Recent advances in rotavirus reverse genetics and its utilization in basic research and vaccine development

Abstract

Rotaviruses are segmented double-stranded RNA viruses with a high frequency of gene reassortment, and they are a leading cause of global diarrheal deaths in children less than 5 years old. Two-thirds of rotavirus-associated deaths occur in low-income countries. Currently, the available vaccines in developing countries have lower efficacy in children than those in developed countries. Due to added safety concerns and the high cost of current vaccines, there is a need to develop cost-effective next-generation vaccines with improved safety and efficacy. The reverse genetics system (RGS) is a powerful tool for investigating viral protein functions and developing novel vaccines. Recently, an entirely plasmid-based RGS has been developed for several rotaviruses, and this technological advancement has significantly facilitated novel rotavirus research. Here, we review the recently developed RGS platform and discuss its application in studying infection biology, gene reassortment, and development of vaccines against rotavirus disease. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-05142-7.

Rotaviruses: From Pathogenesis to Disease Control—A Critical Review

“…The current advances in reverse genetics system for RVs involving the use of an entirely plasmid-based platform has been regarded as a breakthrough and a key technological advancement over the more tedious helper virus-dependent reverse genetics techniques developed for RVs [ 179 ]. In a recent study, a plasmid-based RV reverse genetic system was successfully employed to generate both NSP3 and a fluorescent reporter protein by replacing the open reading frame (ORF) of segment 7 of RV dsRNA with an ORF encoding NSP3 that is fused to a fluorescent reporter protein [ 180 ].…”

“…Further, the use of recombinant rotaviruses as an expression vector of the highly immunogenic protein domain of the SARS-CoV-2 [ 181 ], is suggestive of broader usefulness when fully developed. Studies have shown that the reverse genetic system not only permits the regulation of the experimental conditions, but also the preferred combination of several RV gene segments with concurrent mutations and the production of mutants with a reduced interferon response [ 179 , 181 ]. All these features of the reverse genetic system-based approach may be beneficial in advancing the development of next-generation rotavirus vaccines.…”

Genetic and biological characteristics of species A rotaviruses detected in common shrews suggest a distinct evolutionary trajectory

“…This might indicate a low reassortant potential of the common shrew strain with other mammalian strains. However, the applied system is only based on the SA11 strain, and recently developed reverse genetics systems for other RVA strains ( Uprety, Wang, and Li 2021 ) should therefore be tested in future. In addition, it has been shown that the applied system is restricted to the presence of a specific receptor-binding region in the analysed VP4 molecules ( Falkenhagen et al.…”

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

“…The plasmid-only reverse genetics system has been optimised to study the functions of RV proteins, to generate RV reassortants and RV reporter expression systems, and conceptualise novel vaccine platforms [43][44][45]. Using the plasmid-only reverse genetics system, recombinant RVs harbouring chimeric VP4 proteins that showed efficient replication in cell culture and neutralising activity in mice have also been engineered [46][47][48]. Protection from infection with RV is primarily mediated by heterotypic neutralising antibodies that target VP4 and/ or VP7.…”

“…Thus, the potential for VP4 to express heterologous epitopes from different RV strains may provide a delivery platform for expression of different vaccine antigens, though peptide insertions into VP4 has not previously been tested. Additionally, the plasmid-only reverse genetics system has been utilised to generate a repertoire of recombinant RVs expressing fluorescent reporter proteins [48, 51]. The C-terminus of the SA11 NSP3 open reading frame (ORF) was fused to a porcine teschovirus translational 2A element followed by various reporters including UnaG, mKate, mRuby or TagBFP to successfully yield two uncoupled proteins without compromising virus replication [51].…”