Fig. 5

The TCID50 assay. A Schematic of a 96-well viral titration plate. An original CCHFV sample was serially diluted as indicated and inoculated onto SW-13 CO2 + cells. The peripheral wells, represented with light pink color, were excluded from usage for the assay, due to an edge effect on cell morphology as illustrated in the microscopic image. Each dilution condition was assigned onto one column of wells, consisting of six replicates. CCHFV positive wells (CPE+, represented in red) or negative wells (CPE−, orange) were distinguished by the presence or absence of typical morphological changes characteristic of CPE, such as monolayer disruption and cellular deformation (detailed in text of Results and Discussion). Medium colors were a quick accessory indicator of CPE status, based on phenol red, a pH indicator. Extended culture, with accumulation of metabolites, led to a color change from red to orange (pH decrease) in CPE- wells, whereas the color remained red (sometimes slightly purplish) in CPE+ wells, where metabolism was inhibited. Colors used here, for illustration purpose, may not precisely reflect the real colors seen in experiments. B Highlight of the TCID50 calculator. This is in an excel spreadsheet format. Represented in green were customizable design parameters and numbers of positive (CPE+) wells such as those observed on the titration plate in A. Based on these inputs from the user, the viral titer in TCID50/ml was automatically calculated