Construction characterization and application of Flavivirus infectious clones

Abstract

The topics in this thesis revolve around a group of plus-strand RNA viruses that belong to the Flavivirus genus, a general introduction of which is presented in Chapter 1. The experimental chapters in this thesis mainly focus on the construction and characterization of flavivirus infectious clones and the application of these useful tools to address fundamental questions on virus biology or to explore novel vaccine platforms based on the yellow fever virus vaccine strain (YFV-17D). Chapter 2 describes the construction and characterization of recombinant YFV-17D viruses expressing Lassa virus (LASV) glycoprotein coding sequences as bivalent vaccine candidates against wildtype YFV and LASV that circulate in the same region in West Africa. Chapter 3 describes the construction and characterization of an alternative YFV-17D vaccine candidate based on a DNA launched YFV-17D infectious clone, and the study of its immunological characteristics. Chapter 4 describes the construction and characterization of the first NKV (no known vector) flavivirus infectious clone, which was further utilized to study the functional significance of RNA elements present in Modoc virus (MODV) 3__-UTR (untranslated region), as a representative of the NKV flaviviruses. A recently discovered common feature for all flaviviruses is the production of a small flavivirus RNA (sfRNA) in infected cells or animals that results from the incomplete degradation of the viral genome by the host exoribonuclease, XRN-1. XRN-1 was predicted to be stalled by a non-H-type RNA pseudoknot structure that is present is the 3__-UTR of every flavivirus studied thus far. Chapter 5 presents a detailed mutagenesis study of many of the unique sequence and structural characteristics of the XRN-1 stalling site in mosquito-borne flaviviruses using YFV-17D as a model. Chapter 6 describes the establishment of mammalian cell lines that constitutively expressed sfRNA from YFV-17D and WNV. This cell line approach could be valuable to further our understanding of sfRNA function within flavivirus life cycle. Chapter 7 of this thesis is a short epilogue, in which the findings presented in the experimental chapters are summarized and discussed in the context of recently published findings and future research directions are suggested to improve the YFV-17D based vaccine platforms or to address the yet-to-be-solved questions about sfRNA production and function.