Deciphering BVDV entry by targeted functional genomics

Bovine viral diarrhea virus (BVDV) is an important, notifiable animal pathogen causing amongst others - reproductive failure and immunosuppression in cattle. It is a small virus (50nm diameter) with a lipid envelope and is distantly related to Hepatitis C virus and Flaviviruses. BVDV enters cells by interacting with cellular surface proteins. Subsequently, it is taken up by the cell and releases its genetic material after fusion with the host cell membrane in the endosomal compartment. While some factors relevant for BVDV entry, such as clathrin, CD46 and ADAM17, have been identified, lots of pieces are still missing to completely understand the interactors and events involved in BVDV entry. To identify novel factors required for BVDV entry and to understand the mechanism of virus entry, this project employs state of the art CRISPR-CAS9 technology. Using this approach, we can inactivate (knock-out) specific genes in bovine cells and assess the effect of such a knock - out on the survival after infection with a BVDV that is able to kill its host cells. By checking several genes preselected based on already available evidence in parallel, we can screen for promising candidates whose knock-out inhibits BVDV infection. Once such candidates are identified, their exact role in BVDV entry will be determined by different biochemical, cell biological and imaging methods. With this new evidence, we can - bit by bit - fill the knowledge gaps associated with BVDV entry. The knowl edge gained within this project will greatly advance our understanding of mechanisms of virus infections and support the breeding of disease resistant animals and the optimization of treatment and prevention strategies.