Modeling Flavivirus Pathogenesis in a Stem Cell Derived Blood-Brain Barrier Endothelial Cell System

Flaviviruses are positive sense single-stranded enveloped RNA viruses, most are mosquito-transmitted viruses that cause systemic infection as they disseminate through the bloodstream in vertebrate hosts. The members of the flavivirus genus are highly similar in structure and genome organization, however, some viruses have been associated with neurological complications in humans, like Zika virus (ZIKV) and West Nile Virus (WNV); while others have been linked to hemorrhagic fever like Dengue virus (DENV) and Yellow Fever virus (YFV). In the context of brain neuroinvasion, viral entry into the brain can occur through different mechanisms, for example, direct infection of brain microvascular endothelial cells (BMECs) or breakdown of the blood-brain barrier (BBB) homeostasis. The specifics of how flaviviruses gain access to the central nervous system (CNS) remain incompletely understood, highlighting the need for researching the viruses' phenotype in the context of the BBB and CNS. In our lab, we use human pluripotent stem cell derived BMECs (iBMECs) and different CNS cellular units including primary astrocytes and stem cell derived forebrain organoids to model the brain microenvironment in vitro. Comparing viral infection phenotypes between ZIKV, DENV and other flaviviruses in the BBB we seek to understand the different neuroinvasion mechanisms used by flaviviruses. We have evaluated how a variety of neurotropic and non-neurotropic flaviviruses might use different mechanisms to access the brain, which is typically protected by blood-brain barrier (BBB), we utilized iPSC-derived human brain microvascular endothelial cells (iBMECs) to study virus-BBB interactions. These iPSC-derived cells exhibited functional and physiological BBB properties and faithfully recapitulated the in vivo neuroinvasiveness of flaviviruses. We further developed a BBB model consisting of cocultured iBMECs and astrocytes in transwell setups. This model exhibited high TEER value and selectively blocked non-neurotropic flaviviruses from crossing the barrier. We next focused on developing bacterial-virus co-infection models to understand how bacterial cell wall components may alter barrier function and viral permeability. To this end, I have discovered an unknown mechanism where bacteria cell wall components may increase BBB permeability and allow for traditionally non-neurotropic viruses to establish infection in the iBMECs and the brain Overall, we were able to effectively model flavivirus neuroinvasion using iBMECs. Our research led to the description of brain-invasion mechanisms that modulate the ability of viruses to infect this physiologically relevant protective brain barrier. Highlighting the importance of virus' ability to directly infect the iBMECs and the relationship between the cellular response to inflammation and virus permissiveness.