Enping Liu

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: ⁵² DMKRLHLDDEKSSNL ⁶⁶ and ¹⁷⁷ VAPGPPALEWSATNE ¹⁹¹ . Alanine‐scanning mutagenesis revealed that residues D52, M53, R55, L56, and L58 were critical for the recognition of epitope ⁵² DMKRLHLDDEKSSNL ⁶⁶ by mAbs 2D2 and 18G7. Residues G180, P181, and W186 were essential for recognition of epitope ¹⁷⁷ VAPGPPALEWSATNE ¹⁹¹ 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.

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.

Pseudorabies virus (PRV), an important member of the Alphaherpesvirinae subfamily, shares high evolutionary homology with other members of the same subfamily, including human herpes simplex virus (HSV) and varicella‐zoster virus (VZV). However, significant differences exist among them in terms of host range, pathogenicity, and immune evasion strategies. This review systematically compares the roles of PRV, HSV type 1 (HSV‐1), and VZV in modulating host innate immune responses, encompassing the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) signaling, RIG‐I‐like receptor (RLR) signaling, NOD‐like receptor (NLR) signaling, Toll‐like receptor (TLR) signaling, Janus kinase (JAK)‐signal transducer and activator of transcription (STAT) pathway, apoptosis, and autophagy. By summarizing and comparing the interactions of these three viruses with the host innate immune response, this review aims to provide a theoretical reference for understanding the conserved and unique mechanisms of immune evasion among alphaherpesviruses, and to offer insights for the development of novel antiviral strategies.

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.

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.

Flurogestone acetate (FGA) is a highly effective synthetic progesterone analog extensively utilized to regulate estrus synchronization, enhance superovulation, and improve the economic productivity of female livestock. However, FGA is associated with potential toxicity, and its improper application can lead to environmental residues, resulting in adverse effects on both humans and animals. These residues pose a significant threat to human health, underscoring the critical need for the development of rapid and accurate detection methods. In this study, the complete antigen of FGA was successfully synthesized, and a high‐affinity monoclonal antibody against FGA was developed. Utilizing this antibody, a direct competitive ELISA (dc‐ELISA) method based on HRP and monoclonal antibody coupling was established for the efficient detection of FGA residues in milk. The assay exhibits a validated quantitation range of 0.0034–0.97 ng mL ⁻¹ (IC ₅₀ = 0.058 ng mL ⁻¹ ; LOD = 0.0013 ng mL ⁻¹ ). In milk (20‐fold dilution to mitigate matrix effects), recoveries at 0.2, 0.5, and 0.8 ng mL ⁻¹ were 95.63%–116.44%. The anti‐FGA monoclonal antibody displayed no cross‐reactivity ≥ 10% ( n = 6) among tested analogs (highest: medroxyprogesterone acetate, 7.46%). Samples whose calculated concentrations exceed the upper limit of 0.97 ng mL ⁻¹ are reported as positive (above range) without assigning a numeric value. These attributes support the use of this HRP‐labeled dc‐ELISA as a rapid screening method for FGA residues in milk. In conclusion, this study presents a simple and effective method for detecting residual flurogestone acetate in animal‐derived foods. Compared to indirect competitive ELISA (ic‐ELISA), the developed method significantly reduces the reaction time and enhances the detection efficiency.

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.

The spike (S) glycoprotein of porcine respiratory coronavirus (PRCV) plays a critical role in viral entry, with its receptor binding domain (RBD) on the S1 subunit responsible for interacting with the host receptor porcine aminopeptidase N. In this study, the PRCV RBD protein was successfully expressed, and a monoclonal antibody (mAb), 2E6, was produced using hybridoma technology. The specificity of 2E6 was confirmed by western blot and indirect immunofluorescence assay (IFA). To identify the linear B‐cell epitope recognized by 2E6, the RBD was initially divided into three overlapping fragments, cloned into pET‐32a vectors, and expressed in Escherichia coli BL21 (DE3). Dot blot analysis revealed that 2E6 reacted with the RBD‐1 fragment (amino acids 299–357). Further subdivision into RBD1‐1, RBD1‐2, and RBD1‐3, followed by expression using both pET‐32a and pEGFP‐C1 vectors, enabled Dot blot and IFA validation of specific recognition of RBD1–3 (amino acids 329–347). Subsequent peptide mapping using synthetic overlapping peptides (RBD‐P1, P2, P3) confirmed that the minimal linear epitope lies within RBD‐P2 (amino acids 334–343). This epitope is surface‐exposed and conserved among PRCV strains, making it a promising candidate for diagnostic assay development and epitope‐based vaccine design.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the pathogenic agent leading to COVID-19. Due to high speed of transmission and mutation rates, universal diagnosis and appropriate prevention are still urgently needed. The nucleocapsid protein of SARS-CoV-2 is considered more conserved than spike proteins and is abundant during the virus’ life cycle, making it suitable for diagnostic applications. Here, we designed and developed a fluorescent immunochromatography assay (FICA) for the rapid detection of SARS-CoV-2-specific antibodies using ZnCdSe/ZnS QDs-conjugated nucleocapsid (N) proteins as probes. The nucleocapsid protein was expressed in E.coli and purified via Ni-NTA affinity chromatography with considerable concentration (0.762 mg/mL) and a purity of more than 90%, which could bind to specific antibodies and the complex could be captured by Staphylococcal protein A (SPA) with fluorescence displayed. After the optimization of coupling and detecting conditions, the limit of detection was determined to be 1:1.024 × 10⁵ with an IgG concentration of 48.84 ng/mL with good specificity shown to antibodies against other zoonotic coronaviruses and respiratory infection-related viruses (n = 5). The universal fluorescent immunochromatography assay simplified operation processes in one step, which could be used for the point of care detection of SARS-CoV-2-specific antibodies. Moreover, it was also considered as an efficient tool for the serological screening of potential susceptible animals and for monitoring the expansion of virus host ranges.