Protocol for quantification of intercellular connection transmission in ebolavirus infections using ImageJ

Previous work demonstrates that ebolaviruses can spread to neighboring cells through intercellular connections. Here, we present a protocol to quantify the intercellular spread of ebolaviruses via immunofluorescence. We describe steps for cell plating, Bundibugyo virus infection, and adding a neutralizing antibody. We detail procedures for quantitative microscopy assay using ebolavirus immunodetection. Strong virus accumulation around the plasma membrane leads to high fluorescence signal preventing quantification of viral spread based on signal intensity. This protocol minimizes the impact of this bias. For complete details on the use and execution of this protocol, please refer to Santos et al.¹.