Examination of CHLA and PUG treatment on virus cell-to-cell spread. (A) Schematic of the experiment (left) with the virus concentration (PFU/well) and step-wise incubation periods (i, ii, iii) indicated for each virus in the table on the right. Virus infections were established (i) in the different cell types by direct inoculation (HCMV, DENV-2, MV, and RSV) or electroporation of viral RNA (HCV; *), and the cell monolayers were washed with citrate buffer or PBS before being covered with an overlay medium that prevents secondary infection. Initial virus plaques were allowed to form in the subsequent infections (ii), and then the test compounds were added to the overlay medium for an additional time of incubation (iii) before analysis of viral plaque size by immune fluorescence microscopy. Five random virus-positive plaques at the endpoint of the experiment were evaluated for each treatment group of viruses, and the data was plotted as “fold change of plaque area” against the size of the initial viral plaques formed prior to test compound treatment. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. The S29 cell line and the FIP inhibitor were included as controls for HCV and MV, respectively. Results shown are means ± SEM from three independent experiments and representative micrographs of the evaluated plaques are provided in Additional file 1 Figure S1, Additional file 2 Figure S2, Additional file 3 Figure S3, Additional file 4 Figure S4 and Additional file 5 Figure S5. See text for details.