Table 1.
Summary of biological and physical titers of wild type HAdVs used in this study.
Figure 1.
Plaque morphology of HAdVs on A549 cells.
HAdVs (HAdV-C5, D9, D51, E4, and F41) were serially diluted with medium containing 2% FBS and A549 cells were infected with HAdV at the range of dilution of 5.0×10−8 and 5.0×10−9 for 1 hour. We performed plaque assay as described in the Materials and Methods section. At 14 days post-infection, we stained cells with 2 ml of medium containing 0.75% agar and 0.033% neutral red in order to visualize individual single plaques on A549 cells. Photographs of plaque morphology of HAdVs were taken with a digital camera.
Table 2.
Host cell lines used for viral propagation and plaque sizes of HAdVs.
Figure 2.
Release of HAdV-D9 from infected cells to culture medium.
A549 cells were infected with HAdV-C5 or HAdV-D9 at an MOI of 10 PFU/cell and harvested at various time points. Samples to measure the infectious titer of HAdVs were prepared from infected cells harvested along with culture medium (A), a fraction extracted from infected cells without culture medium (B), and culture medium (C). Infectious titers of HAdVs contained in each fraction were measured by triplicate TCID50 assays. Data points represent mean+standard error of the mean (n = 3). Unpaired student t-test analysis was performed with respect to HAdV-C5 at each time point and significance is indicated by *P<0.05.
Figure 3.
Cellular uptake of HAdV-D9 independently of human CAR.
Cells (CHO and CHO-hCAR) were infected with HAdV-C5 or HAdV-D9 at an MOI of 100 genomes/cell, and total cell DNA extracted from infected cells were used for qPCR analysis. (A) Cellular uptakes of HAdVs in CHO and CHO-hCAR were represented as a fold of genome transfer in HAdV-C5-infected CHO cells at 1 hour post-infection and normalized by cellular uptake of HAdV-C5 in CHO cells; HAdV-C5 (black bars) and HAdV-D9 (white bars). (B and C) Cellular uptakes of HAdVs in a time-dependent manner in CHO (B) and CHO-hCAR (C). Total cell DNA was extracted from infected cells at 0, 30, and 60 min post-infection and used for qPCR analysis; HAdV-C5 (black squares) and HAdV-D9 (white squares). Data points represent mean+standard error of the mean (n = 3). CHO; human CAR negative CHO cells and CHO-hCAR; CHO cells stably expressing human CAR. (D) CHO-hCAR cells were treated with the HAdV-C5 fiber knob protein at a final concentration of 0, 0.5, 5.0 or 50 µg/ml at 4°C for 1 hour and then infected with HAdV at an MOI of 100 genomes/cell at 37°C for 1 hour. HAdV-C5 (black bars) and HAdV-D9 (white bars). (E) Cellular uptakes of HAdV-D9 in cancer cells which express little or no hCAR. Cells were infected with HAdV-C5 or HAdV-D9 at an MOI of 100 genomes/cell for 1 hour post-infection, and total cell DNA extracted from infected cells was analyzed by qPCR. HAdV-C5 (black bars) and HAdV-D9 (white bars). Cellular uptakes of HAdVs in cell lines were represented as (A) a fold of genome transfer, (B, C and D) a percentage of genome transfer, and (E) Genome copy numbers per 50 ng of total DNA of each HAdV. Data points represent mean+standard error of the mean (n = 3).
Figure 4.
Analysis of hCAR expression and cell killing activity of HAdV-D9 in cancer cell lines.
(A) Analysis of hCAR expression in various cancer cell lines by flow cytometry. Filled, gray histograms indicate stained cells; open, white histograms indicate unstained control cells. (B) Two-dimensional cell viability assay. Cancer cell lines were infected with HAdV-C5 (black squares) or HAdV-D9 (white squares) at indicated MOIs. Cell survival in each well was measured at 6 days post-infection using an MTS assay and plotted on y-axis as the percentage of the control values obtained from uninfected cells. (C) Three-dimensional cell viability assay. Spheroids were infected with HAdV-C5 (black squares) or HAdV-D9 (white squares) at indicated MOIs. Cell survival in each well was measured at 12 days post-infection using an MTT assay and plotted on y-axis as the percentage of the control values obtained from uninfected cells. Data points represent mean+standard error of the mean (n = 3).