Lab Protocols for Coxsackievirus B3 Infection Using Brain-Like Endothelial Cells Derived from Induced-Pluripotent Human Stem Cells.

Coxsackievirus B3 (CVB3) is an enterovirus known to be a leading cause of viral aseptic meningoencephalitis worldwide. Therefore, the virus may interact with and cross the blood-brain barrier (BBB). The BBB is a highly specialized endothelial barrier that helps protect the brain from foreign bodies and infection, primarily being composed of brain endothelial cells. Despite the fact that CVB3 can be fatal, particularly in young children, the mechanism through which CVB3 is able to penetrate the BBB and gain access to the central nervous system is not fully characterized and is poorly understood. However, there are challenges when modeling the BBB, as in vivo modeling has morphological differences and in vitro models tend to fall short when it comes to retaining vital BBB phenotypic properties. Modeling the BBB with induced-pluripotent stem-cell-derived brain-like endothelial cells (iBECs) has been shown to be very promising, as iBECs display and retain brain-like phenotypes and have recently been utilized to model CVB3 infection of the BBB. This chapter describes methodologies using iBECs to study the interaction between CVB3 and the BBB as a meningeal human pathogen, including cell differentiation and maintenance, Western blotting, immunostaining, plaque assays, infection of cells, use of antivirals, and viability assays.