Hong Tian

To prepare monoclonal antibodies (mAbs) with high specificity and affinity against enhanced green fluorescent protein (EGFP), we used purified EGFP emulsified with Freund{L-End} 's adjuvant to immunize Balb/c mice. Mouse splenocytes were then fused with SP2/0 myeloma cells. Hybridoma cell lines stably secreting antibodies were obtained through indirect ELISA screening and subcloning with the limited dilution method. The epitope specificity, reactivity, and affinity of the antibodies were systematically analyzed via an additivity assay, isotype identification, Western blotting, and surface plasmon resonance (SPR). The results demonstrated the successful screening of two hybridoma cell lines, 1F11 and 3C11, which stably secreted antibodies specifically targeting EGFP, with isotypes of IgG2a and IgG2b, respectively. The additivity assay showed an additivity index (AI)>40%, indicating recognition of distinct antigenic epitopes. Antibody titers reached 1:512 000. Western blotting confirmed that the prepared antibodies could specifically recognize EGFP with a size of about 27 kDa. SPR analysis revealed affinity constants of 3.806×10^-10 mol/L and 2.631×10^-10 mol/L, respectively, both within the high-affinity range. These findings indicate that the prepared mAbs possess excellent specificity, reactivity, and high affinity, providing a specific and important biological tool for the immunological detection of EGFP-tagged proteins and the in vivo monitoring of vaccine strains.

This study aims to establish a double-antigen sandwich ELISA method for detecting serum antibodies against caprine parainfluenza virus type 3(CPIV3), thereby supporting the clinical diagnosis of CPIV3 infection. According to the reference nucleocapsid (N) gene sequence (NCBI accession number: MT756864.1) published in NCBI, the N gene was synthesized and inserted into pET-30a to construct the recombinant plasmid pET-30a-N. The N protein was expressed in Escherichia coli and purified by Ni²⁺-affinity chromatography via its His tag. Reactivity of the purified N protein was verified by indirect ELISA and Western blotting. The His tag was removed with thrombin (1:1 000, W/W). The optimal coating concentration of the capture antigen, dilution of the HRP-conjugated detection antigen, and serum dilution were determined by checkerboard titration. Ultimately, a double-antigen sandwich ELISA method was established for clinical CPIV3 samples. Indirect ELISA and Western blotting confirmed that the N protein exhibited excellent antigenicity. The His tag was completely removed without altering the reactivity of the N protein. Checkerboard assays established the following optimal parameters: coating antigen concentration of 0.5 µg/mL, HRP-conjugated antigen dilution of 1:10 000, and serum dilution of 1:20. The cut-off value for the assay was set at 0.237 (OD₄₅₀). No cross-reactivity was observed with positive sera against foot-and-mouth disease virus type O (FMDV-O), sheep/goat pox virus (GTPV), orf virus (ORFV), or Brucella. The sensitivity of the developed ELISA reached 1:512, being significantly higher than that of a commercial kit. Both intra- and inter-assay coefficients of variation were below 10%. Parallel testing of 150 field sheep sera with the Shengbo indirect ELISA kit yielded an overall concordance rate of 87%. The established double-antigen sandwich ELISA method demonstrates high specificity, sensitivity, and reproducibility. It provides an effective technical tool for large-scale surveillance, sero-epidemiological investigations, and disease control of CPIV3 infection.

This study aims to establish a colloidal gold immunochromatographic test strip for the detection of glycoprotein B (gB) antibodies of porcine pseudorabies virus (PRV), thus providing a simple and rapid technique for the detection of PRV gB antibodies. The monoclonal antibodies to gB were prepared by the hybridoma technology. The gB protein was coupled with colloidal gold, and then gB and its monoclonal antibodies were separately sprayed onto a nitrocellulose membrane as the test line and control line. After optimization of conditions, a colloidal gold immunochromatographic test strip for detecting PRV gB antibodies was successfully developed. The specificity, sensitivity, stability, and concordance of the test strip were subsequently validated. The sensitivity of the test strip was 1:128. There was no cross-reactivity with positive sera of porcine reproductive and respiratory syndrome virus, classical swine fever virus, senecavirus A, and foot-and-mouth disease virus. The test strip could tolerate 37 °C for at least 72 d. In the test of 96 clinical pig serum samples, the concordance rate of the test strip results with the results of the commercial ELISA kit was 92.71%. The colloidal gold immunochromatographic test strip prepared in this study for PRV gB antibodies has good specificity, high sensitivity, and simple result interpretation, being suitable for rapid detection of porcine pseudorabies in the field.

African swine fever (ASF), an acute infectious disease with a high mortality rate, poses considerable challenges to the development of the global swine ind

African swine fever (ASF), a highly fatal disease often termed the “number one killer” of pigs, presents clinical symptoms indistinguishable from classical swine fever (CSF), such as fever, diarrhea, and vomiting, complicating on‐site differential diagnosis. As both ASF and CSF are notifiable diseases under the World Organisation for Animal Health (WOAH), rapid and accurate identification is crucial for effective outbreak management. In this study, we developed a multicolor lateral flow immunoassay (LFIA) based on latex microspheres (LMs) for the simultaneous detection of antibodies against ASF virus (ASFV) and CSF virus (CSFV). The assay enables visual differentiation within 15 min, with red indicating ASFV antibodies and blue indicating CSFV antibodies. After optimization, the LFIA demonstrated a sensitivity of 1:256, equivalent to that of a commercial ASFV ELISA kit and four‐fold higher than that for CSFV (1:64). The assay exhibited high specificity, showing no cross‐reactivity with other common swine pathogens and bovine viral diarrhea virus (BVDV). When applied to 180 clinical serum samples and compared with commercial ELISA kits, the LFIA achieved Cohen’s kappa values of 0.986 for ASFV and 0.918 for CSFV, indicating excellent agreement. Additionally, intra and interbatch evaluations confirmed its robust repeatability. Overall, the multicolor LM‐LFIA offers a rapid, sensitive, specific, and cost‐effective tool for point‐of‐care testing (POCT) of ASFV and CSFV antibodies, holding promise for routine field surveillance and disease control.

