We have performed selection using a tissue culture infectious dose 80 % (TCID₈₀) of virus . Thus, this section identifies the dilution factor of a stock concentration of virus needed to achieve 80 % infection of cells. We then try to maintain ~90 % inhibition for each round of selection (Subheading 3.5) using inhibitor concentrations determined in Subheading 3.4.

Culture 293 cells to confluency in 12-well culture dishes.

Dilute 5 μL purified HAdV-5.polF421Y from Subheading 3.1, step 12, into 1.5 mL complete DMEM.

Create ten serial threefold dilutions of this virus by adding 0.5 mL of the previous dilution to 1.0 mL complete DMEM and mixing thoroughly by gentle vortexing.

Remove media from each well of 293 cells and replace with 1 mL/well serially diluted virus . Replace one well with fresh complete DMEM without virus as a negative control. Incubate at 37 °C, 5 % CO₂.

Quantitate eGFP expression 18–24 h p.i. by using flow cytometry to determine the percent of eGFP-positive cells in each well compared to uninfected cells or by gently washing the 12-well plate with PBS, adding 1 mL PBS/well, and scanning the plate with a 488 nm laser using the 520 bp 40 filter on a Typhoon 9400, 9410, or Trio variable mode imager. For Typhoon images, we use ImageJ software to quantify the integrated density of relative fluorescence units (RFUs) for each well above background fluorescence in the uninfected control well.

Plot either the percent positive cells from flow cytometry or RFUs from the Typhoon scan against a log transformation of the dilution factor for each well.

Perform nonlinear regression analysis using Prism software using the function “log(agonist) vs. response—Find ECanything,” constraining “F” to 80. The resulting ECF value will be the dilution of the starting virus stock that will yield 80 % maximal infection of the culture (see Note 6 ).

This assay determines an inhibitor concentration that results in 90 % inhibition of infection (IC₉₀).

Culture 293 cells to confluency in 12-well culture dishes.

Dilute purified HAdV-5.polF421Y from Subheading 3.1 step 12 into 6 mL complete DMEM by a dilution factor equal to half of the value of ECF obtained in Subheading 3.3 step 8. This will yield a 2× virus stock.

Create ten serial threefold dilutions of the inhibitor by first diluting it into a total of 0.75 mL complete DMEM and then adding 0.25 mL of the previous dilution to 0.5 mL complete DMEM and mixing thoroughly by gentle vortexing.

Mix 0.5 mL of each inhibitor dilution with 0.5 mL 2× virus stock (see Note 7 ).

Remove media from each well of 293 cells and replace with 1 mL/well virus plus serially diluted inhibitor. Replace one well with fresh complete DMEM without virus as a negative control. Replace one well with 2× virus diluted to 1× with complete DMEM alone as a positive control. Incubate at 37 °C, 5 % CO₂.

Quantitate eGFP expression 18–24 h p.i as in Subheading 3.3 step 6.

Plot either the percent positive cells from flow cytometry or RFUs from the Typhoon scan against a log transformation of inhibitor concentration for each well.

Perform nonlinear regression analysis using Prism software as in Subheading 3.3, step 8, constraining “F” to 10. The resulting ECF value will be the IC₉₀ of the inhibitor.

Culture 293 cells to confluency in two 12-well culture dishes.

Dilute purified HAdV-5.polF421Y from Subheading 3.1, step 12, into 2 mL complete DMEM by a dilution factor equal to half of the value of ECF obtained in Subheading 3.3 step 8. This will yield a 2× virus stock. Dilute 0.1 mL of 2× virus with 0.9 mL complete DMEM to yield 0.2× virus (see Note 9 ).

Dilute inhibitor in 0.5 mL complete DMEM to a concentration equal to twice the IC₉₀. This will yield a 2× inhibitor stock.

Mix 0.5 mL 2× virus with 0.5 mL complete DMEM (no inhibitor control). Mix 0.5 mL 2× virus with 0.5 mL 2× inhibitor (treated sample).

Mix 0.5 mL 0.2× virus with 0.5 mL complete DMEM to create a sample in the absence of inhibitor that will mimic the infection level of the inhibited sample (passaging control).

Incubate all samples under the conditions used in Subheading 3.4, step 5, (see Note 7 ).