APPLICATIONS OF REVERSE GENETICS SYSTEMS TO UNRAVEL SPECIFIC ASPECTS OF THE REPLICATION CYCLE OF ROTAVIRU
The focus is on combining reverse genetics and structural insights to generate rationally designed rotaviruses to study viral entry into mammalian host cells to better understand the replication cycle, tissue tropism and possibly pathogenicity, and mechanisms controlling identification and packaging a full set of viral genome segments and mechanisms of genome segment reassortment. This is attempted by the incorporation of genes for reporter proteins in the plasmids for better tracking of viral growth, improving the expression of viral proteins by deactivating inhibitory motifs in some of the genome segments, and finding ways to stimulate viroplasm formation that will in turn increase the viral yield.
The vast number of rotavirus strains with different antigenic regions makes it impractical to implement a RG system for every strain. Reverse genetics of related segmented dsRNA viruses, bluetongue and African horsesickness virus use a universal core particle trans-typed with outer layer proteins from different serotypes. Using this strategy, novel vaccine candidates have been rescued. A similar approach is taken to generate rationally designed rotavirus VP4 antigenic regions for rotavirus RG using the non-pathogenic SA11 as a backbone.
DEVELOPMENT OF NON-LIVE REGIONAL STRAIN BASED ROTAVIRUS VACCINE CANDIDATES FOR HUMANS AND LIVESTOCK FOR LOCAL PRODUCTION
Non-live regional virus-like particles (VLPs) might be good boosters after live attenuated vaccination. We generated chimeric VLPs insect cells using codon-optimised consensus sequences of African rotavirus field strains directly from faeces. Transmission electron microscopy showed that about 30% tRV-VLPs were obtained when all the proteins were derived from the same strain. When the VP2/6 of one strain was co-expressed with the VP4 and VP7 of other strains, only 10-20% of tRV-VLPs were obtained. Therefore, we are doing further research to improve the assembly efficiency of chimeric tRV-VLPs on a suitable universal SA11 VP2/6 backbone.