FFU assay for measuring viral spread

A549 cells were seeded in 6-well plates at a density of 6 × 105 cells per well. The following day, virus stocks were serially diluted in 2% DMEM, and cells were inoculated and fixed as described above. Following fixation, cells were washed with 1× PBS, and an anti-flavivirus envelope antibody, 4G2 (Ab00230-2.0; Absolute Antibody, Oxford, UK), diluted 1:500 in permeabilization/wash/block buffer (see recipe above) was added to the cells. Following incubation with primary antibody, donkey anti-mouse HRP secondary antibody diluted 1:1,000 in permeabilization/wash/block buffer was added and incubated with rocking for 1 h in the dark. Foci were developed and detected as described above. The diameters of the foci were measured in Fiji.