Fig. 4

Plaque assays for CCHFV titration. Cell cultures in 48-well plates were infected with CCHFV as indicated. A carboxymethylcellulose (CMC) immobilizing overlay was added to restrict viral replication to the sites of initial infection, for the formation of plaques, foci of cell degradation (empty spots in monolayer), or foci of viral accumulation in cells that are not degraded (monolayer intact). The detection of these two possible types of plaques was tested using crystal violet staining, which would stain cellular monolayer into a violet color background, contrast-exposing the cellular degradation-based plaques as colorless empty spots, and using immunostaining to show the plaques based on intracellular viral antigen accumulation, respectively. Crystal violet staining failed to identify any plaques in CCHFV-infected SW-13 CO2−, SW-13 CO2+ or Vero E6 cells at any daily time point between 3 and 11 DPI. Immunostaining successfully revealed plaques in CCHFV-infected SW-13 CO2 + cells, at all the four tested time points, 3, 4, 5 and 12 DPIs, while the other cell culture types were not tested. A representative image was shown for each of the staining methods