HBV DNA replicates through a genomic RNA intermediate. The HBV reverse transcriptase lacks proof-reading activity, resulting in a much higher mutation rate for the HBV genome compared with other DNA viruses. HBV DNA thus is often present in quasi-species in an individual. One or more species may be favorably selected by factors like host immune clearance and use of antiviral drugs. / Hepatitis B virus (HBV) infected millions of people worldwide. Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). / In summary, this study developed and validated two platforms for (1) HBV mutation discovery; and (2) HBV mutation detection in viral quasi-species. These tools may be useful for research on HBV drug resistant mutations, clinical instructing and monitoring of antiviral treatment. / In this study, I have developed high-throughput methods for (1) discovery of novel HBV mutations; and (2) highly multiplexed detection of known HBV mutations, both in the background of HBV quasi-species. Patients undergoing long-term lamivudine treatment were used for mutation discovery. For mutation discovery in quasi-species, the MassCLEAVE(TM) technology, a method based on base-specific RNA cleavage and automated Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), was used. I found that MassCLEAVE(TM) can be used to discover mutations present as minorities. Additionally, a synergistic effect was found between direct sequencing and MassCLEAVE(TM) in identifying minority mutations. Multi-PLEX, a method based on single nucleotide extension and automated MALDI-TOF MS, was used to develop a highly multiplexed assay for simultaneous detection of 60 HBV mutations including all functionally known HBV mutations and other frequently observed mutations during antiviral treatment with unknown functions. This multiplex assay was tested on a large cohort of single and multiple drug-resistant patients and was shown to be highly accurate in detecting HBV viral mutations in quasi-species. / Nucleotide and nucleoside analogues (NAs) are widely used for antiviral therapy by effectively suppressing viral DNA replication. However, long-term administration may select for drug-resistant mutant strains, leading to treatment failure and liver disease progression. A number of HBV mutations such as rtM204V/I, rtN236T and rtL180M within the HBV reverse transcriptase are known to confer drug resistance. Detection of these known mutations is useful genotypic markers for monitoring antiviral treatment. In addition, novel drug resistant mutations continue to be discovered. / by Luan, Ju. / Adviser: Chunming Ding. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344398 |
| Date | January 2009 |
| Contributors | Luan, Ju., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xiii, 149 leaves : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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