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Title: Norovirus replication and attenuation
Author: Thorne, Lucy
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Human norovirus is a major cause of viral gastroenteritis worldwide, yet there are no licensed antivirals or a vaccine. This is mainly due to the lack of an efficient cell culture system for human norovirus which has prevented full understanding of norovirus replication. Murine norovirus (MNV), which can be grown in permissive cells and manipulated by reverse genetics, now provides a model for studying norovirus replication. Here, to firstly identify the essential viral factors required for replication, the entire MNV genome was subjected to transposon-mediated insertional mutagenesis and insertion profiling. Validation of the technique and genome-wide profiling of over 2000 insertions revealed the essential regions of each protein and probed the importance of known functional motifs. Identification of tolerated insertion sites led to the generation of the first epitope-tagged noroviruses, carrying the FLAG tag in three proteins of unknown function. This facilitated SILAC-based proteomic studies to identify host cell factors involved in norovirus replication, which may represent potential antiviral targets. With the availability of a small animal model of infection, MNV also provides the opportunity to develop rational approaches to norovirus attenuation. One approach that has been successful for attenuating other RNA viruses involves harnessing the regulatory capacity of the cellular microRNA machinery to control viral replication and tropism. This approach was applied to MNV by inserting a microglial-specific microRNA target sequence into the MNV genome which successfully attenuated norovirus replication in permissive cell lines expressing the cognate microRNA. A second rational approach to attenuation was based on manipulating the rate and fidelity of the viral polymerase, which has been shown to affect RNA virus pathogenesis and can lead to attenuation. To this aim a panel of MNV polymerase mutants were engineered and characterised for their growth and fidelity and their ability to establish acute and persistent infections in vivo.
Supervisor: Goodfellow, Ian Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
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