Poxviruses are a family of large, double-stranded DNA viruses that infect a wide range of organisms, from insects to humans. They possess a complex structure and an intricate life cycle that takes places entirely in the cytoplasm. Additionally, their genome is remarkably large for viruses, encoding over 200 proteins. Historically, one representative of the orthopox genera – variola virus, the causative agent of smallpox – has claimed hundreds of millions of humans lives. Despite its eradication, smallpox, and some zoonotic poxviruses, are still considered as potential threats, and new ways of combating these viruses are in demand. To this end research has continued on vaccinia virus (VACV), the prototypic orthopox virus which was used as smallpox vaccine. In this project we intend to screen a library of 1280 FDA approved molecules for cell based VACV inhibitors using a high-throughput microscopy based screen, following the principles of drug repurposing. After confirming initial hits using focused secondary screens and assays we will investigate the mechanism by which inhibition occurs. The project will involve biochemistry, cell biology, virology, and microscopy as main tools, and may result in discovery of potential new anti-poxviral drugs, with the possibility of testing those drugs on other viruses. Additionally we hope to learn more about the host-pathogen interaction.