Development of high-content imaging assays for lethal viral pathogens.

Filoviruses such as Ebola (EBOV) and Marburg (MARV) are single-stranded negative sense RNA viruses that cause acute hemorrhagic fever with high mortality rates. Currently, there are no licensed vaccines or therapeutics to counter filovirus infections in humans. The development of higher throughput/high-content primary screening assays followed by validation using the low-throughput traditional plaque or real-time PCR assays will greatly aid efforts toward the discovery of novel antiviral therapeutics. Specifically, high-content imaging technology is increasingly being applied for primary drug screening. In this study, the authors describe the challenges encountered when optimizing bioassays based on image acquisition and analyses for the highly pathogenic filoviruses Ebola and Marburg. A number of biological and imaging-related variables such as plating density, multiplicity of infection, the number of fields scanned per well, fluorescence intensity, and the cell number analyzed were evaluated during the development of these assays. Furthermore, the authors demonstrate the benefits related to the statistical analyses of single-cell data to account for heterogeneity in the subcellular localization and whole-cell integrated intensity of the viral antigen staining pattern. In conclusion, they show that image-based methods represent powerful screening tools for identifying antiviral compounds for highly pathogenic viruses.