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Title: Development of in vitro trans-complementation methodologies for investigating polymerase function in clinically significant hepatitis B virus phenotypes
Author: Beale, M. A.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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In chronic hepatitis B virus (HBV) infection, the loss of hepatitis B e antigen (HBeAg) and seroconversion to anti -HBe coincide with a decrease in viral load and reduction in clinical hepatitis. This thesis describes a programme of work undertaken to investigate viruses causing an unusually aggressive phenotype of anti-HBe positive chronic hepatitis with high viral load. The thesis is broadly divided into two sections. The first describes a genomi cs approach to investigating these viruses. A large multiple alignment of whole genome H BV sequences was generated using sequences retrieved from GenBank. This alignment was used for phylogenetic studies, as well as providing the reference set for developing a whole genome HBV next generation sequencing technique. A scheme for whole genome sequencing was developed using the SEQUENOM M assCLEAVE protocol, in which RNA transcribed PCR products are subjected to base-specific cleavage and MALDI-ToF mass spectrometry, followed by computer-aided pattern matching. Specific issues encountered with this technology will be described in depth. Phylogenetic and bioinformatic analysis of DNA sequencing information obtained for clinically significant viruses will also be described. The replication cycle of HBV remains poorly understood, partially due to the limitations of existing in vivo and in vitro models. The second half of the thesis describes the development of in vitro culture methods for investigating HBV replication. Mammalian expression vectors were designed and generated to express elements of the HBV. Transfection of individual vectors into human hepatoma cell lines was performed, and protein expression and viral DNA characterised using a number of techniques, including immunofluorescent microscopy, ELISA, western blotting and qPCR. This was followed by co-transfection experiments to demonstrate trans-complementation of the viral genome and restoration of viral replication. Once fully optimised, these techniques will allow comparisons of replication efficiency between viruses to be performed in a safe and flexible manner.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available