Optimization of rotavirus reverse genetics and the rescue of rationally designed rotaviruses

Abstract

Rotaviruses are the leading cause of diarrhoea related deaths in children under the age of five, despite the availability of vaccines. The currently available rotavirus vaccines are up to 30% less effective in the developing countries of South-East Asia and Sub-Saharan Africa than in the developed countries. The lower vaccine efficacy may be due to various reasons including differences in circulating rotavirus strains and the individuals presenting different cell receptors in these regions. A region-specific rotavirus vaccine covering specific circulating strains may reduce the burden of disease in developing countries. The spike (VP4) and outer capsid proteins (VP7) are the main proteins used in vaccine design as antibodies against them have been proven to protect against severe diarrhoea. However, there are unresolved restrictions to reassortment of VP4 that affects not only the reassortment of VP4 but also VP7 and hampers the rational design of region-specific vaccine candidates. The recently developed plasmid-only rotavirus reverse may prove a valuable tool in the identification of the restrictions of VP4 reassortment and the rational design of region-specific vaccine candidates. The goals of this project were to implement a plasmid-only rotavirus reverse genetics system, evaluate it with the rescue of GS8(NSP2) reassortants and to rescue SA11 reassorted with various VP4 spikes on a conserved SA11 VP4 foot. To implement the pSMART-SA11-N5 rotavirus reverse genetics system the integrity of the plasmids was evaluated before attempting rescue. During the evaluation of plasmid integrity, insertions and deletions that would prevent rotavirus rescue were detected in four of the eleven plasmids. During the evaluation of the stability of the pSMART-SA11-N5 plasmid set, plasmid instability was observed. It was further observed some of the detected problems with the plasmids were not due to plasmid instability but rather problems during their construction. The problems with the plasmids were corrected and plasmid stability was improved by re-orientating the transcription cassettes with regards to the plasmid origin of replication. Using the corrected pSMART-SA11-N5 plasmid set rescue was still not possible despite testing various transfection reagents, transfection strategies, co-seeding strategies and mycoplasma free cells. Rescue was only successful when transfecting the Japanese pT7-SA11-L2 reverse genetics plasmid set with TransIt-LT1 and passaging into mycoplasma free cells.

The reassortment ability of the pT7-SA11-L2 plasmid-only reverse genetics system was evaluated by the rescue of GS8(NSP2) reassortants that were previously not rescuable or hard to rescue with a helper-virus reverse genetics system. Rescue of SA11 reassorted with AU-1 GS8(NSP2) that was previously not possible was successful using the plasmid-only reverse genetics system, demonstrating the benefits of a plasmid-only reverse genetics system in the rescue of hard to rescue reassortants. To assist in the rescue of reassortants of other rotavirus genome segments, O-agent GS8(NSP2) with its inhibitory motif inactivated (r.5U>A) was reassorted into SA11-L2. O-agent GS8(NSP2) was selected as it was previously observed to outcompete the SA11-N5 GS8(NSP2) during passaging and seemed to have a replicative advantage. However, SA11-L2 reassorted with the O-agent GS8(NSP2) had a replicative disadvantage when compared to rSA11-L2. Comparisons of the predicted secondary structures of the (+)ssRNA of the O-agent GS8(NSP2) revealed significant structural changes in terminal ends of the genome segment that may alter RNA/RNA interactions during the selection of genome segments resulting in observations that was different to what was previously reported. The effects of the inhibitory motif on genome segment selection and rotavirus replication must be further studied. Chimeric GS4s(VP4) were designed to have the spikes of either the GR10924 VP4 or the Bov-1604 VP4 on a SA11 VP4 foot domain. Rescue was then attempted with SA11-L2 reassorted with either the full-length GR10924 GS4(VP4) and Bov-1604 GS4(VP4), or the chimeric GS4s (VP4). Rescue of the SA11-L2 reassorted with the full length Bov-1604 VP4 was successful, but SA11-L2 reassorted with the GR10924 GS4(VP4) could not be rescued despite its rescue being previously reported. The addition of the SA11 VP4 foot domain to GR10924 VP4 however, allowed the rescue of SA11 with the GR10924 spike. The SA11 VP4 foot on the Bov-1604 VP4 spike also significantly increased its rate of replication. The improvement in the replication indicates the presence of restrictions of reassortment in the foot domain of VP4. Amino acid alignment of the VP4 foot domains from various strains showed two variable regions that may act as restrictions to VP4 reassortment. The identified restrictions of reassortment may be used for the optimization of GS4s(VP4) for reassortment in the development of rationally designed vaccine candidates.