Quantifying Cell-to-Cell Spread of HSV-1

To determine cell-to-cell spread of HSV-1 in polarized epithelial cells, a viral plaque assay was used as described previously [7 (link)]. Polarized cells were infected with HSV-1 from the basolateral surface at an m.o.i. of 0.01 p.f.u. per cell, and cells were incubated for 2 h at 37 °C. Cells were washed and overlaid with serum-free medium containing 0.5 % methylcellulose from apical and basolateral chambers. After 3 days, cells were fixed and immunostained with mouse antibody to HSV1/2 gD (Santa Cruz Biotechnology). Cells were washed and incubated for 25 min with secondary anti-mouse antibody conjugated with DyLight 488. Cell nuclei were stained with DAPI. Cells were analyzed using a Nikon Eclipse E400 fluorescence microscope (Nikon).
Viral plaques were counted on a minimum of three filter inserts for each experiment, and average plaque numbers were expressed per insert. HSV-1 cell-to-cell spread was evaluated by quantitative analysis of HSV-1-infected plaques. Foci containing five or more infected cells were considered plaques, and cell numbers in each plaque were counted. At least 30 plaques were evaluated for each experimental condition, and the average number of cells per plaque was expressed.