RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses

Abstract

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.

Fig. 1. Production of rMP12 using Pol I- and T7-promoter driven rescue system

A) Kinetics of rMP12 production after transfection of plasmids expressing L, M and S segments under control of Pol I or T7 promotor together with the RVFV L and N expression plasmids pTM1-L and pTM1-N in BHK/T7-9 cells. B) Sequence analysis of the rMP12 S segment compared to authentic MP12 S segment carried out with total RNA extracted from infected cells.

Fig. 2. Properties of rescued recombinant RVFV

A) Plaque assay showing authentic ZH548 and the different recombinant viruses: rZH, rZHΔNSs, rZHΔNSs-GFP and rZHΔNSs-FLuc at passages 0 and 3. B) Growth curves of rZH and rZHΔNSs in comparison to ZH548 after infection of Vero cells at a moi=3. C) Expression of GFP in Vero cells infected with rZHΔNSs-GFP at passage 0 or 4 and in non-infected cells (NI). D) Kinetics of FLuc expression in Vero cells infected with rZHΔNSs-Fluc at a moi= 0.1 or 0.5. Luciferase activity is expressed in arbitrary units per 2.5×10⁴ cells. Each data point is the mean of triplicate assay.

Fig. 3. Analysis of virus stability for foreign gene expression

A) Titer of rZHΔNSs-GFP and rZHΔNSs-Fluc after rescue (P0) or passaged one to four times (P1–P4) on Vero cells at a moi=0.001 and recovered at day 3. B) Luciferase activity in Vero cells infected with different passages of rZHΔNSs-FLuc at a moi=3, expressed in arbitrary units per 2.5×10⁴ cells. Mean of triplicate assays. C) Gel analysis of RT-PCR products representing the full-length S segment of rZHΔNSs-GFP and rZHΔNSs-Fluc at P0 and P4. RT-PCR was performed using total RNA extracted at 15h p.i. from Vero cells infected by these viruses or ZH548 and rZHΔNSs.

Fig. 4. Analysis of the virulence associated with the NSs of RVFV MP12

A) Characterization of S segment by Taq1α digestion. S segment RT-PCR carried out on total RNA extracted at 15h p.i. from Vero cells infected by ZH548, MP12, rZH, rZH-SMP12 or rZH-NSsMP12 at a moi=3. Schematic representation of the position of Taq1α in the S segment of ZH548 and MP12. The size of digestion products for ZH548 and rZH are indicated. B) Inhibition of the IFN-β mRNA transcription by these viruses except for the rZHΔNSs. IFN-β- and GAPDH-specific RT-PCR performed on total RNA extracted at 8h p.i. from L929 cells infected at a moi=3 by ZH548, MP12, or the recombinant RVFV. C) Survival curves of 4 to 5 week old mice (Swiss) infected with 10⁴ pfu of each RVFV. The experiment was carried out using lots of 12 mice.

Fig. 5

Schematic diagram of the pPolI-SZH plasmid and the generation of pPolI-SΔNSs in which the NSs sequence was deleted and replaced by a XhoI/BbsI cloning site. The terminal noncoding (NC) and intergenic (IGR) regions are indicated as well as the restriction sites utilized to generate the constructs.