Single molecule vRNA FISH of NSR infected cells.

<p>(A) Schematic presentation of the experimental setup. (B) Maximal titers and SDs of wild-type RVFV and NSR stocks. (C) Spatio-temporal distribution of genome segments in NSR infected cells. Vero cells were infected at MOI 0.1 with NSR and cells were fixed at 2,4,6,8 and 10 hpi. Cells were subsequently probed against the S segment (N gene, red) using quasar 670 labelled probes and against the L-segment (polymerase gene, green) using quasar 570 labelled probes. Cell nuclei were visualized with dapi (blue). Images were taken using a wide-field microscope. Magnified images of the squared regions are shown at the right of each panel. The merge images show the spatial relationship between all the different channels.</p

Growth of codon shuffled RVFV variants.

<p>(A) Schematic presentation of the viruses with shuffled or codon-optimized genes. (B) Part of the shuffled M segment and codon-optimized N gene sequence. (C) Growth curve of the indicated viruses in Vero cells infected at MOI 0.01. Supernatants were harvested at different time points and titrated on Vero cells.</p

Colocalization coefficient of vRNAs in RVFV infected cells.

<p>Vero cells were infected at MOI 0.1 with RVFV and cells were fixed at 5 hpi. Cells were subsequently probed against (A) the S segment (N gene, green) using fluorescein labelled probes, the M segment (Gc gene, red) using quasar 670 labelled probes and the GAPDH mRNA using quasar 570 labelled (blue) probes or against (B) the S segment (N gene, green) using fluorescein labelled probes, the M segment (Gc gene, red) using quasar 670 labelled probes and the M-segment using quasar 570 labelled (Gn gene, blue) probes or against the (C) S segment (N gene, green) using fluorescein labelled probes, against the M segment (Gn gene, red) using quasar 670 labelled probes and against the L-segment (polymerase gene, blue) using quasar 570 labelled probes. Cell nuclei were visualized with dapi (cyan). Images were taken using a wide-field microscope. The level of colocalization is determined by calculation of the Pearson’s colocalization coefficient. Bars represent means and SDs of 4 independent measurements.</p

Single molecule vRNA FISH of multiple RVFV infected cells.

<p>RVFV infected Vero cells (MOI 0.1) were fixed at 7 hpi. Cells were subsequently probed against the S segment (N gene) using fluorescein labelled probes (green), against the M segment (Gn and Gc gene) using quasar 670 labelled probes (red) and against the L-segment (polymerase gene) using quasar 570 labelled probes (blue). Cell nuclei were visualized with dapi (cyan). The picture shows that the molar ratios of different vRNAs vary among cells. Most likely the presented cells were infected with either a particle containing a single copy of each genome segment, a particle lacking the M-segment, and a particle with an additional M-segment, respectively. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005800#ppat.1005800.s002" target="_blank">S2 Fig</a>, additional images are presented.</p

Genome segment composition of immobilized virions.

<p>(A) Schematic presentation of the experimental setup. (B) Control experiment to validate the visualization of immobilized RVFV virions and their genome segments. Immobilized virions were incubated in the presence (left image) or absence (right image) of the 4-39-cc mAb targeting the Gn glycoprotein followed by incubation with a DyLight 350 labelled conjugate. (C) Validation of the ability to determine segment colocalization inside immobilized virions. Immobilized RVFV virions were hybridized with a quasar 570 labelled Gn gene-specific probe set (green) and a quasar 670 labelled Gc gene-specific probe set (red). Since the Gn and Gc coding regions are both present on the M-genome segment, Gn and Gc gene-specific spots should show a high level of colocalization (in yellow). Colocalization percentages were on average 80% (D) Immobilized RVFV virions were hybridized with S segment specific probe sets (N gene, fluorescein, green), M segment specific probes sets (Gn and Gc, quasar 670, red) and L segment specific probe sets (polymerase, quasar 570, yellow) and incubated with the 4-39-cc Gn specific mAb in combination with the DyLight 350 labelled conjugate (blue). In each channel, spots were subsequently detected with the ComDet plugin of ImageJ and merged images of the four different channels are presented. (E) Quantification of the different genome compositions inside virions. About 800 virions were analysed for their genome content using the ComDet plugin of ImageJ.</p

Humoral and cellular immune responses elicited by vaccination of mice with NSR or NSR-Gn.

<p>(A) VNT titers in sera collected from mice vaccinated with NSR or NSR-Gn before vaccination (DPV -1) and 13, 22 and 29 DPV. Bars represent average titers (n = 6) of each group with standard deviation. The detection limit of the assay is depicted by the interrupted line. (B) Detection of IFN-γ-producing splenocytes isolated from mice vaccinated with NSR or NSR-Gn. Splenocytes were isolated and seeded at a density of 5×10⁵ cells/well in triplicate and stimulated for 12 hours with the indicated peptides or the ectodomain of Gn. Bars represent an average number of IFN-γ producing cells (n = 4) per group with standard deviation. The non-parametric Mann-Whitney test was used for statistical analysis and statistical significance between the groups is depicted by asterisks (*p<0.05; **p<0.01).</p

Detection of viral RNA in plasma by qRT-PCR.

<p>Plasma samples were collected daily at the first 7 days post challenge (DPC) and subsequently on DPC 9, 11, 14 and 21. Viral RNA copy numbers detected in individual animals of the mock-vaccinated group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) and high-dose group (H1–H7) are depicted.</p

Construction of the S-Gn segment and expression of Gn.

<p>(A) Schematic representation of the S segment of RVFV (upper panel) and the S segment in which the NSs gene is replaced for the codon-optimized Gn gene (lower panel). Distribution of Gn in Rep-Gn cells (B and E) and in BHK cells infected with NSR-Gn at an MOI of 0.5 (C and F). Panels D and G represent BHK control cells. Upper panels represent permeabilized cells and lower panels represent nonpermeabilized cells. The cells were stained with an anti-Gn monoclonal antibody and a Texas Red-labeled secondary antibody. Nuclei were visualized by DAPI staining.</p

Detection of anti-N antibodies by ELISA.

<p>Sera were obtained 7, 14 and 21 days post vaccination (DPV) and at 0, 7, 14 and 21 days post challenge (DPC). Titers are expressed as percentage competition ratio of the optical densities (OD) of the sample and the OD of the negative control (%S/N). All values lower than 40% are considered positive, between 40–50% are considered doubtful and above 50% are considered negative. The 40% and 50% cut-offs are represented by solid and interrupted lines, respectively. Results obtained from analysis of each individual animal from the mock-group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) or high-dose group (H1–H7) are depicted.</p

Rectal temperatures of vaccinated and mock-vaccinated lambs before and after challenge with RVFV.

<p>Fever was defined as a rectal body temperature above 40.5°C (interrupted line). Body temperatures of mock-vaccinated lambs (C1–C8) and lambs vaccinated with a low dose (L1–L8), medium dose (M1–M8) or high dose (H1–H7) of NSR-Gn are depicted individually.</p