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After 1 h incubation, the inoculum was removed and the wells were washed with PBS to remove free virus (inoculum and washes are referred to as unabsorbed virus in Figure ?Figure6A).6A). Thereafter, the respective drug media (or vehicle control) was put back onto the wells and culture supernatants were collected and viral titers were determined by plaque assay. As shown, sorafenib prevented an increase in viral titers over the entire experimental time course. Figure 6 Comparison between experimental data and the computational model for HSAEC infection. (A) Virus in the culture supernatant (2�C24 hpi) after the addition of sorafenib (gray bars) or DMSO-vehicle (black bars). Data are average pfu/ml �� standard ... A computation model of RVFV infection was developed that included 4 variables and 5 parameters (see Materials and Methods Section for details). A schematic of the model is shown in Figure  ?Figure6B.6B. Model results obtained using these initial conditions and parameter values (as discussed in Materials and Methods Section) are shown in Figure ?Figure6C.6C. As shown, there is excellent agreement between the model and actual pfu/ml data. Indeed, the model predictions at 8, 16, and 24 hpi were 5.75 �� 101, 4.85 �� 105, and 1.02 �� 106 pfu/ml, respectively, and the actual pfu/ml data at these time points (from Figure ?Figure6A)6A) were 3.10 �� 103, 1.40 �� 105, and 1.05 �� 106 pfu/ml. To account for biological restrictions, the minimum pfu/ml value was bounded to 1 in the data plotted on a log10 scale (Figure ?(Figure6C).6C). Additionally, 700 pfu/ml was subtracted from all data points prior to plotting Figure ?Figure6C6C to account for background levels of virus detected in the culture supernatant at all time points. These background levels are not related to the infection dynamics, but are experimental artifacts that are primarily the result of (i) virus particles settling on plastic rather than cells that are easily washed off, (ii) particles that are loosely associated with cell surfaces that become dislodged during washing, and (iii) from HSAECs (with internalized viruses) that are http://www.selleckchem.com/products/ulixertinib-bvd-523-vrt752271.html actually dislodged during washing. Including this background level of virus has negligible impact on the later time points where the titers are 105 pfu/ml or higher, but in contrast these artifacts can greatly distort data at the early time points where very little de novo synthesized virus is expected to be in the culture supernatant. Because these results suggest the model captured the natural infection dynamics very well, the model was used to begin addressing what step in the virus life cycle could be altered by sorafenib. To this end, pfu/ml over time was modeled after each parameter was manually adjusted. This modeling technique is a type of preliminary sensitivity analysis and begins to test different hypotheses for the drug mechanism of action.