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.

Small ruminant lentiviruses (SRLVs) cause lifelong persistent infections in sheep and goats, impairing productivity and restricting livestock trade. Serological enzyme‐linked immunosorbent assay (ELISA) testing is the primary method for SRLV epidemiological monitoring, and most commercial ELISAs use capsid (CA), envelope, or a combination of both proteins as antigens. We previously developed a CA‐based indirect ELISA (iELISA) to detect SRLV antibodies. Here, we expressed full‐length recombinant SRLV Gag protein and further developed an iELISA using Gag as the coating antigen. The cut‐off value of the established Gag‐based iELISA was determined to be 0.093 based on sample‐to‐positive (S/P) ratio analysis of 100 SRLV‐negative sera. Parallel testing of 228 sera using the Gag‐iELISA, CA‐iELISA, and a commercial ELISA showed that the positive detection rate of the Gag‐iELISA was 35.96 percentage points higher than that of the CA‐iELISA and 4.39 percentage points higher than that of the commercial ELISA. All samples that tested positive by either the CA‐iELISA or the commercial ELISA were also positive by the Gag‐iELISA. Samples testing positive by the Gag‐iELISA but negative by the CA‐iELISA or the commercial ELISA were confirmed as SRLV antibody‐positive by Western blot (WB). Concordance analysis of the Gag‐iELISA and commercial ELISA showed 95.61% overall agreement. Endpoint dilution assays demonstrated that the Gag‐iELISA showed twice the analytical sensitivity of the CA‐iELISA and sensitivity comparable to that of the commercial ELISA. Furthermore, testing of 761 ovine sera from Hulunbuir during 2024–2025 revealed an overall SRLV seropositivity rate of 54.80%, with regional positive rates ranging from 0.00% to 84.44%. Collectively, this Gag‐based iELISA is a sensitive and practical tool for large‐scale SRLV serological surveillance.

Small ruminant lentiviruses (SRLV) cause lifelong persistent infections in sheep and goats, impairing productivity and restricting livestock trade. Serological ELISA testing is the primary method for SRLV epidemiological monitoring, and most commercial ELISAs use capsid (CA), envelope, or a combination of both proteins as antigens. We previously developed a CA-based indirect ELISA (iELISA) to detect SRLV antibodies. Here, we expressed full-length recombinant SRLV Gag protein and further developed an iELISA using Gag as the coating antigen. The cut-off value of the established Gag-based iELISA was set at 0.093 based on S/P ratio analysis of 100 SRLV-negative sera. Parallel testing of 228 sera using the Gag-iELISA, CA-iELISA, and a commercial ELISA showed that the Gag-iELISA had a 2.71-fold higher positive rate than the CA-iELISA and 4.39 percentage points higher than the commercial ELISA. All samples that tested positive by either the CA-iELISA or the commercial ELISA were also positive by the Gag-iELISA. Samples testing positive by the Gag-iELISA but negative by the CA-iELISA or the commercial ELISA were confirmed as SRLV antibody-positive by Western blot. Concordance analysis of the Gag-iELISA and commercial ELISA showed 95.61% overall agreement. Endpoint dilution assays showed that the Gag-iELISA had two-fold higher analytical sensitivity than the CA-iELISA and comparable sensitivity to the commercial ELISA. Furthermore, testing of 761 ovine sera from Hulunbuir during 2024–2025 revealed an overall SRLV seroprevalence of 54.80%, with regional positive rates ranging from 0.00% to 84.44%. Collectively, this Gag-based iELISA is a sensitive and practical tool for large-scale SRLV serological surveillance.