Ab35958

To evaluate the ability to inhibit RSV infection, EO was serially diluted to generate dose-response curves and added to cells. After 2 h, EO was removed and a mixture of EO dilutions and RSV (MOI 0.01 PFU/cell) was added to A549 cells grown as monolayers in a 96-well plate at a density of 7 x 10⁴/well. After 3 hours of incubation at 37°C, monolayers were washed and overlaid with 1.2% of methylcellulose medium containing serial dilutions of EO. Three days post-infection, cells were fixed with cold methanol and acetone for 1 min and subjected to RSV-specific immunostaining using an RSV monoclonal antibody (Ab35958, Abcam, Cambridge, UK) and the UltraTech HRP streptavidin-biotin detection system (Beckman Coulter, Marseille, France). Immunostained syncytia were counted and the percent inhibition of virus infectivity determined by comparing the number of plaques in treated wells with the number in untreated control wells. 50% effective concentration (EC50) values and 95% CIs were determined using Prism software. All data were generated from duplicate wells in at least three independent experiments.

The neurovirulent strains LV (21 (link)) and MS (ATCC VR-540) of HSV-1 and HSV-2, were propagated in Vero cells at 37°C (22 (link)). HRhV 1A (ATCC VR-1559) was propagated in HeLa cells, at 33°C. HSV-1, HSV-2, and HRhV titers were determined by the standard plaque method and expressed as plaque-forming unit (PFU)/ml. A bacterial artificial chromosome (BAC)-derived HCMV strain Towne incorporating the green fluorescent protein (GFP) sequence (23 (link)) was propagated on HFF-1 and viral titres were determined by fluorescent focus assay. RSV strain A2 (ATCC VR-1540) was propagated in Hep-2 and titrated by the indirect immunoperoxidase staining procedure using an RSV monoclonal antibody (Ab35958, Abcam) (24 (link)). Human HRoV strain Wa (ATCC VR-2018) was activated with 5 μg/ml of porcine pancreatic trypsin type IX (Sigma) for 30 min at 37°C and propagated in MA104 cells by using MEM containing 0.5 μg of trypsin per ml. HCMV, RSV and HRoV titers were expressed as focus-forming units (FFU)/ml. Virus stocks were maintained frozen (-80°C).