Many flaviviruses are adapted to dual-host transmission and maintained in cycles between hematophagous arthropods (i.e. mosquitoes and ticks) and vertebrates. An example of mosquito-borne flavivirus (MBFV) is Zika virus (ZIKV), which is a human pathogen of global concern. Other flaviviruses, such as Long Pine Key virus (LPKV), replicate in mosquitoes and phylogenetically affiliate with MBFVs, but lack the capacity to infect vertebrate cells. These viruses are known as dual-host affiliated insect-specific flaviviruses (dISFs). The precise sequence elements and virus-host interactions that modulate the differential host ranges of MBFVs and dISFs have not been defined. Identification of these sequences and virus-host interactions would provide key insight into why some flaviviruses infect and cause devastating disease in humans while others are insect-specific. In the initial funding period, we identified the broad genetic determinants that modulate the differential host ranges of MBFVs and dISFs. Through the construction and characterization of chimeric viruses, we demonstrated that ZIKV loses its vertebrate-infecting tropism when its 5’ untranslated region (UTR), adjacent capsid protein (C) gene, and 3’ UTR are replaced with those of LPKV, while its mosquito-infecting phenotype is retained. The UTRs contain highly structured elements that interact with NS3 (the viral helicase) and NS5 (the viral RNA polymerase) to regulate genome replicate. The UTRs, NS3, and NS5 of MBFVs also interact with many host proteins, but the host proteins that comprise the dISF replication complex are unknown. The overall goal of this continuation is to dissect the 5’- and 3’-terminal ends of the flavivirus genome to pinpoint the precise sequences responsible for flavivirus host-specificity and to compare the virus-host interactions that occur during MBFV and dISF replication through the identification and functional characterization of host proteins that bind to their UTRs, NS3, and NS5. T