Article Figures & Data
Figures
- Fig. 1.
RVFV genome organization and expression strategy of the M genome segment. (A) Schematic representation of the RVFV small (S), medium (M), and large (L) genome segments in antigenomic orientation. (Inset) S-based minigenome containing the eGFP gene used in this work. (B) Coding strategy of the M genome segment. The mRNA encoded by the M segment is translated into a polyprotein that is processed into the NSm1, NSm2, Gn, and Gc proteins.
- Fig. 2.
Expression of the N protein from the antigenomic-sense S segment. BSR-T7/5 cells (A) or FP-T7-infected BHK-21 cells (B) were transfected with plasmid pUC57-S, containing the RVFV S genome segment in antigenomic-sense orientation. Expression of the RVFV N protein was detected using an N-protein-specific MAb and horseradish peroxidase-conjugated anti-mouse IgG antibodies.
- Fig. 3.
Production of RRPs by the three-plasmid system. (A) BHK cells were infected with FP-T7 and subsequently remained untreated (mock) or were transfected with plasmid pUC57-S-eGFP (S-eGFP) only, in combination with plasmid pUC57-L encoding the RVFV L genome segment (S-eGFP/L), or with the aforementioned plasmids and pCAGGS-M (GP), encoding the structural glycoproteins (S-eGFP/L/GP). The percentage of eGFP-positive cells was determined by flow cytometry (values are means and standard deviations for three replicates). (B) Schematic representation of the three-plasmid system. BHK cells were first infected with FP-T7 (step 1) and subsequently transfected with transcription plasmids pUC57-L (L) and pUC57-S-eGFP (S) and expression plasmid pCAGGS-M (step 2). After 24 h, the culture medium containing the RRPs was collected (step 3). Transcription from the expression plasmid is controlled by a CAG promoter (CAGp), and transcription from the transcription plasmids is controlled by a T7 promoter (T7p). Untranslated regions are depicted as black boxes.
- Fig. 4.
(A) RVFV glycoprotein expression in BHK-GnGc cells. BHK cells were transfected with plasmid pCIneo-GnGc, encoding the RVFV structural glycoproteins Gn and Gc. The BHK-GnGc cells were cloned by limiting dilution. Expression of Gn and Gc was detected by staining of BHK-GnGc cells with polyclonal antibodies specific for the Gn and Gc proteins. (B) Similarly treated BHK parent cells.
- Fig. 5.
Cartoon representing the construction of the BHK-Rep (A) and BHK-Rep2 (B) cell lines. Construction of the BHK-Rep cells started with a BHK cell line constitutively expressing small amounts of the Gn and Gc proteins (BHK-GnGc), whereas wild-type BHK cells were used for production of the BHK-Rep2 cell line. Stable glycoprotein expression was achieved in these cells by introducing the pCIneo-M plasmid. The S-eGFP and L genome segments were introduced in the BHK-Rep cell line by FP-T7-driven transcription from plasmids, whereas these genome segments were introduced into the BHK-Rep2 cells by infection with RRPs. Transfection with pCAGGS-M was used to produce RRPs, which assist in spread of the genome segments among cells.
- Fig. 6.
RRP infection of mammalian cells (A) and insect cells (B). BHK cells, human embryonic kidney 293T cells (HEK293T), Drosophila S2 cells, and Aedes albopictus C6/36 cells were infected with RRPs at an MOI of 1. The number of positive cells was determined by flow cytometry at 42 (BHK and HEK293T) or 72 (S2 and C6/36) h postinfection. Histograms show averaged results of three independent measurements, with standard deviations.
- Fig. 7.
Schematic representation of the one-plasmid systems. Transfection of BHK-Rep cells with pCAGGS-M results in the production of RRPs. Alternatively, RRPs can be produced by infection of potentially any type of mammalian cell, such as HEK293T cells, by infection with RRPs followed by transfection with pCAGGS-M.
- Fig. 8.
Northern blot analysis of the viral RNA segments present in BHK-Rep cells. Total RNA of BHK-Rep and BHK-Rep2 cells was extracted, separated by electrophoresis using the glyoxal-dimethyl sulfoxide system, and transferred to positively charged nylon membranes. Hybridization was performed with DIG-labeled S, M, and L probes, and hybridization was detected by phosphatase-conjugated anti-DIG antibodies. RNA extracted from recombinant RVFV 35/74 was used as a positive control and a reference for size. Wild-type BHK cells were used as a negative control. The specificity of the probe used for hybridization of each blot is indicated below each panel. The positions of the S, M, and L segments are indicated by arrows.
- Fig. 9.
Characterization of RRPs. (A) BHK-Rep cells were either left untreated (−GP) or transfected with pCAGGS-M (+GP), and RRP titers in the collected supernatant were determined at different time points posttransfection. (B) To demonstrate that RRPs are nonspreading, BHK cells were infected with RRPs, and after 2 days, eGFP expression was observed in infected cells (left). Fresh BHK cells were incubated with the collected supernatant and monitored for eGFP expression after 3 days (right). (C) Electron micrograph of RRPs. Concentrated RRPs were stained with 1% PTA and analyzed by TEM. Bar, 50 nm. (D) To visualize RRP proteins, culture medium of BHK-Rep cells (−GP) or of BHK-Rep cells transfected with pCAGGS-M (+GP) was ultracentrifuged at 100,000 × g for 2 h. The proteins present in the pellets were separated in 4 to 12% bis-Tris gels and subsequently transferred to nitrocellulose blots. Specific proteins were detected by an anti-Gn (α Gn) or anti-Gc (α Gc) peptide antiserum or a MAb specific for the N protein (α N). The positions of the NSm, Gn, Gc, and N proteins are indicated by arrows. Molecular weight standards are indicated on the right, in thousands.
- Fig. 10.
Vaccine efficacy of RRPs. Mice were either left unvaccinated (n = 9; mock) or vaccinated (n = 10) either once (1×) or twice (2×) via the intramuscular route (IM) or subcutaneous route (SC) with 106 TCID50 of RRPs. Mice were challenged with a known lethal dose of RVFV strain 35/74 via the intraperitoneal route. The mortality rates were determined until 21 days postchallenge (d.p.c.).
