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Title: Development of an experimental paradigm for the investigation of innate immune pathways restricting hepatitis delta virus replication
Authors: Wu, Evelyn
Advisors: Ploss, Alexander
Department: Molecular Biology
Certificate Program: Global Health and Health Policy Program
Class Year: 2019
Abstract: HDV is a single-stranded, negative-sense RNA satellite virus, dependent on HBV envelope proteins to complete its replication cycle. HBV/HDV co-infection increases the likelihood, severity, and rate of liver disease onset. HDV can only establish robust infection in humans and chimpanzees, which poses a challenge to studying immune responses to HDV and assessing the efficacy of antiviral treatments. The discovery of the human sodium co-transporting polypeptide (hNTCP) as the functional receptor for HBV and HDV has helped overcome the barrier to entry in non-susceptible cells and has led to the generation of various in vitro and in vivo models. However, work remains to be done to develop a small animal model that accurately recapitulates HDV pathogenesis. To better understand HDV viral-host interactions, we first established a system for studying immune responses to HDV independently of HBV infection in vivo. We showed that supplying HBV surface proteins (HBsAg) in trans via an AAV vector supported HDV infection in both liver chimeric mice and a mouse model that transgenically expresses hNTCP on a bacterial artificial chromosome (NRG-hNTCP/BAC). Using this system, we observed that HDV persisted in engrafted human hepatocytes of liver chimeric mice for at least 8 weeks while it was cleared in the hepatocytes of our NRG-hNTCP/BAC model under mono-infection conditions. To study the mechanisms of the observed difference in persistence, we generated knockouts of innate immune pathways that could restrict HDV replication in murine hepatocytes by using recombinant AAV to deliver CRISPR/Cas9 components to NRG-hNTCP/BAC mice. Greater HDV RNA copy numbers in NRG-hNTCP/BAC mice that harbored a partial knockout of MAVS suggests a role for RIG-I-like receptor (RLR) signaling in HDV clearance. However, the challenge of generating a homogenous, efficient knockdown in vivo precludes any definitive conclusions and remains to be resolved. The findings in this study contribute to establishing an experimental framework for studying innate immune pathways that might be involved in antagonizing HDV replication in murine hepatocytes. Identification of such pathways would contribute to creating a better small animal model that recapitulates natural HDV infection, identifying a point of intervention for treatment, and a better understanding of the immune response to the virus.
Type of Material: Princeton University Senior Theses
Language: en
Appears in Collections:Molecular Biology, 1954-2022
Global Health and Health Policy Program, 2017-2022

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