Virus yield reduction assays

The effectiveness of the compounds was evaluated by virus yield reduction assay using Vero E-6 cells (American Type Culture Collection, Manassas, VA). The cells were maintained in Modified Eagle’s media (MEM) with 10 % fetal bovine serum (FBS), and 1X GlutaMax (Invitrogen, Carlsbad, CA). RVFV and LASV were evaluated using 90 % confluent cells in 6-well plates. Medium was removed from cells which were infected at an MOI of 0.1 in 200 μl of medium (MEM) containing 5 % FBS and no antibiotics that contained various concentration of compound. Plates were incubated 1 h at 37 °C/5 % CO₂ with rocking every 15 min. Media containing virus was removed and plates were washed 3X with media or phosphate buffered saline (PBS; Invitrogen). After washing, media that contained various concentrations of compound was added and plates were incubated at 37 °C/5 % CO₂. Supernatant was collected on day 1 and 2 post-infection (PI) for viral titer determination by plaque assay.