A tale of two viruses: molecular investigations of flavivirus infection in Jamaican fruit bat cells

Abstract

Flaviviruses, including mosquito-borne pathogens such as dengue (DENV), West Nile (WNV), and Zika (ZIKV), cause significant morbidity and mortality across the globe. Many species of bats can be infected with flaviviruses, but bats typically do not develop severe disease. There has been very limited study of flavivirus infection in bats, so why bats are infected by flaviviruses but do not get disease remains unclear. Bats are vectors for several human viral pathogens and therefore it is important to clarify their role in viral transmission cycles between different species and their potential as reservoirs for future spillover events. To explore why bats can be infected with flaviviruses but not develop severe disease, we need a solid understanding of the molecular mechanisms occurring during flavivirus infection of bat cells. This body of work represents the first detailed molecular investigation of mosquito-borne and no-known vector flavivirus infection in bat cells. The research described in this dissertation provides new insights into how flaviviruses control host processes during infection and how bat cells respond to these pathogens. Chapter 1 is a literature review of publications describing flavivirus infections in live bats and bat cells. Studies in live bats with flaviviruses have occurred sporadically over the last 100 years and have often left more questions than answers in regard to the interaction of flaviviruses and bats. The history of in vivo and in vitro bat studies with flaviviruses is explored and the problems with data interpretation that has plagued the field discussed. This chapter serves as an introduction into the paucity of research available that focuses on flavivirus and bat infections and how technological advancements have improved our ability to study flavivirus infection dynamics in bats. Chapter 2 is an investigation into West Nile virus (WNV) infection of Jamaican fruit bat cells. In this study we found that WNV productively infects primary Artibeus jamaicensis bat cells and ablation of subgenomic flaviviral RNAs (sfRNAs) does not impact viral replication. Through an RNA-seq experiment and subsequent bioinformatic analysis we determined that sfRNAs impact RNA splicing in bat cells, similar to WNV's effects on other mammalian flavivirus hosts. We observed that the RBM39 gene is alternatively spliced during infection to produce a premature stop codon that feeds into the nonsense mediated decay (NMD) pathway, which may have implications into antiviral responses in bat cells. We explore how production of WNV subgenomic flavivirus RNAs (sfRNAs) influence gene expression and could play a role in immune evasion of these flaviviruses in bat cells, leading to persistence. This chapter encompasses the first investigation of West Nile infection in primary bat cells and suggests that sfRNAs play an active role in modulating host responses to infection. Chapter 3 represents the first molecular exploration of Entebbe bat virus (ENTV), a no-known-vector flavivirus that has been isolated only twice in nature. We propose ENTV as a model no-known-vector flavivirus for study in bats due to its unique position of having a recorded passage history, recent isolation, and newly available infectious clone. Interestingly, we observed that ENTV displays a low genomic RNA to plaque forming units (PFU) ratio compared to WNV and discuss how this this ratio could act as a marker of severe infection in bats. An RNA-seq experiment of ENTV in primary bat cells demonstrated a large modulation in gene expression seen at the same timepoints for WNV, indicating differing responses between viruses. Additionally, we detected and began characterizing an exoribonuclease resistant RNA (xrRNA) stall site in ENTV for the first time, laying the groundwork for future studies into the structure of the ENTV 3' untranslated region (UTR) and sfRNA generation. Overall, this work described in this dissertation represents the first molecular investigation of West Nile virus and Entebbe bat virus infection in Jamaican fruit bat cells and opens the field for further investigation into the pathogen:host dynamics between flaviviruses and bats.