Date of Award

Fall 12-12-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Margo Brinton

Abstract

Members of the genus Flavivirus are positive-stand RNA viruses that can cause a variety of human diseases, including fever, encephalitis, hepatitis and hemorrhagic fevers. Well-known mosquito-transmitted members are West Nile virus (WNV), Dengue virus, and Zika virus. The aim of this study was to characterize flavivirus infections at the molecular level using different models such as mouse cells (embryofibroblasts and neurons), induced pluripotent stem cell (iPSC) derived human neurons, and human brain organoids. Some mouse strains show innate resistance to disease caused by a flavivirus infection that is mediated by the Oas1b gene but how the OAS1b protein establishes this resistance is unknown. We found that (i) resistance is not dependent on an increased generation of defective viral genomes by resistant mouse embryofibroblasts infected with one of three WNV strains at a low multiplicity of infection; (ii) a WNV Eg101 mutant that partially overcomes the resistance phenotype contains 12 non-synonymous genome substitutions, some of which could be involved in overcoming the resistance; (iii) resistant mouse neurons can be infected by WNV NY99, but intracellular viral proteins are no longer detectable by 10 days post infection (dpi) and infectious extracellular virions are not detected by 20 dpi; transcriptomic analysis of resistant cells showed a complex response to infection; and (iv) a transgenic resistant mouse strain was constructed, which has a floxed-Abcf3 and a tamoxifen-inducible Cre recombinase gene under the control of a brain-specific neural promoter, to study the effect of the loss of the OAS1b-ABCF3 interaction on the flavivirus-resistant phenotype. We found that human brain organoids support WNV NY99 infection but that there is preferential tropism for cortical layer neurons SATB2+ and CTIP2+ independent of the iPSC line or age of the organoid. Also, 9- week old iPSC-differentiated human neurons support infection by neuroinvasive WNV NY99 and non-neuroinvasive WNV MAD78, and transcriptome analyses showed evidence of differential cellular responses to infection by these two viruses. Finally, we predicted the presence of canonical and non-canonical interferon response elements and NF-κB motifs in the promoters of a subset of upregulated interferon-stimulated genes in interferon receptor knockout mouse embryofibroblasts infected with WNV.

DOI

https://doi.org/10.57709/32645202

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