Ebola virus (EBOV) infection causes a severe disease in humans and leads to widespread liver necrosis, a dysregulated cytokine response, and coagulopathy. However, little is known about the specific liver response to EBOV infection in humans. Here we present the utilization of an induced pluripotent stem cell (iPSC)-derived hepatocyte platform to define the host response to EBOV infection. We demonstrate that iPSC-derived hepatocytes are a suitable platform for investigating innate immune responses to viral infections. We compared the host response to EBOV infection in iPSC-derived hepatocytes, immortalized hepatocytes, and primary human hepatocytes and identified minimal transcriptomic changes 1 day post infection (dpi). Between 2-3 dpi, EBOV infection led to a significant upregulation of interferon-beta (IFN-β) and select interferon-stimulated genes (ISGs) in iPSC-derived hepatocytes. In addition, the acute phase response and coagulation cascade was downregulated in these hepatocytes, mimicking known liver dysfunction in EBOV disease. Using fluorescent in situ hybridization (RNA-FISH), we showed at single cell resolution that EBOV-infected iPSC-derived hepatocytes express IFN-β, indicating that infected cells mount an antiviral response to EBOV infection. This platform can be utilized to investigate therapeutic targets in human hepatocytes that may attenuate EBOV infection in patients. In addition, we present in this dissertation the development of a minigenome system for the filovirus Lloviu virus (LLOV). LLOV is closely related to EBOV and is known to circulate in bats throughout Europe. The complete sequence of LLOV has yet to be resolved, and therefore investigation of LLOV biology is limited. As part of this work, we established a functional LLOV minigenome system based on sequence complementation of other filoviruses. We demonstrate that the LLOV replication and transcription strategy is generally more similar to ebolaviruses than marburgviruses. We show that a single nucleotide at the 3ꞌ end of the LLOV genome determines specificity of the LLOV polymerase complex. This minigenome system can now be used to elucidate replication and transcription mechanisms employed by this novel filovirus. / 2023-02-19T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/42076 |
| Date | 19 February 2021 |
| Creators | Manhart, Whitney Ann |
| Contributors | Muhlberger, Elke, Mostoslavsky, Gustavo |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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