Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against African Horse Sickness Virus Serotype 1 VP2 Protein

African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV-1 to -9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV-1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV-1 VP2. In this study, a recombinant VP2 protein was expressed in <i>Escherichia coli</i> and used as an immunogen, and three monoclonal antibodies (mAbs), designated 7D11, 10A9, and 9E7, against AHSV-1 VP2, were generated. These three mAbs were then successfully used in IFA, WB, and ELISA for the detection of AHSV-1 VP2. Two overlapping linear epitopes, ⁶⁷⁰NEFDFE⁶⁷⁵ (E670-675) recognized by 9E7 and ⁶⁷⁰NEFDF⁶⁷⁴ (E670-674) recognized by 7D11 and 10A9, were identified through truncation of GST-fused VP2. Amino acid sequence alignment shows that the ⁶⁷⁰NEFDFE⁶⁷⁵ motif is completely conserved within AHSV-1 but is highly divergent in other AHSV serotypes. Our studies provide an important tool for basic research into AHSV-1 and for the diagnosis of AHSV-1.