A549 human epithelial [America Type Culture Collection (ATCC), Manassas, VA, USA] cells were cultured in T25 flasks using F12-K media supplemented with 10% Fetal Bovine Serum (FBS) and 1 µg/mL gentamicin. Trypsin EDTA (0.25%) was used to subculture cells. The cells were maintained at 37˚C and 5% CO₂.

A recombinant strain of HSV-1, GHSV-UL46, which contains the sequence for green fluorescent protein (GFP) fused to the tegument protein pUL46, was used for all experiments [33] (ATCC, Manassas, VA, USA). Virus was passaged in T25 flasks and A549 cells were allowed to reach complete cytopathic effect (CPE). The viral media containing virus was then collected, centrifuged, and the supernatant stored at −80˚C.

EGCG (>90%) purchased from Pulimeidi Biotechnology Co., Ltd. (Hangzhou, China) and EGCG-S (US Patent 20120172423) modified by and purchased from Camellix, LLC, Augusta, GA, were dissolved in dimethyl sulfoxide (DMSO) and ethanol to prepare initial 5 mM stock concentrations. EGCG and EGCG-S were then diluted in F12-K media to desired concentrations of 12.5, 25, 50, 75, and 100 µM.

A549 cells were plated in 6 well plates, grown for 24 hours and treated with various concentrations (25, 50, and 75 µM) of EGCG and EGCG-S. After 1 hour the EGCG and EGCG-S were removed by aspiration and the cells were washed with phosphate buffered saline (PBS). Media was then added to the wells and cells were incubated at 37˚ under 5% CO₂ and observed every 24 hours for a period of three days after treatment. Cells were examined, using an inverted microscope, at 400× magnification to observe morphological changes in the cells.

A549 cells were plated in 6 well plates and treated with appropriate concentrations of EGCG and EGCG-S for 1 hr. The viable cells were then stained and counted using trypan blue and hemocytometer. After 1 hr, the EGCG and EGCG-S were aspirated and the cells were washed with PBS. F12K media was then placed in each well and cells were incubated for 24 hrs. Cells, including controls of ethanol (ETOH) and DMSO, were trypsinized and harvested. Cells were then stained with trypan blue, which stains dead cells blue while live cells are not stained, and counted using a hemocytometer. Cell viability was determined by the proportion of viable cells to control levels at different treatments, and illustrated as relative cell viability with the controls as 100% viable.

Cell viability (%) = total viable cells (unstained)/total cells (stained and unstained) × 100

A549 cell suspensions (100 µL) were plated in separate wells in a 96 well plate and after 24 hours cells were treated with appropriate concentrations of EGCG and EGCG-S for one hour, then aspirated (as described in section 2.5). After 24 hours, 10 µL of WST-1 reagent (Roche Diagnostics, Indianapolis, IN, USA) was then added to the samples; the plate was gently rocked, then incubated at 37˚C under 5% CO₂ for 30 minutes. The absorbance level for each well of the plate was measured at 450 nm in a 96 well plate reader. The assay was performed in triplicate.

A549 cells were plated in a 96 well plate and after 24 hours, 100 µL of virus was treated with 100 µL EGCG-S (final concentrations are 25, 50, 75, and 100 µM) for one hour. After treatment, cells were infected with treated and non-treated virus and incubated for one hour at 37˚C and 5% CO₂. Any unabsorbed virus was aspirated and replaced with 100 µL of F12-K media. After 72 hours, 10 µL of ToxGlo reagent (Promega, Madison, WI, USA) was added to all wells containing samples (controls included 100 µL of 10% FBS-F12-K media both with and without the ToxGlo reagent) then incubated at 37˚ and 5% CO₂ for 15 minutes. The plate was then read through a luminometer and relative light unit (RLU) values of each well were recorded. The assay was performed in triplicate.

The equation for calculating the percent of inhibition is shown below:

% inhibition = [(Cells + treated HSV1) − (Cells + HSV1)]/[Cells only − (Cells + HSV1)]

A549 cells were plated in 6 well plates and were infected with 200 µL of various dilutions of treated or non treated HSV-1 for one hour incubation, with intermittent rocking at 37˚C and 5% CO₂. After one hour, unabsorbed virus was aspirated and 2.5 mL of media was added to each well and incubated at 37˚C and 5% CO₂. Morphological changes were observed at day 3 post-infection.

