*2.5. Bromodeoxyuridine (BrdU) Incorporation Assay*

Cells were grown to ~90% confluency on 8-well chamber well slides (NUNC™ Lab-Tek™), then serum-starved for ~18 h, after which genistein treatment (0–100 μM) or 0.1% DMSO (diluent control) was added to cells in serum-containing medium with BrdU labeling reagent (Invitrogen 00-0103, Carlsbad, CA, USA). Cells were incubated for 4 h, then fixed and stained for BrdU following the manufacturer's protocol (ThermoFisher, Waltham, MA, USA). Region of interest (ROI)analysis was performed using Zeiss AxioVision rel 4.8 software.

#### *2.6. DNA Isolation and RT-PCR*

DNA was isolated from cells using the manufacturer's protocol with slight modification. Viral DNA was detected by real-time PCR using a 6-carboxyfluorescein/tetramethylrhodamine (FAM/TAMRA)-labeled probe with primers to glycoprotein G (F 5- 3- R 5- 3- ). DNA isolated from Vero-infected cells was used as a positive control. Average CT values were calculated from replicate groups then compared to the uninfected control group.

### *2.7. Indirect Virus Yield Assay*

HFFs or RMFs were grown to confluency in 12-well plates, then infected with 150 PFU of B virus for 1 h in the presence of 0–100 μM genistein or DMSO control. After absorption, the media was removed and fresh media added. After 48 h, cells/supernatants were collected, serially diluted, and added to Veros grown to confluency in 12-well plates for standard plaque assay.

#### *2.8. Plaque Reduction Assay*

Cells were grown to confluency in 12-well plates then infected with 150 PFU of B virus for 1 h in the presence of 0–100 μM genistein or DMSO control. After absorption, the media was removed and cells overlaid with 0.5% methylcellulose with the indicated concentration of genistein. To assay the effects of the drugs on virus replication, the old methylcellulose was replaced with new methylcellulose containing 50 μM genistein at 0, 2, 4, 6, and 8 h post-infection. For combination plaque assays, infected cells were treated with 50 μM genistein and 0–100 μM ACV or 0–40 μM GCV, both during and after the absorption period. In all cases, at 48 h post-infection (hpi), cells were fixed with 100% methanol and plaques were visualized and counted either by immunohistochemistry using BV+ macaque sera as the primary antibody and HRP-conjugated goat-antihuman as the secondary antibody with DAB detection or by staining with crystal violet. IC50 values were calculated using logarithmic regression line generated from a semi-log dose–response curve.

#### *2.9. Direct Inactivation Using Modified Plaque Reduction Assay*

150 PFU of B virus were incubated with 0 μM or 50 μM genistein at 37 ◦C for 15, 30, 60, 90, and 120 min. At the end of the pre-incubation period, the virus inoculum was removed, diluted 10-fold, then used to infect Veros. All treatments were carried out in duplicate. After 1 h adsorption, cells were overlaid with methylcellulose and incubated at 37 ◦C for 48 h. Plaques were then counted and the titer calculated.

#### *2.10. Virus Yield Assay*

Vero cells were grown to confluency in 6-well plates then infected with B virus at a multiplicity of infection (MOI) of 1 and incubated for 1 h to allow for viral attachment. Cells were washed with 1 mL of 50 mM citrate buffer for 2 min to remove unabsorbed viruses, then rinsed twice with sterile PBS. Next, 0–100 μM of genistein or 0.1% DMSO was added to the infected cells and infection was continued for 24 h. The infected cells were harvested by scraping and lysed by three cycles of alternating freezing and thawing. The total infectious virus yields in the presence of each drug concentrations tested were determined by titration on Vero using a standard plaque assay.

#### *2.11. Cell-Based ELISA Assay*

HFFs and RMFs were grown to confluency in 12-well plates, then infected with 150 PFU of B virus for 1 h in the presence of 0–100 μM genistein or DMSO control. After 1 h absorption, the media was removed and fresh media added. After 48 h, cells/supernatants were collected, serially diluted, and added to Vero grown to confluency in 96-well plates. A standard curve was generated from 10-fold serial dilutions of B virus (1:100–109). After 1 h absorption, the inoculum was removed, fresh media added and infection was continued for 24 h. Next, cells were fixed with 85% acetone, washed three times with borate-buffered salt solution containing 0.05% Tween 20 (BBST), blocked for one hour in ELISA-Blotto (BBST + 2.5% dry milk), and incubated with rabbit anti-gB sera (generated from injection of rabbits with a recombinant B virus gB protein, as previously described [36]) at 1:50 dilution overnight at 4 ◦C. The primary antibody was removed, cells were washed three times in BBST, incubated with goat anti-rabbit IgG-alkaline phosphatase for 1 h and washed again three times in BBST. For detection, 200 μL of p-nitrophenyl phosphate (pNPP) substrate was added to each well and incubated for 30 min at room temperature. OD values were generated by reading absorbance

on an ELISA microplate reader at 405 nm and 490 nm wavelengths. The average OD values were calculated from quadruplicates per experiment. Net OD values were obtained by subtracting the average OD values of the background control (ODbkg) from the average OD values of the test samples (ODts). A titration curve was constructed by plotting the net OD values of the virus standard versus the experimental virus dilution. The titer of the virus was determined from the curve using a cutoff valued that is determined from the background controls. We used the average of the background control OD values plus three standard deviations as the cutoff. The titer of the experimental samples was calculated from the standard curve.
