Background

The spread of virus infections relies both on the dissemination of free virus particles and direct cell-to-cell transmission. In an infected host, cell-associated transmission overcomes multiple barriers imposed to diffusion of free viruses, such as epithelial and mucosal barriers, or antibody neutralization (Sattentau, 2008; Mothes et al., 2010). In vitro, spread of infection in the presence of virus neutralizing antibodies serves as a model to study direct cell-to-cell transmission. Virus spread from a producer cell to a target cell is usually studied in the presence of neutralizing antibodies. Different protocols have been previously devised that measure foci size in non-dividing cell cultures (Barretto and Uprichard, 2014), that use a viral infection-activated split-intein-mediated reporter system to light the expression of fluorescent reporters of contrasting colors in co-cultured producer and target cells (Zhao et al., 2018), or that require immunofluorescence staining of viral antigens in co-cultures where target cells express a fluorescent protein (Yang et al., 2015). In addition, luciferase-based assays have been developed to measure cell-associated spread of HIV in co-cultures of cells expressing an HIV reporter genome and target CD4⁺ T cells (Zhong et al., 2013; Agosto et al., 2014). We have recently shown that bovine viral diarrhea virus (a pestivirus in the family Flaviviridae) spreads by cell-to-cell transmission in cell culture using an approach that relies on the use of a co-culture of producer cells infected with a reporter bovine viral diarrhea virus expressing mCherry fluorescent protein, and target cells expressing GFP (Merwaiss et al., 2019). In this setup, numbering cells positive for both mCherry and GFP scores virus transmission from producer to target cells. Quantification is performed by automated image analysis of fluorescence microscopy images, and spreading is expressed as the percentage of (mCherry and GFP positive cells)/(GFP positive cells). The protocol provides an unambiguous method to quantify virus spread and has the main advantages of not requiring immunofluorescence staining, and of relying only on the use of any susceptible cell line. In addition, it can be adapted to fully automated image acquisition and analysis, live cell imaging or flow cytometry setups.