Figure 2
From: Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever
Characterization of rVSV vectors. (A) Single-cycle growth kinetics comparing recombinant vesicular stomatitis virus (rVSV) wild-type (Isolate: Indiana) tagged with green-fluorescent protein (GFP) (rVSV-GFP), the replication competent pseudotype (ΔGrVSV-CCHFV-GPCΔ), and Crimean-Congo hemorrhagic fever virus (CCHFV) (Isolate: IbAr10200) all at a multiplicity of infection (MOI 0.1) in baby hamster kidney (BHK) cells. Data shown are a mean ± standard deviation from three biological replicates, titrated by plaque assay in duplicate. (B) Fluorescence microscopy of ΔGrVSV-CCHFV-GPCΔ at MOI 0.01 infected human hepatocarcinoma (HuH-7) cells at 20x objective, with a nuclear stain of 4′,6-diamidino-2-phenylindole (DAPI) and GPC antigens stained with α-CCHFV-Gc monoclonal antibody (MAb) 8A1 and α-CCHFV-PreGN MAb 13G8 using a Fluorescein isothiocyanate (FITC) conjugated secondary, fixed and permeabilized at 24 hpi, respectively. (C) Coomassie of approximately 100 ng of loaded 20% sucrose cushioned/semi-purified and gradient purified particles on 4–16% gradient Mini-PROTEAN TGX (Tris-glycine precast) gels. 1: rVSV-GFP, 2: ΔGrVSV-CCHFV-GPCΔ, and 3: CCHFV. Particle preps were stained with Coomassie Fluor Orange. (D) Western blot of approximately 100 ng of loaded 20% sucrose cushioned/semi-purified and iodixanol gradient purified particles on 4–16% gradient TGX gels. 1: rVSV-GFP, 2: ΔGrVSV-CCHFV-GPCΔ, and 3: CCHFV. Particle preparations were stained with α-CCHFV-GC MAb 11E7 using an HRP-conjugated secondary. (E) Transmission electron micrographs of rVSV-GFP and replication competent ΔGrVSV-CCHFV-GPCΔ particles from BHK cells, semi-purified using a 20% sucrose cushion or purified using iodixanol gradient centrifugation, negatively stained with 2% aqueous uranyl acetate, immunolabeled with α-CCHFV-Gc MAb 11E7 and a secondary 15 nm gold labeled secondary. Samples were fixed with 2% glutaraldehyde. Images were adjusted for brightness, contrast, and formatted for size for display purposes. Coomassie gel and western blot images were cropped from the same image file at same imaging parameters for ease of viewing. Gel and blots originals are available in supplemental information. Other original raw data files available upon request.
