West Nile virus (WNV) encodes two envelope proteins, prM and E. While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation in flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. My thesis work has focused on examining the impact of prM and E glycosylation on WNV assembly, infectivity, and tropism. I found the absence of the prM or E glycosylation site in either a lineage I or II strain dramatically decreased viral particle release. Infectivity analysis of these viral particles bearing combinations of glycosylated and non-glycosylated forms of prM and E found all could infect mammalian and avian cells to comparable levels, while the absence of E glycosylation greatly enhanced infection of mosquito cells. Further investigation found this enhanced infection could be attributed to more efficient binding to mosquito cells. Interestingly, this phenotype is dependent on the location of the glycan on the E protein, but independent of the glycan structure as the same pattern was observed whether the glycan was high mannose or complex in nature. This suggests WNV can interact with a unique or highly expressed molecule on the surface of mosquito cells that is blocked when the E N-linked glycan is present. This result may begin to provide some insight to the benefits, and thereby the existence of non-glycosylated E WNV strains in the circulating population. In summary, my thesis research highlights the multiple roles N-linked glycosylation of WNV prM and E can play in pathogenesis.