Fig 1: Type I IFNs do not potently protect neurons from RVFV infection.(A-D) Human U2OS cells (A,C), or rat cortical neurons (B,D) were treated with vehicle or universal IFNα for 4h before infection with RVFV (24hpi, U2OS: MOI 1, neurons: MOI 0.3). Immunofluorescence and automated microscopy were used to detect RVFV Gn (green) or neuronal MAP2 (magenta). A and B show representative images of cells treated with vehicle or 1.2 x 104 Units / mL at 10x magnification, scale bars = 350μm. C and D show quantification of relative infection (red squares) and survival (black circles) in U2OS (C) or neurons (D). Automated image analysis was used to determine the percentage RVFV Gn positive U2OS cells, or the percentage of neurons (MAP2 positive cells) positive for RVFV Gn. Infection and survival normalized to % infected or total cell number in vehicle treated, infected cells. IC50 values were determined by nonlinear regression. n = 3. (E,F) U2OS were treated with vehicle or 1.2 x 104 U/mL uIFNα for 4h before RVFV infection (MOI 1). At 15hpi viral titers in the supernatant were measured by plaque assay (E) or at 24hpi viral RNA was quantified by qPCR (F). LoD = limit of detection. *P = .0167, **P = .0028 (G-H) Relative RNA expression of interferon stimulated genes in U2OS treated with 1.2 x 104 U/mL of uIFNα for 4h was measured by qPCR. *P = .0202, **P = .0017 (I-J) Neurons were treated and analyzed as in E and F, but infected at an MOI of 0.1. I: *P = .0137, J: *P = .041 (K-L) Neurons were treated and analyzed as in G and H. **P = .0013, ***P = .0005 (M) Quantification of relative RVFV infection in neurons treated with rat IFN alpha (dark blue circles), rat IFN beta (teal squares), or rat IFN gamma (light blue triangles). Images acquired as in (D), n = 3. Dotted line represents 50% reduction relative to control. For all qPCR experiments, expression was calculated relative to vehicle, normalized to GAPDH (human) or Gapdh (Rat) as a loading control. Symbols and bars represent mean, error bars = SEM. Statistical analyses were performed using student’s t test on log10 transformed titers (E,I) or Welch’s t test (F-H,J-L).