Experiments with recombinant EBOV were approved by the Institutional Biosafety

Committee (IBC) and performed in BSL4 containment at the Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), ZD6474 datasheet National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), following standard operating procedures. TCID50 assays were performed by infecting Vero cells in 96-well format with a tenfold dilution series of samples, infecting 4 wells per sample and dilution step (for stock titrations 8 wells per sample and dilution step were infected). CPE-based TCID50 assays were read after 18 days, to ensure a definitive distinction between infected and uninfected wells even at higher dilutions. Luminescence-based TCID50 assays were read by measuring luciferase activity at the

indicated time points, as Selleck IOX1 described above. Wells were deemed positive when reporter activity was at least 1 log10 higher than in uninfected control samples and not more than 2 log10 lower than directly neighboring wells, to compensate for cross-talk between different dilution steps. To further eliminate the possibility of crosstalk between different samples, at least one column was left empty between these samples when measuring luciferase activity. Titers were calculated using the Spearman–Kaerber method (Wulff et al., 2012). For the luminescence-based direct titration (LBT) assay, 50 μl of undiluted and 1:1000 diluted unknown samples were used to infect Vero cells in 96-well format in a total volume of 100 μl, along with known virus standards (5 × 105, 5 × 104, 5 × 103, 5 × 102 TCID50/ml). All infections were done in triplicate. 48 h post-infection, luciferase activity Glutamate dehydrogenase was measured as described above, and a linear regression curve based on the virus standard samples was used to calculate

the titer of the unknown samples based on their luciferase activity. For testing of neutralizing antibodies, 100 TCID50 of rgEBOV-luc2 were incubated with the previously characterized neutralizing antibodies 133/3.16 or 226/8.1 or the non-neutralizing antibody 42/3.7 (Takada et al., 2003) at the indicated concentrations in a total volume of 100 μl in a 96-well plate. After 1 h, 2 × 104 Vero cells in 100 μl were added to each well. After 2 days luciferase activity was determined as described above. For testing of siRNAs, 293 cells at a confluency of ∼50% were transfected with the indicated amount of L-specific Dicer substrate siRNA (DsiRNA) duplex (5′-rGrArUrCrArArUrUrUrArUrArUrArCrArGrCrUrUrCrGrUrArCrArA-3′, 5′-rGrUrArCrGrArArGrCrUrGrUrArUrArUrArArArUrUrGrArTrC-3′; Integrated DNA Technologies) or control DsiRNAs (NC1 and DS Scrambled Neg, Integrated DNA Technologies). To this end, the DsiRNA was diluted in 5 μl Opti-MEM (Invitrogen; all amounts are per well), and 0.3 μl Lipofectamine 2000 (Invitrogen) in 5 μl Opti-MEM was added to the diluted DsiRNA.