Engineering and validation of single-cycle alphavirus replicon particles

Abstract

MAYV, RRV, ONNV, and BFV are understudied alphaviruses, often investigated using tools developed for model viruses, such as SINV and CHIKV. This study aimed to develop and evaluate VRP systems for a panel of alphaviruses using ZsGreen and mCherry reporter replicons and a split-helper system in BHK-21 cells. The VRPs were analysed by second-round infection using fluorescence microscopy. The results showed that VRP production differed between alphavirus backbones. SINV and MAYV produced relatively high titers of VRPs, whereas RRV and BFV were less efficient. Removal of the capsid enhancer sequence improved ONNV VRP production but reduced RRV VRP titers and abolished detectable BFV VRP titers. Second-round infection confirmed that the generated VRPs were single-cycle. Overall, this thesis demonstrates that alphavirus VRP production is virus-specific and that regulatory elements optimised for model alphaviruses cannot be assumed to function universally across the genus.