ABSTRACTAfrican horse sickness virus (AHSV) is a virus species in the genusOrbivirusof the familyReoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species ofCulicoidesbiting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replicationin vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.IMPORTANCEAfrican horse sickness virus is transmitted by species ofCulicoidesbiting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replicationin vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since thein vitrorelease of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.
Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus
