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Title: Investigating the molecular determinants of mumps virus pathogenesis
Author: Bamford, Connor Gavin George
ISNI:       0000 0004 5369 1044
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2014
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Abstract:
Mumps virus (MuV) is an enveloped, non-segmented, negative-sense, RNA virus, genus Paramyxoviridae. MuV causes an acute, systemic infection in humans characterised by parotitis, orchitis and aseptic meningitis. Despite widespread vaccination, outbreaks continue to occur. The biology of MuV infection and pathogenesis in humans remains poorly understood, in part due to a lack of physiologically-relevant virus and host cell model systems. Of particular interest is how MuV interacts with epithelial cells during infection. Therefore, a reverse genetics system was generated based on the consensus genomic sequence of unpassaged, clinical material from a genotype G MuV infection from the New York 2009 outbreak. The resulting viruses were referred to as recombinant (r) MuVG09. Establishment of this reverse genetics system allowed the generation of rMuVG09 expressing the enhanced green fluorescent protein (EGFP) in the third position, rMuVG09EGFP(3) to allow real -time tracking of virus replication in vitro. Surprisingly, recovered viruses had mutations, suggesting genetic instability of MuVG09 during growth in vitro. However, using this system, it was demonstrated that MuV could infect transformed and primary polarised epithelial cells from the apical and basolateral surfaces. Infection of differentiated, normal, human, bronchial, epithelial (dNHBE) cells showed that MuV could infect ciliated and non-ciliated, non-goblet cells. MuV was released from the apical and basolateral surfaces. In dNHBE cells this was associated with cell -to-cell spread and cell -to-cell fusion. MuV cell -to-cell fusion was controlled by haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. Mutation of a single amino acid (I278T) in the F glycoprotein ablated cell -to-cell fusion while preserving virus-to-cell fusion, which was associated with increased virion stability. Analysis of growth kinetics in vitro showed that the non-fusogenic virus was growth restricted in interferon (IFN) competent cell lines. This was correlated with an increase in interferon stimulated gene (ISG) expression during infection, suggesting a role for fusogenicity in evading the human IFN response.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.669545  DOI: Not available
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