Local implementation and optimization of rotavirus reverse genetics systems

Abstract

Reverse genetics (RG) is one of the most powerful tools for the study of viral replication, pathogenesis and for the generation of rationally designed vaccine candidates. The main bottleneck in rotavirus (RV) research has been the lack of a robust, traceable, helper-virus independent RV RG system (Desselberger, 2014). The first viral RG system was developed in 1976 to rescue the dsDNA virus, ?-phage, from cultured monkey kidney cells . In 1981 the first RNA virus, poliovirus, was rescued from cell culture through the transfection of viral genome transcripts generated in vitro from cDNA plasmids. In 2006, 25 years later, the first RV RG system was developed. It was a helper-virus based system that relied on the segmented genome of the RV to undergo reassortment during co-infection and depended on a selection system for the isolation of recombinant viral progeny. In 2017, 41 years since the development of the first viral RG system, a Japanese group (Kanai et al., 2017) published the first, plasmid only, helper-virus independent, pT7_SA11-L2 RV RG system. The pT7_SA11-L2 RG system, and its subsequent adaptations and optimizations (Komoto et al., 2018; Komoto et al., 2019), has opened up a new era of targeted, rationally guided RV research opportunities. The main goal of this project was to establish a plasmid-based, helper-virus independent RV RG system at the NWU. To accomplish this, the project had three main objectives, namely: 1) To obtain and implement the Japanese pT7_SA11-L2 RV RG system, and to optimize it through the incorporation of insights gained from the bluetongue virus (BTV) and African horsesickness virus (AHSV) RG systems. 2) To finalize and implement the locally developed, consensus sequence-based pSmart_SA11-N5 RV RG system with all the optimizations used during the pT7_SA11- L2 RV RG systems implementation, and 3) to perform a comparative analysis of the pSmart_SA11-N5 and pT7_SA11-L2 RV RG systems and their various optimizations throughout the project via TCID50 assay. The original pT7_SA11-L2 RV RG system was obtained from AddGene and implemented at the NWU with initial difficulty. Several aspects of the BTV and AHSV RG systems were incorporated into the original pT7_SA11-L2 RG system.