Powassan virus is the only known tick-borne flavivirus (TBFV) circulating in North America and has two distinct genetic lineages. Prototypical Powassan virus, lineage I (POWV), was isolated in 1958 and known to be primarily vectored by Ixodes cookei ticks. Lineage II Powassan virus is also known as Deer tick virus (DTV) and was initially isolated in 1995 and is vectored primarily by Ixodes scapularis ticks. Powassan virus and DTV share approximately 84% nucleotide sequence identity and 94% amino acid identity but are serologically indistinguishable. POWV can be deadly, with a 10 to 15% case fatality rate and long-term neurological damage in 50% of survivors. While there have been advances in the study of POWV neuroinvasive disease, the underlying pathology is not entirely defined, especially in relation to the similarly pathogenic DTV. While there have been advances in the study of POWV neuroinvasive disease, the underlying pathology is not entirely defined, especially in relation to the similarly pathogenic DTV. Although two distinct lineages circulate in North America, clinical differentiation is not typically performed, and pathology has been assumed to be similar between lineages. We recently demonstrated with foot-pad inoculation that POWV (lineage I) and DTV (lineage II) demonstrated distinct differences in clinical presentation and neuro-immunopathology. These differences suggest these two viral lineages do not utilize the same routes of neuroinvasion and stimulate distinct neuroimmunological responses. This comparative analysis was performed on mice that reached the moribund stage due to virus infection and did not use the natural route of virus transmission (tick bites) to humans. Herein, we hypothesize that tick-transmitted POWV and DTV will utilize both neuronal and hematogenous routes of neuroinvasion and that a longitudinal and natural tick transmission model will allow us to unravel: (1) spatiotemporal kinetics of the routes of neuroinvasion from the skin transmission site to CNS; (2) the neurotropism (spatial/cellular) and neurovirulence in the brain from the time the virus is delivered in the skin to when the virus establishes infection in the brain. The outcome of the proposed study will fill the critical gaps in our current understanding of the routes of neuroinvasion and neurovirulence of these two closely related emerging viruses of human health concern.