Abstract
Different viruses rely on direct cell-to-cell transmission to propagate infection within the infected host. Measuring this mode of transmission in cultured cells is often complicated by the contribution of cell free viruses to spread, and the difficulty to distinguish between primary infected cells that produce the virus and neighboring cells that are the target of spreading. Here, we present a protocol to quantify cell-to-cell transmission of the model pestivirus bovine viral diarrhea virus that is based on the co-culture of producer cells that are infected with a reporter virus expressing mCherry and target cells that stably express GFP. Spread of cell-free viruses is blocked by the presence of a neutralizing antibody in the cell culture medium, and cell-associated transmission is unequivocally quantified by numbering cells that are positive for both GFP and mCherry using automated analysis of fluorescence microscopy images.