5, 0.05, 0.005 and 0.0005, respectively), and measured luciferase activity after 1, 2, 3, 4, 7 and 10 days. We did not test a lower infectious doses of 100 TCID50 per well, since at such a low dose stochastic effects would start to play an unacceptably large role, resulting in only 50% of the tested wells being infected. For comparison, infections were also performed using rgEBOV-eGFP, using eGFP fluorescence as a read-out, and rgEBOV-WT, using CPE as a read-out. For rgEBOV-eGFP, the first isolated eGFP-positive cells appeared after 2 days in wells receiving the highest dose, and after 4 days using

102 TCID50 (Fig. 3A). However, the eGFP-positive cells were initially very rare and locating them required extensive scanning of the well. A robust eGFP-signal throughout most of the well became apparent after 3 to 4 days using higher doses Bosutinib chemical structure (104 and 103 TCID50), but only after 7 days at lower doses (102 TCID50). Similar results were obtained using rgEBOV-WT, with mild isolated CPE becoming apparent between 3 and 7 days post-infection, depending on the infectious dose, and clear CPE throughout the well being visible at day 4 post-infection using the highest dose, and 7 to 10 days post-infection for the other doses (Fig. 3B). In contrast, an increase in reporter activity was already detected using rgEBOV-luc2

for all infectious doses at day 1 Trametinib post-infection (Fig. 3C). When determining the Z′-factor (Zhang et al., 1999), infectious doses of 103 TCID50 or higher yielded Z′-factors of >= 0.5 already at day 1, indicating a very robust assay, whereas the lower doses of 102 and 101 TCID50 yielded a Z′-factor of >= 0.5 at days 2 and 3 post-infection,

respectively (Fig. 3D). When comparing this to the results obtained with rgEBOV-eGFP and rgEBOV-WT, it becomes apparent that rgEBOV-luc2 allows much quicker turnaround times for screening assays, and represents an extremely robust assay even at low infectious doses (Fig. 3D). For comparison, all drug-screening efforts with eGFP-expressing EBOV have thus far used high infectious doses (MOI = 5), with readout 2 days post-infection (Panchal et al., 2010 and Panchal et al., 2012). As a proof-of-concept that rgEBOV-luc2 is feasible for use as an antiviral www.selleck.co.jp/products/erastin.html screening tool, we assessed the effect of two well-characterized neutralizing antibodies as well as the effect of a DsiRNA directed against the viral polymerase L. For testing of the neutralizing antibodies, 100 TCID50 (equivalent to an MOI of 0.005) of rgEBOV-luc2 were preincubated with the previously characterized neutralizing antibodies 133/3.16 and 226/8.1 or the non-neutralizing antibody 42/.37, and then used to infect Vero cells. After two days, reporter activity was measured. As expected, there was a clear drop in reporter activity for both neutralizing antibodies, with 226/8.1 showing a 2.