Characterization of Flavivirus-Infected Neurons

Powassan virus (POWV) is a tick-borne flavivirus (TBFV) that can cause severe encephalitis in humans with a case fatality rate as high as 15%. Patients who survived severe encephalitic disease can develop long-term sequelae that can be debilitating and life-long. In this dissertation, a primary human fetal brain neural stem cell system (hNSC) was characterized, which can be differentiated into neuron and astrocyte co-cultures to serve as a translational in vitro system for infection with POWV and a comparative mosquito-borne flavivirus (MBFV), West Nile virus (WNV). It was found that both viruses were able to infect both cell types in the co-culture and that WNV elicited a strong inflammatory response characterized by increased cytokines IL-4, IL-6, IL-8, TNF-α, IL-1β and apoptosis. POWV infection resulted in fewer cytokine responses, as well as less apoptosis, while neurons infected with POWV exhibited aberrations forming in the dendrites. These anomalies were consistent with previous findings using tick-borne encephalitis virus (TBEV) infected murine primary neurons and the formation of laminal membrane structures (LMS). Tissue clearing protocols for infected tissues were established and optimized and applied to prove that structural aberrations were also recapitulated in tissue samples from virus-infected mice. Overall, the studies showed that POWV was capable of infecting human primary neurons and astrocytes without causing widespread apoptosis, while forming punctate structures consistent with LMS in primary neurons and in vivo. This is significant as it is the first time that LMS formation has been indicated in human cells and in animal models. The findings will contribute to the knowledge of TBFV neuropathogenesis by defining the inflammatory response in human neuronal cells and indicating structural changes that could contribute to long-term sequelae.