Investigating the coding capacity of rotaviruses using a newly developed reverse genetics system

Bovine rotavirus (RV) infection causes severe diarrhoea in young dairy calves and has a significant economic impact on livestock production as a result of high morbidity and mortality caused. Development of technologies to engineer infectious RV using an entirely plasmid-based reverse genetics (RG) system has proven challenging. A breakthrough was made when Kanaiand co-authors (PNAS, 2017)developed a plasmid-only-based RG system for the simian RV strain SA11.We are currently developing an analogous RG system for the bovine RF RV strain. Having parallel systems for different RV strains will help to validate phenotypic changes induced by site-directed mutagenesis (SDM) within the RV genome.

The coding capacity of the 11-segmented dsRNA RV genome has been largely unexplored. Using bioinformatic analyses, we have identified four segments with up to five putative alternative initiation codons which are in moderate or strong Kozak context. Furthermore, some occur in segments for which the canonical start codon occurs within 15 nucleotides of the start of the segment, further suggesting the possibility of alternative translation start sites to generate coding diversity. We are now applying our RG systems to investigate RV coding capacity using TnT transcription assays, radiolabelling and SDM.