirus threatening swine industries and human health, exploits glycoprotein D (gD)-nectin-1 interactions for host cell entry. Here, we report a dual-target neutralizing strategy combining a novel gD-specific monoclonal neutralization antibody (3F7) with nectin-1-blocking mAb. In vitro neutralization assays demonstrated that 3F7 alone achieved potent cross-species inhibition of PRV infection in porcine kidney cells (PK15), human neuroblastoma cells (SK-N-SH), and human embryonic kidney cells (HEK 293 T) (IC50: 0.56-5.39 μg/mL). Co-administration with nectin-1-targeting mAb synergistically enhanced neutralization efficacy by 0.9-25.8 %, revealing a cooperative mechanism between viral glycoprotein blockade and host receptor interference. This study presents the initial evidence indicating that antibodies targeting both the pathogen and its host entry factor, nectin-1, significantly enhance the anti-PRV activity. These findings highlight a novel and effective strategy for mitigating zoonotic PRV transmission, especially among high-risk populations such as swine workers. Moreover, the study provides a promising avenue for the development of next-generation biologics against emerging zoonotic herpesviruses, offering significant implications for both veterinary and human public health.

African swine fever virus (ASFV) is the only member of the family Asfarviridae and can cause African swine fever, a disease with a consistently high mortality rate. The pE248R protein, a myristoylated integral membrane protein of ASFV, is required for virus infectivity and some early postentry event, making it a key target for studying the prevention and treatment of ASFV. In this study, BALB/c mice were immunized with purified recombinant pE248R protein, leading to the generation of five monoclonal antibodies (mAbs). Selected mAbs were subsequently subjected to further characterization. By identifying the reactivity of different pE248R protein peptide segments with these monoclonal antibodies, we screened and identified a linear B cell epitope (87QEVALTQWMDAG98) on the pE248R protein. These results provide a new theoretical basis for analyzing the structure and function of pE248R protein, particularly contributing to the construction of a comprehensive B-cell epitope map for ASFV immunogens.

The production of carbapenemase is the primary mechanism of bacterial resistance to carbapenem antibiotics. This resistance seriously compromises the efficacy of carbapenem antibiotics in treating infections and poses a significant challenge to clinical anti-infective therapy. Oxacillinase-48 (OXA-48) is a class D carbapenemase, whose genes are usually located on transferable elements and can spread between different strains and genera. Therefore, from the perspective of infection control, effective monitoring and prevention of OXA-48 are imperative. In this study, CdSe/ZnS quantum dots (QDs) were coupled with an anti-OXA-48 monoclonal antibody (mAb) as a fluorescent signal probe to establish a quantum dot-based fluorescent immunochromatographic strip. The test strip contains a Test line (T-line) coated with an anti-OXA-48 monoclonal antibody and a Control line (C-line) coated with Staphylococcus protein A (SPA). Based on a double-antibody sandwich detection mode, it can complete highly sensitive detection of OXA-48 within 10 min. The visual detection limit of this test strip for OXA-48 recombinant protein was 3.13 ng/mL, and there was no cross-reaction with other common carbapenemases (IMP-1, NDM-1, KPC-2, VIM-2, OXA-23). It can be positioned as a rapid and reliable OXA-48 detection tool, providing a novel method for the rapid identification of OXA-48-producing resistant bacterial strains in clinical practice.

A novel electrochemical immunosensor for the sensitive detection of monensin (MON) was developed. The sensor platform utilized anti-MON monoclonal antibodies and a nanocomposite (Ce-MOF@AgAuNPs), composed of cerium-based metal-organic frameworks decorated with silver-gold nanoparticles. The physicochemical properties of the Ce-MOF@AgAuNPs nanocomposite were investigated using transmission electron microscopy, energy dispersive X-ray, UV-Vis spectrophotometry, and fourier transform infrared techniques. The stepwise fabrication process and electrochemical performance of the immunosensor were investigated through electrochemical impedance spectroscopy and cyclic voltammetry. The sensor exhibited excellent analytical performance for MON detection, with a wide linear response range from 0.05 to 250 ng mL- 1 and a low LOD of 0.008 ng mL- 1. Additionally, the immunosensor exhibited high accuracy, with recovery rates ranging from 97.4% to 103.2% in chicken liver and 96.0%-104.7% in milk. This developed immunosensor shows great potential for food safety applications, demonstrating high specificity, stability, and reproducibility in the detection of MON.

