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. 2019 Sep 8;11(9):834. doi: 10.3390/v11090834

Figure 1.

Figure 1

Rescue of RVFV BJ01 and a mutant lacking the NSs gene. (A) Schematic of the T7 RNA polymerase-dependent reverse genetics system for construction of recombinant viruses Adapted from the Box 2 figure of Orthobunyaviruses: recent genetic and structural insights [18]. (B) Cytopathic effect (CPE) of rescued virus in BHK-21 cells. BHK-21 cells were mock treated or infected with rWT at an MOI of 5, images were captured at 48 h p.i. Bars, 20 μm. (C) Construction of the plasmid encoding the S segment in which the NSs gene was replaced by eGFP. (D) Fluorescence microscopy analysis to verify the successful rescue of r△NSs-eGFP. BHK-21 cells were infected with r△NSs-eGFP (MOI = 5) and the fluorescence images were acquired at 72 h p.i. Bars, 20 μm. (E) Western blot analysis of infected cells with anti-N, anti-NSs and anti-Tubulin antibodies. Vero cells were infected with WT, rWT and r△NSs-eGFP at an MOI of 1 and cell lysates were harvested at 24 h p.i. (F) Plaque assay of RVFV WT and rescued viruses (rWT and r△NSs-eGFP) on Vero cells. After 7 days of incubation, plaques were stained with crystal violet.