Assembly of replication-incompetent African horse sickness virus particles: rational design of vaccines for all serotypes

African horse sickness virus, an orbivirus in the Reoviridae family, with nine different serotypes causes devastating disease in equids. The virion particle is composed of seven proteins, organized in three concentric layers, an outer made of VP2 and VP5, a VP7 middle layer and inner layer of VP3 that encloses a replicase complex of VP1, VP4 and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious vaccine candidates against all AHSV serotypes, taking into account not only immunogenic and safety properties, but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established highly efficient reverse genetics (RG) system for AHSV1 and subsequently, VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required exchange of two to five RNA segments to achieve equivalent titers of particles. In the VP6-complementary cells, all reassortant defective viruses could be amplified and propagated to high titers, but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor knock-out mice. Thus, these defective viruses have potential for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis and pathogenesis.