The advent of globalization and the continuing effects of climate change have exposed an even greater percentage of the world to disease agents carried by vectors which were once thought to be restricted to tropical or subtropical areas. Arthropods vectors such as mosquitoes and ticks harbor a number of emerging and re-emerging pathogens of public and veterinary health importance worldwide. These pathogens include viruses from four major families including the Flaviviridae, Bunyaviridae, Togaviridae and Reoviridae families. The Flavivirus genus is one of four genera in the Flaviviridae family, with over 70 species, the great majority of which are maintained in horizontal transmission cycles between hematophagous arthropod vectors and vertebrate hosts. However, within this genus also exists two smaller subsets of viruses which are restricted to either insects (insect-specific flaviviruses) or vertebrates (no known vector flaviviruses). Since it is likely that dual-host flaviviruses may have evolved from single-host precursors, investigative studies into the genetic elements which modulate flavivirus host specificity may elucidate the evolutionary hurdles necessary to make the leap from a single- to a dual-host flavivirus. Such studies could have major implications for efficacious vaccine or antiviral drug development.

In this dissertation, I report the creation and characterization of the first bat-associated flavivirus chimeras, designed to investigate host restriction in no known vector (NKV) flaviviruses at the level of attachment and entry. These chimeras were created using two representative dual-host flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV) whose attachment and entry proteins (premembrane and envelope) were substituted with that of a bat-associated NKV flavivirus, Rio Bravo virus (RBV). The result was the generation of two different chimeric viruses, both of which were able to replicate in both insect and vertebrate cell lines, indicating that NKV flavivirus restriction is dictated by a post-attachment/entry event. Additional studies examining restriction at downstream stages in the viral life cycle may prove valuable in determining the genetic elements that modulate host restriction in this group.

In the chapters that follow, we also report the discovery and identification of four novel and two previously identified viruses from mosquitoes collected in the Yucatan Peninsula of Mexico using metagenomics. We also report full sequence data for two insect-specific flaviviruses (ISFs) and for the large segment of two bunyaviruses for which full sequence data was not available.