The high abortion rate associated with Salmonella Abortusequi ( S . Abortusequi) infection in equids has re-emerged over the past 10 years and has caused serious economic losses to China. Our previous studies showed that the flagellin FljB gene could distinguish S . Abortusequi from most Salmonella serotypes. In this study, the flagellin antigen was used to develop a competitive enzyme-linked immunosorbent assay (cELISA) that could be used to detect both horse and donkey serum samples using a monoclonal antibody (MAb) that was found to bind to FljB. A cELISA was established using the purified MAb coating of the plate and incubation of the mixture of horseradish peroxidase (HRP)-conjugated FljB antigen with the undiluted serum sample. The performance of the cELISA and the tube agglutination test (TAT) assay was compared with respect to sensitivity and specificity, by testing a panel containing 660 S . Abortusequi-positive and 515 S . Abortusequi-negative serum samples, all of which had been characterized by Western blotting. Receiver operator characteristic (ROC) analyses were performed to determine the cutoff value and estimate the detection specificity (Sp) and sensitivity (Se). ROC analysis showed that the area under the ROC curve (AUC) values of cELISA [AUC = 0.9941; 95% confidence interval (CI), 0.9898–0.9984] were higher than those of TAT (AUC = 0.7705; 95% Cl, 0.7437–0.7972). A cutoff value of 39.5% was selected with Sp and Se values of 100 (95% Cl, 99.26–100.00) and 97.58 (95% Cl, 96.10–98.50), respectively. The cELISA has excellent futures compared with TAT, such as shortened detection time, no need for pre-treatment of sera, and easy interpretation of the results, and is more suitable for disease surveillance.

Enzootic nasal tumor virus (ENTV) is the etiological agent responsible for enzootic nasal adenocarcinoma (ENA), a chronic and contagious disease predominantly affecting sheep and goats. ENTV is classified into two distinct types: ENTV‐1, which infects sheep, and ENTV‐2, which infects goats. ENA has been globally reported in small ruminant‐rearing regions, causing significant mortality and substantial economic impacts in affected flocks. There is currently no standardized detection method for ENA. In this study, we successfully generated a monoclonal antibody (mAb) and a polyclonal antibody (pAb) against the ENTV‐2 capsid protein (p27), and identified the epitope of the mAb, which was found to be highly conserved among different ENTV‐2 isolates. An antigen capture ELISA (acELISA) was then successfully developed using the mAb as the capture antibody and the pAb as the detection antibody to specifically detect p27 of ENTV‐2 in nasal secretions. The cut‐off value of the acELISA was determined to be 0.1052 by analyzing S/P values. The detection limit of this assay was 0.16 ng/mL of rp27 protein and equivalent to 844 copies/μL of ENTV‐2 RNA. Specificity tests showed that the method had no cross‐reaction with other prevalent small ruminant pathogens. The coincidence rates of the developed acELISA compared with western blotting and qRT‐PCR assays were 98.95% (189/191) and 96.34% (184/191), respectively. Furthermore, the acELISA was applied to assess ENTV‐2 in 1228 clinical nasal swab samples collected from seven provinces in China. The results demonstrated that the positivity rate varied between 0.00% and 28.21%. In conclusion, we successfully developed an acELISA with high specificity, sensitivity and reproducibility for the detection of ENTV‐2 antigen. This high‐throughput method for the detection of ENTV‐2 represents a significant advancement in the field and may contribute to the prevention and control of ENTV‐2.

African horse sickness (AHS) is a lethal vector-borne disease caused by African horse sickness virus (AHSV) and represents a major threat to equine health and the horse industry. In 2020, outbreaks of AHS caused by AHSV serotype 1 (AHSV-1) were reported in Thailand, increasing the risk of AHS introduction into China. Given the safety issues associated with currently available live attenuated AHS vaccines, the development of safer and more effective vaccination strategies is urgently needed. In this study, we constructed a recombinant fowlpox virus (rFPV) expressing the AHSV-1 VP2 protein as a candidate vaccine, designated rFPV-VP2. The recombinant virus was verified by PCR and Western blot analysis, which confirmed the successful expression of VP2. Preliminary immunization trials were conducted in both mice and horses, and immune responses were evaluated via an indirect enzyme-linked immunosorbent assay (iELISA) and immunofluorescence assay (IFA). The results revealed that VP2-specific antibodies were successfully induced in the serum of rFPV-VP2-immunized animals. Notably, serum from immunized horses showed specific reactivity with AHSV-1, confirming the induction of AHSV-1-specific immune responses. Therefore, these results demonstrate that rFPV-VP2 is a promising candidate vaccine for AHSV-1 and provide a scientific basis for the development of safer preventive strategies.

