This would provide an advantage since CPE-based TCID50 assays require a relatively ZD1839 mouse long incubation to allow a clear distinction between infected and uninfected wells, particularly at higher dilutions. To this end, we performed TCID50 assays on a virus stock with known concentration, measured luciferase activity after 1, 2, 3, 4, 7 and 10 days to determine infected vs. uninfected wells, and then calculated a TCID50 titer based on these data (Fig. 2A). While at 1 and 2 days after infection

the calculated titer did not concur with the actual titer, after 3 days and at all later time points the luminescence-based TCID50 matched the actual titer as previously determined by CPE-based TCID50 analysis, indicating that this assay reliably allows rapid titration of rgEBOV-luc2 within 3 days, and is able to detect single infectious particles (as determined by conventional TCID50) with the same sensitivity as conventional TCID50 assays.

When analyzing the data from the TCID50 assay, we observed that the reporter signal declined about 1 log10 for each of the 10-fold dilution steps (data not shown), which lead us to explore the possibility of a linear relationship between reporter activity and input virus titer. To this end, we performed a 0.5 log10 dilution series of our virus stock, and determined reporter activity Natural Product Library cost for each Palmatine sample 2 days post-infection (Fig. 2B). Our data show that there is a clear linear relation between the input titer and luciferase activity in the range between 102.7 TCID50/ml and 105.2 TCID50/ml. At higher titers we no longer observed an equivalent increase in reporter activity, most likely due to the fact that these

signals exceeded the linear dynamic range of the luminometer, whereas at lower titers we observed occasional samples that showed only background activity, suggesting that at these low concentration stochastic effects (i.e. an increasing probability that a sample of a highly diluted virus contains no infectious particles) start to significantly influence the outcome of the assay. Based on these findings, we developed a luminescence-based direct titration assay, in which the luminescence of an unknown sample is compared to a known standard dilution series. In order to increase the linear range of this assay, we measured both undiluted and 1000-fold diluted samples, to circumvent the fact that higher titers exceeded the linear dynamic range of the luminometer. To evaluate this assay, unknown samples were titered both using luminescence-based TCID50 assays and LBT assays, and both titration methods showed good concurrence (Fig. 2C), indicating that the LTB assay can be used to accurately titer rgEBOV-luc2 samples within 2 days. One obvious application for the rgEBOV-luc2 virus is in the screening for antivirals.