Microscopy has been instrumental in the discovery and characterization of microorganisms. Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular systems, both at the population and at the single-cell level. High-Content Imaging (HCI) has been used to characterize host-virus interactions in genome-wide reverse genetic screens and to identify novel cellular factors implicated in the binding, entry, replication and egress of several pathogenic viruses. Here we present an overview of the most significant applications of HCI in the context of the cell biology of filovirus infection. HCI assays have been recently implemented to quantitatively study filoviruses in cell culture, employing either infectious viruses in a BSL-4 environment or surrogate genetic systems in a BSL-2 environment. These assays are becoming instrumental for small molecule and siRNA screens aimed at the discovery of both cellular therapeutic targets and of compounds with anti-viral properties. We discuss the current practical constraints limiting the implementation of high-throughput biology in a BSL-4 environment, and propose possible solutions to safely perform high-content, high-throughput filovirus infection assays. Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of possible cellular biomarkers of virus infection
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