Autonomously replicating RNAs of Bungowannah pestivirus : E^RNS is not essential for the generation of infectious particles

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1 and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiency of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2 or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These “defective in third cycle” BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc-protein and the receptor binding domain of the Middle-East Respiratory Syndrome Coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPV are able to infect a wide variety of target cell lines, allowing the expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have the potential as broad spectrum viral vectors.