Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus

African horse sickness virus (AHSV) causes a fatal disease in horses. The v irus capsid is composed of a double protein layer, the outermost of which i s formed by two proteins: VP2 and VP5. VP2 is known to determine the seroty pe of the virus and to contain the neutralizing epitopes. The biological fu nction of VP5, the other component of the capsid, is unknown. In this repor t, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specifi c monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure o f AHSV VP5, the protein was cloned in Escherichia coil using the pET3 syste m. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antis era, with 17 overlapping fragments from VP5 was analyzed. The most immunodo minant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120). The epit opes were further defined by PEPSCAN analysis with 12mer peptides, which de termined eight antigenic sites in the N-terminal half of the molecule. Neut ralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize blueto ngue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techn iques. These data will be especially useful for vaccine development and dia gnostic purposes. (C) 1999 Academic Press.