Reverse genetics technology for Rift Valley fever virus: current and future applications for the development of therapeutics and vaccines

Abstract

The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.

Fig. 1. RVFV genome organisation

A: Schematic representation of the three genomic segments and coding strategy. The arrows indicate the open reading frames in each segment with the cleavage sites generating the mature glycoproteins. B: RVFV M segment based mRNA with the five in frame AUG start codons at its 5′ terminus. The proteins expressed from the first and the second AUG are displayed.

Fig. 2

Confocal microscopy of uninfected (MOCK) or infected L929 cells (ZH: with RVFV ZH548). Cells were fixed at 18 h p.i. and stained with antibodies against SAP30, NSs or YY1 and NSs.

Fig. 3. Comparison of RVFV minigenome rescue systems

RVFV M segment-based minigenome plasmids under the control of the cellular RNA polymerase I are transfected into suitable mammalian cells (e.g., HEK293). The viral proteins needed for minigenome transcription and replication are provided by either superinfection with RVFV helpervirus (upper panel) or co-transfection of RVFV L and N expression plasmids (lower panel). Cells are harvested and reporter gene expression levels analyzed with suitable detection tools (e.g., CAT assays).

Fig. 4. RVFV infectious clone system

Plasmids containing the sequence of the three RVFV genome segments (L, M and S) under the control of the cellular RNA polymerase I are transfected into suitable mammalian cells (e.g., HEK293). Bunyaviral genome segments can be transcribed into genomic vRNA (as shown) or antigenomic cRNA, depending on the orientation within the plasmid. The viral proteins needed for RNA genome transcription and replication are provided by co-transfection of RVFV L and N expression plasmids. Supernatant is harvested and recombinant infectious RVFV can be used for subsequent applications (e.g., vaccine candidate).

Fig. 5

Plaques generated by RVFV rescued from plasmids using reverse genetics. rZH: rescued from wild type ZH548-based plasmids; rZHΔNSs: rescued with a mutated S segment, lacking the NSs coding sequence.