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Title: Using label free proteomics and RNA sequencing to investigate the human respiratory syncytial virus and the effects of the antiviral ribavirin
Author: Aljabr, Waleed A.
ISNI:       0000 0004 6059 754X
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Human respiratory syncytial virus (HRSV) is a known cause of severe lower respiratory tract infection (LRTI) in infants and young children worldwide. HRSV can cause illness in all ages especially those people at high risk including the immunocompromised and the elderly. Globally, HRSV infection leads to a significant healthcare and economic burden due to the lack of an approved vaccine and costly antiviral therapies that are potentially ineffective in some cases. Ribavirin is the only therapeutic licensed for the treatment of severe HRSV infection. It is a synthetic nucleoside with broad spectrum of antiviral activity encompassing both DNA and RNA viruses. The mechanism of action of ribavirin is unclear. It is thought to inhibit the replication of HRSV and lead to a reduction in viral load. How it does this is unknown and the subject of this thesis. This study focused on investigating the effect of the anti-viral ribavirin on cells in general and then infected with HRSV using both label free quantitative proteomics and transcriptomics. This allowed the investigation of the mutation frequency in HRSV and cellular protein abundance, which encompass several mechanisms by which ribavirin is postulated to work. This study was demonstrated that treatment of cells with ribavirin resulted in the increased transcription of selected cellular mRNAs including those involved in mediating anti-viral signalling. Additionally, ribavirin treatment caused a decrease in viral mRNA and proteins. In the absence of ribavirin, HRSV specific transcripts accounted for up to one third of total RNA reads from the infected cell RNA population. Ribavirin treatment resulted in a greater than 90% reduction in reads mapping to viral mRNA, while at the same time no such drastic reduction was detected for the abundance of cellular transcripts. The presented data revealed that ribavirin significantly increased the frequency of HRSV-specific RNA mutations in the viral genome, suggesting direct influence on the fidelity of the HRSV polymerase. The presented data shows transition and transversion substitutions occur during HRSV replication, and that these changes occurred in 'hot spots' along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, the data indicated that in the continuous cell types used, and at the time points analyzed, the abundance of some HRSV mRNAs did not reflect the order in which the mRNAs were transcribed. Overall, the work describes a mechanism of action for ribavirin in the context of viral infection, that has not previously been elucidated for HRSV.
Supervisor: Hiscox, Julian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral