Time of addition experiments. Vero cells (3 × 105 cells) plated in 6-well plates were infected with NDV, treated with fucoidan at different times of infection, and analyzed by PFU inhibition assays and immunofluorescence assays. A) Fucoidan was added at 60 min preinfection and 0, 15, 30, 60, and 120 min postinfection. The data are expressed as relative PFU (%) compared to that of untreated virus-infected control cells (VC), which was defined as 100%. The data shown are the mean ± SD of triplicate experiments. The asterisks indicate a significant difference between the treatment and viral control (*p<0.05). B) Fucoidan was added at 60 min preinfection and 0, 15, and 60 min postinfection with NDV, stained with a mouse monoclonal anti-NDV HN antibody, and analyzed by indirect immunofluorescence as detailed in the Methods.