The internal ribosome entry site (IRES) is a cis‐acting element found in certain RNA viruses. In virus‐infected cells, the Senecavirus A (SVA) IRES can directly recruit the small ribosomal subunit to an internal initiation codon on the mRNA, enabling translation initiation independently of both the 5 ^′ cap structure of the viral genome and the host cell eukaryotic initiation factor 4F (eIF4F). As a small RNA virus, SVA frequently accumulates genetic mutations during transmission. This study aimed to characterize the mutational and evolutionary patterns of the SVA genome during its transmission within host cells exhibiting enhanced innate immunity. SVA was serially passaged for 80 generations in preactivated 3D4/21 cells that had been established in an antiviral innate immune state. During serial passaging, two stable single‐nucleotide mutations were consistently identified in the IRES region of the viral genomic RNA from passages 60–80: a uridine (U) insertion at genomic position 109, and a guanine (G)‐to‐adenine (A) substitution at position 265. Furthermore, the rescued SVA mutants harboring these mutations exhibited significantly higher replication titers than the parental strain. Notably, our results indicated that the mutant virus was unable to evade host antiviral innate immunity, whereas it significantly enhanced the translational activity of the SVA IRES. Collectively, these findings provide a foundation for understanding how single nucleotide polymorphisms (SNPs) in the 5 ^′ untranslated region (UTR) region influence viral IRES activity and the replication capacity of recombinant viruses.

Foot‐and‐mouth disease virus (FMDV) serotype Southern African Territories 2 (SAT2), historically endemic to sub‐Saharan Africa, has recently expanded its geographical range to the Middle East, posing a significant transboundary threat to global livestock industries. Characterized by high genetic diversity encompassing 14 distinct topotypes, SAT2 poses a challenge to reliable serological diagnosis and underscores the need for conserved, serotype‐specific antigenic targets. In this study, we aimed to identify such targets by mapping linear epitopes in the VP1 capsid protein using three monoclonal antibodies (mAbs; 3B2, 6F2, and 2F11) raised against SAT2 VP1. Epitopes were mapped through sequential truncation and alanine‑scanning mutagenesis, and their conservation was assessed across VP1 sequences from 53 SAT strains (35 SAT2, 13 SAT1, and 5 SAT3). The epitope recognized by mAb 3B2 was identified as ²⁰⁴ RFDAPIGVE ²¹² . It displayed 88.9% sequence identity within SAT2 topotypes I, II, III, and VI, and 100% identity in all other topotypes, while showing minimal similarity with SAT1 and SAT3 VP1 proteins—confirming its serotype specificity. Through systematic alanine scanning, Asp206 was defined as a critical binding residue and was 100% conserved across all SAT2 topotypes, including recent outbreak strains. Structural analysis further indicated that this epitope is surface‑exposed and possesses high solvent accessibility. In contrast, the ⁴⁷ TSFVVDL ⁵³ epitope (recognized by mAb 6F2) was predominantly conserved only within SAT2 topotype VII, with limited conservation in other topotypes. The ⁷ GAD ⁹ core recognition sequence (targeted by mAb 2F11) showed variable conservation (66.7%–100%) among SAT strains. Collectively, the broad conservation across topotypes, combined with favorable structural features, positions the ²⁰⁴ RFDAPIGVE ²¹² epitope as a promising candidate for developing robust, SAT2‑specific serological diagnostics.

African swine fever virus (ASFV) poses a significant threat to the global pig industry due to high mortality rates and complex genetic variation. Live attenuated vaccines (LAVs) provide protection against ASFV. Previously, MGF505-2R was identified as a potent inhibitor of innate immunity in vitro. This study evaluates the pathogenicity of a recombinant Eurasian genotype II strain with the MGF505-2R gene deleted (ASFV-ΔMGF505-2R) in piglets. Twelve five-week-old crossbred piglets were divided into two groups, with one group of eight piglets inoculated with ASFV-ΔMGF505-2R (n = 8) and the other group of four piglets inoculated with the same dose of parental ASFV CN/GS 2018 (n = 4). Clinical symptoms, viral loads, and immune responses were monitored over 30 days. ASFV-ΔMGF505-2R-inoculated piglets exhibited transient fever and low viremia only in the beginning of the challenge, while the control group developed high levels of viremia and hyperthermia at day 2 and 8 post-challenge, respectively. Meanwhile, the control group demonstrated more severe post-mortem signs and immuno-histochemistry injury when compared to the ΔMGF505-2R group. ELISA analysis displayed higher levels of IFN-β and IL-1β in the ΔMGF505-2R group, further solidating the immunosuppressive role of MGF505-2R. All ASFV-ΔMGF505-2R-inoculated piglets developed high titers of ASFV-specific P30 antibodies at 10 days post-challenge. These findings rationalized the potential of ASFV-ΔMGF505-2R as a live attenuated candidate for ASF infection.