2.5. Virus Cell-Binding Assays

HCE cells were detached with pre-warmed PBS containing 0.05% ethylenediaminetetraacetic acid (EDTA). The cells were counted and reactivated in 10% growth medium for 1 h at 37 °C (in suspension). The cells were then pelleted in a V-bottom 96 well plate (1 × 10⁵ cells/well) and washed once with binding buffer (BB; DMEM supplemented with 20 mM HEPES, PEST, and 1% bovine serum albumin). ³⁵S-labeled HAdV-D37, -D53, and -D64 viruses (10000 vp/cell, diluted in BB) were added to cells and incubated for 1 h at 4 °C on ice. To remove unbound viruses, cells were washed three times with BB. Cell-associated radioactivity was measured by using Wallac 1409 liquid scintillation counter (Perkin-Elmer, Waltham, MA, USA). The assay was performed with the following variations: (i) HCE cells were treated with 20 mU/mL of neuraminidase for 1 h at 37 °C before incubating with viruses and (ii) viruses were incubated with increasing concentrations of ME0462 (diluted in BB) for 1 h at 4 °C before incubating with HCE cells. Untreated cells and viruses were used as control.