A novel electrochemical immunosensor was proposed for rapid determination of virginiamycin M1 (VIR M1). To enhance the sensitivity, anti-VIR M1 monoclonal antibody (mAb) and silver nanoparticles-Zinc 2-methylimidazole MOF (AgNPs-ZIF-8) nanocomposite were used as bio-recognition element and electrode modification material for signal amplification, respectively. The characterization of nanocomposite was performed using ultraviolet visible spectrophotometry (UV-Vis), X-ray diffraction (XRD), fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Under optimal conditions, proposed immunosensor achieved quantitative detection of VIR M1 in range from 0.4 to 20 ng/mL, with a detection limit (LOD) of 0.32 ng/mL. The immunosensor was applied to feed and food of animal origin with recoveries ranging from 90.51 % to 101.17 %, featuring RSDs within 2.8 % to 8.8 %. Therefore, the immunosensor could be a promising tool for determining VIR M1 levels in feed and residues in food of animal origin.

This study aimed to develop and evaluate a bispecific single-chain variable fragment (bsscFv) targeting B-cell activating factor (BAFF) and interleukin-17 (IL-17) for the treatment of systemic lupus erythematosus (SLE). The bsscFv was engineered by linking single-chain variable fragments (scFvs) specific for BAFF and IL-17 with a flexible peptide linker. It was expressed in E. coli BL21 and purified using affinity chromatography. Binding affinities to BAFF and IL-17 were assessed by enzyme-linked immunosorbent assay (ELISA). In vitro neutralization assays were conducted using Raji and HT-29 cell cultures. In vivo therapeutic efficacy was evaluated in an MRL/lpr mouse model of SLE, with 10 mice per group. Statistical significance was determined using a Student’s t-test for comparison of two groups. The bsscFv demonstrated strong binding to both BAFF and IL-17 in ELISA. In vitro, it inhibited BAFF-induced B-cell survival, proliferation, and immunoglobulin production, as well as IL-17-induced inflammatory cytokine secretion in HT-29 cells. In the MRL/lpr mouse model, bsscFv treatment significantly reduced autoantibody levels (p < 0.05), proteinuria, renal pathology, and cytokine expression in a dose-dependent manner compared to controls. The bsscFv exhibited potent neutralizing activity in vitro and therapeutic efficacy in vivo, suggesting it as a promising bispecific therapeutic agent for the treatment of SLE. Further studies are warranted to explore its clinical potential.

African swine fever virus (ASFV) is a highly contagious pathogen causing African swine fever in wild boars, warthogs and domestic pigs. The disease leads tosubstantial economic losses to the global pork industry and poses a grave threat to biodiversity. The early-encoded structural protein p22, owing to its immunodominant characteristics and high conservation across most genotypes, represents a promising diagnostic target and subunit vaccine candidate. In this study, the soluble extracellular domain of p22 protein (aa 30-177) was successfully expressed and purified, yielding 1.220 g/L. Eleven strains of monoclonal antibodies against p22 were generated, with four selected for B-cell epitope screening. Bioinformatic prediction-guided design was employed to generate overlapping truncations and peptides for epitope mapping. Based on those strategies, three novel linear B-cell epitopes were identified to be 30KKQQPPKK37, 130WGTDDCTG137 and 150YVYNNPHH157 by monoclonal antibodies. Sequence alignment across ASFV isolates revealed 100% evolutionary conservation in genotypes I/II, with minor variation in genotypes IV/VIII/XX/XXII. This study provided valuable data for broadening the ASFV antigen spectrum and identifying immunological targets for subunit vaccine formulation strategies.