Fig. 9. NSs forms nuclear filaments in the brain of infected animals.

a BALB/c mice were inoculated intraperitoneally with 100 pfu of either RVFV or its natural mutant clone 13 that lacks NSs expression (RVFV ΔNSs C13). When the first disease symptoms appeared, animals were sacrificed and brains collected, fixed, and subjected to immunohistochemistry staining against NSs or N. High magnification images of areas where viral replication occurs are shown. Results are representative of three independent experiments. Scale bar, 50 µm. b Shows a whole slide of a mouse brain infected with RVFV as analyzed in a. The black dashed box indicates the area of the brain where N and NSs were expressed. Note that only N staining is presented here. Scale bar, 1 mm. Brainstem tissues exposed to either RVFV or RVFV ΔNSs C13 were subjected to immunofluorescence staining against N c and NSs d and visualized by high-speed confocal microscopy (left panels). Series of Z-stacks were used to generate 3D-reconstructions with IMARIS software (right panels). The images show the cellular location of N c and NSs d in infected brainstem cells. Nuclei appear in blue and the proteins N c and NSs d in red. Experiments were repeated independently three times with similar results. Scale bars, 10 µm. e Brain tissues from mice infected with RVFV and RVFV ΔNSs C13 were permeabilized with a buffer containing Triton X-100 (0.5%) and Tween-20 (0.5%) before staining with ThS (1%) and Abs against NSs. Tissues were imaged with a fluorescence wide-field microscope. ThS appears in blue and NSs in red. White arrowheads show colocalizing ThS and NSs signals. Higher magnifications of NSs filaments are shown (white numbers and squares). Images are representative of three individual animals. Scale bars, 10 µm.