A549 cells were grown on glass cover slips within 12 well plates and were infected with treated or non-treated virus for one hour. Twelve hours post infection, cells were stained with 300 µL of 300 nM DAPI (4,6-diamidino-2-phenylindole) stain for 5 minutes at 37˚C in the dark. Cells were then fixed with polyethylene glycol solution for 15 minutes at 20˚C. The glass cover slip containing cells were then glued to a slide using a drop of clear nail polish. Cells were then visualized and were examined under a Leica SP5 scanning confocal microscope under 10× or 63×/1.4 NA water Plan Apo objectives at Vassar College (Poughkeepsie, NY).

To study the effect of ECGC and EGCG-S on A549 human epithelium cells, different concentrations (25, 50 and 75 µM) of EGCG and EGCG-S were used. The morphology was observed at 24, 48 and 72 hours after the treatment as shown in Figure 1. There were no observed effects of dissolving in ethanol or DMSO; therefore, the study was continued using DMSO as the solvent. Since the polyphenols were dissolved in DMSO (the media contained < 2% DMSO in solution) the effect of DMSO was also studied. The results indicated that DMSO at these concentrations (0.5%, 1%, and 1.5%, respectively) had no effect on cell morphology; DMSO treated cells did not appear different from the control cells (Figure 1(a) and Figure 1(b)). The alterations of cell morphology study demonstrated that tested concentrations of both EGCG and EGCG-S are not toxic to A549 cells. Concentrations of 25 μM, 50 μM, and 75 μM of EGCG and EGCG-S do not show any morphological changes and no cytopathic effects in growing A549 cells (Figure 1(c)). These results suggested that the cells can tolerate EGCG and EGCG-S at concentrations up to 75 µM.

The cell viability was determined using the Trypan blue assay. After treatment with Trypan blue, cells were counted using a hemocytometer. The percent (%) of viability was calculated as explained in the Materials and Methods. The results are shown in Figure 2. It is indicated that ETOH and DMSO have no effect on

cell viability, with % viability of 98% and 100%, respectively, when compared with the control. For EGCG the % of viability ranges from 89% to 95%; for EGCG-S, the % of viability ranges from 83% to 87%. The cell proliferation assay using WST-1 was carried out to analyze the proliferation activity of cells. The results are shown in Figure 3. The data indicated that concentrations of 25, 50 and 75 µM of EGCG and EGCG-S do not affect cell proliferation. These concentrations could be used to treat the HSV-1 virions and would not affect the cells. The cell viability assay indicates that there is a slight decrease in the number of viable cells with increasing concentrations of EGCG and EGCG-S. However, EGCG and EGCG-S do not show any effect on A549 cells and the cell proliferation assay confirms that EGCG and EGCG-S do not affect the growth of A549 cells at the concentrations tested.

The ToxGlo antiviral assay was used to measure cell viability in infected cells in response to EGCG-S treatment of the virus. The calculated percentage of inhibition ranged from ~80% at the highest concentration of EGCG-S while the maximum viral inhibition occurred at a concentration of 75 µM (>90%) (Figure 4). Antiviral assay using luminescence suggests that EGCG-S treatment inhibits HSV-1 by up to 90%.

The equation for calculating the percent of inhibition is given in Section 2.7 of the materials and methods.

A549 cells were plated in 6 well plates and were infected with treated and non treated HSV-1 for one hour. After an hour, unabsorbed virus was aspirated and media was added. Morphological changes were assessed at three days post-infection. The results are shown in Figure 5. The uninfected cells showed the typical flattened, elongated morphology characteristic of epithelial cells (Figure 5(a)). The cells infected with HSV-1 demonstrate properties of cytopathic effects; the cells are rounded and lifted from the monolayer (Figure 5(b)).

HSV-1, treated with 75 µM of EGCG-S, infected cells showed very similar cell morphology to the untreated control cells (c). This study suggests that 75 µM of EGCG-S was able to inhibit HSV-1 infection in cultured A549 cells thus the cells’ morphology remained similar to the morphology of the uninfected control.

HSV-1 used in this study contains the sequence for GFP fused to the sequence for the tegument protein, UL-46 [33]. GFP expression indicates a late stage in the HSV-1 replication cycle. DAPI stain was used to determine the morphology and number of cell nuclei. Figure 6(a) shows uninfected A549 cells. Figure 6(b)

indicates extensive HSV-1 infection. However, when HSV-1 is treated with 75 µM EGCG-S there is minimal GFP expression as shown in Figure 6(c). The confocal microscopy study suggests that 75 μM EGCG-S treatment of HSV-1 results in a drastic reduction in GFP expression and hence, inhibition of HSV-1 when observed at 12 hours post infection.