The most powerful and direct way to get insight into complex biological systems is to remove individual components and observe the consequences. Cultured human cells are widely used to model and study aspects of human disease. Although human cells can be analyzed and manipulated in numerous ways, their genome has remained refractory to efficient mutagenesis-based analysis.We have developed an insertional mutagenesis-based approach in haploid and near-haploid human cells. We have shown that this approach enables the generation of null alleles for most human genes and can be used to pinpoint genes that are involved in phenotypes of interest. We have made use of parallel sequencing approaches to generate high-density genetic overviews of genes that are required for phenotypic cell states. In a variety of genetic screens we have identified host factors required for infection of cells by influenza virus, the first entry receptor for a Clostridium Difficile toxin and a set of host factors that play a role in the entry of Ebola virus. Importantly, we can carry out and analyze a genetic screen in a period of weeks in a cost-effective manner.This application outlines experiments aimed at identification and detailed characterization of host factors that play a role in Filovirus infection. Moreover, we propose a number of experiments to gain mechanistic insight in the role of the Niemann-Pick C1 cholesterol transporter in entry, tropism and the fusion process of Ebola virus. Furthermore we propose to develop refinements in our screening approach. Finally, we will apply our haploid screening platform to generate a “Host Factors Atlas’ for 50 diverse viruses that infect the human population. We foresee that the outlined experiments will provide an detailed and accurate overview of the unusual entry-route used by Filoviridae, will make our screening platform more powerful and will generate a much-needed overview of host factors used by a compendium of viruses.