All lentiviruses encode a regulatory protein, Rev, which mediates nuclear export of viral mRNAs and is required for viral replication, and multimerization of Rev is thought to be critical for its function. The lentivirus equine infectious anemia virus (EIAV) Rev is known to mediate the nuclear export of long-length, unspliced, or incompletely spliced viral mRNA encoding the viral structural proteins (Gag, Pol, and Env), like Rev from other lentiviruses. However, EIAV Rev also mediates the nuclear export of a short-length, fully spliced viral mRNA species encoding the functionally unknown protein Mat. Here, we found that disruption of Rev multimerization impaired Rev activity in mediating nuclear export and expression of gag-pol mRNA, but not in mediating nuclear export and expression of mat mRNA. Deficiency of Rev multimerization leads to lower viral replication. Further investigation revealed that the activity of multimerization-deficient Revs in mediating nuclear export and expression of long-length EIAV or HIV-1 mRNAs was significantly lower than that of wild-type Rev, while this difference was significantly reduced or absent between the two in mediating nuclear export and expression of short-length EIAV or HIV-1 mRNAs. This suggests that the differential dependence of the nuclear export of gag-pol mRNA and mat mRNA on Rev multimerization is modulated by the lengths of the mRNA species. Our findings suggest that when targeting short mRNAs, EIAV Rev appears to be able to perform nuclear export activity in a non-multimerized state. IMPORTANCE: Multimerization of lentiviral Rev is thought to be essential for its function, which is to mediate nuclear export of viral mRNA, a necessary step in lentiviral replication. In equine infectious anemia virus (EIAV), the nuclear export of unspliced viral mRNAs encoding structural proteins (Gag/Pol) is dependent on Rev. Moreover, our recent studies have demonstrated that the nuclear export of a fully spliced mRNA species, mat , encoding a small protein (Mat) of unknown function, is also Rev-dependent. In this study, we observed that the expression and nuclear export of EIAV gag-pol mRNA were strictly dependent on Rev multimerization, whereas those of mat mRNA were not. Interestingly, we also determined that the dependence of Rev-mediated nuclear export of EIAV mRNA on Rev multimerization is strongly associated with lengths of these mRNA species. Our data imply that EIAV Rev is able to exert nuclear export activity in a non-multimerized state.

Equine infectious anemia virus (EIAV) is a member of the Lentivirus genus of the Retroviridae family and serves as an important model for studying lentiviral pathogenesis. A novel EIAV-encoded protein, designated Mat, was previously identified in our laboratory, yet its biological functions remained unclear. Here, we demonstrate that disruption of Mat moderately reduced EIAV replication in equine monocyte-derived macrophages, and this defect was restored when Mat was supplied in trans . Further analyses revealed that Mat exhibits nuclear localization and enhances Tat-mediated transcriptional activation of the long terminal repeat (LTR), an essential and conserved step in lentiviral replication, by interacting with viral Tat and the host factor Cyclin T1 (CycT1), but not with the trans-acting responsive RNA element (TAR) within the LTR. Moreover, we found that Mat stabilizes EIAV Tat (eTat) and promotes the association of eTat with both TAR and equine CycT1. These findings indicate that Mat serves as a cofactor of Tat to regulate EIAV replication. IMPORTANCE: In lentiviruses, Tat-mediated activation of the LTR promoter is essential for viral replication. Mechanistically, Tat binds to TAR (a structured RNA at the 5′ end of nascent viral transcripts, corresponding to the R region of the LTR), recruits host factors, including CycT1, and facilitates RNA polymerase II elongation, thereby promoting efficient production of full-length viral transcripts required for lentiviral replication. We recently identified an uncharacterized EIAV protein, Mat. Here, we show that Mat inactivation moderately attenuates EIAV replication in vitro , and this defect is rescued by ectopic Mat expression. Mat enhances Tat-mediated activation of EIAV LTR activity, and we further confirm that Mat interacts with EIAV Tat (eTat) and equine CycT1 (eqCycT1), stabilizes eTat, and promotes the association of eTat with both TAR and eqCycT1. These results suggest that Mat acts as an eTat cofactor in EIAV transcriptional regulation, providing insights into the complex transcriptional regulatory mechanisms of lentiviruses.

As a naturally self-assembling protein nanocarrier, ferritin enables multivalent antigen display and functions as an intrinsic adjuvant to enhance vaccine-induced immune responses. Streptococcus equi subsp. equi (S. equi) is the causative agent of equine strangles, an acute and highly contagious respiratory disease responsible for substantial economic losses worldwide. However, currently available vaccines often show suboptimal immunogenicity and limited protective efficacy. In this study, we developed a recombinant equine ferritin (rHF)-based nanoparticle vaccine, rSE5Mix, presenting five core protective antigens (EQ8, EQ5, CNE, IdeE, EAG). The fusion proteins efficiently assembled into uniform nanoparticles. Immunization of BALB/c mice elicited rapid, high-titer antigen-specific IgG antibodies and a balanced Th1/Th2 immune response without additional adjuvants. Following lethal challenge with S. equi, rSE5Mix-immunized mice showed 100% survival and markedly milder clinical signs. Histopathological analysis demonstrated significantly alleviated organ damage, and bacterial loads in major tissues were reduced to nearly undetectable levels. Importantly, compared with the octavalent tandem vaccine rSE8, rSE5Mix induced faster elevation of partial antigen-specific antibodies, especially for EQ8 and CNE. Their antibody titers were comparable at later stages. This study developed a safe and effective ferritin nanoparticle vaccine candidate against equine strangles and verified that equine ferritin is a promising candidate delivery platform for veterinary bacterial vaccines.