Andrew C. Issekutz, Dalhousie University, Halifax, NS, Canada) [33]. The overlay medium helps limit viral secondary infection, thus allowing monitoring of cell-to-cell spread of virus in the presence or absence of the drugs. The plates were incubated until initial plaque formation, to which the test compounds were then added into the overlay medium and monitored in subsequent incubation

for analysis of viral plaque size by immunofluorescence assay. The fusion inhibitory peptide (FIP, Z-D-Phe-L-Phe-Gly-OH, 200 μM; Sigma) also served as control for MV [46]. Figure 7 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 Caspase phosphorylation 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 Selleckchem HDAC inhibitor 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 Wnt inhibition included as controls for HCV and MV, respectively. Results shown are means ± SEM from three independent experiments and representative micrographs of the evaluated Phosphoglycerate kinase 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. The examination of HCV spread is based on previously described protocol with some modifications [47]. Huh-7.5 cells were electroporated with HCV Jc1FLAG2(p7-nsGluc2A) RNA (10 μg) as described above to establish random productive infections in the cell population, and then mixed with naïve cells at a ratio of 1:1 before seeding in 12-well plates. Assembled HCV particles (within 24 – 48 h post-transfection) would transmit to neighboring cells that do not harbor viral RNA during viral spread and form localized foci in ensuing incubation period [48]. Medium was changed 24 h post-electroporation with an overlay medium containing the test drugs or control and 0.5% methylcellulose, and the plates were further incubated for 5 days before analysis of HCV-positive foci through immunostaining. The S29 cell line (provided by Dr. Rodney S.