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Modification to the immunodominant loop of hepatitis B virus core protein to enhance vector properties of virus-like particlesHean, Justin 08 September 2014 (has links)
Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism.
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High-throughput discovery and detection of viral mutations in hepatitis B virus quasi-species for patients undergoing antiviral therapy. / 高通量發現及檢測抗乙型肝炎病毒治療患者的病毒突變株的方法學研究 / CUHK electronic theses & dissertations collection / Gao tong liang fa xian ji jian ce kang yi xing gan yan bing du zhi liao huan zhe de bing du tu bian zhu de fang fa xue yan jiuJanuary 2009 (has links)
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.
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Functional characterization of novel HBV subgenotypes/mutations associated with increased risk for hepatocellular carcinoma (HCC). / CUHK electronic theses & dissertations collectionJanuary 2009 (has links)
After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined. / Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A). / In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters. / Dong, Qingming. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-154). / 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.
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The role of direct carboxyl-terminal truncated HBx target genes in hepatocellular carcinoma. / CUHK electronic theses & dissertations collectionJanuary 2011 (has links)
Zhu, Ranxu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Virological characteristics of hepatitis B e antigen-negative chronic hepatitis B virus infection in China.January 2007 (has links)
Zhu, Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-118). / Abstracts in English and Chinese. / Contents --- p.I / List of Abbreviations --- p.IV / List of Tables and Figures --- p.V / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Viral Hepatitis --- p.2 / Chapter 1.2 --- Global Epidemiology of HBV --- p.3 / Chapter 1.3 --- Modes of Transmission --- p.4 / Chapter 1.4 --- Diagnostic Tests --- p.5 / Chapter 1.4.1 --- HBeAg and Anti-HBe --- p.7 / Chapter 1.4.2 --- Serum Enzymes --- p.8 / Chapter 1.4.3 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.1 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.2 --- Clinical Applications of DNA Assays --- p.10 / Chapter 1.4.4 --- Histology --- p.13 / Chapter 1.5 --- Natural Course of Chronic Hepatitis infection --- p.18 / Chapter 1.5.1 --- Phases of chronic hepatitis B --- p.18 / Chapter 1.5.2 --- HBeAg-negative chronic hepatitis B --- p.21 / Chapter 1.6 --- Molecular biology of HBV --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Genomic structure and organization --- p.24 / Chapter 1.6.2.1 --- Surface ORF --- p.24 / Chapter 1.6.2.2 --- Precore/Core ORF --- p.25 / Chapter 1.6.2.3 --- Polymerase ORF --- p.25 / Chapter 1.6.2.4 --- X ORF --- p.26 / Chapter 1.7 --- Genetic Variation of HBV --- p.31 / Chapter 1.7.1 --- HBV genotypes --- p.31 / Chapter 1.7.2 --- Predominant genotypes and their subgroups in Asia --- p.33 / Chapter 1.7.3 --- HBV mutations --- p.36 / Chapter 1.7.3.1 --- Precore mutations --- p.37 / Chapter 1.7.3.2 --- Core promoter mutations --- p.38 / Chapter 1.7.3.3 --- Other Mutations associated with clinical outcome --- p.40 / Chapter Chapter Two: --- Methodology --- p.44 / Chapter 2.1 --- Aims and Hypothesis --- p.45 / Chapter 2.1.1 --- Aims --- p.46 / Chapter 2.1.2 --- Hypothesis --- p.47 / Chapter 2.2 --- Patient Recruitment --- p.48 / Chapter 2.3 --- Laboratory Assays --- p.49 / Chapter 2.3.1 --- Preparation of serum HBV DNA --- p.49 / Chapter 2.3.2 --- Quantification of serum HBV DNA --- p.51 / Chapter 2.4 --- Full-genome Amplification of HBV DNA --- p.53 / Chapter 2.5 --- Full-genome Sequencing of HBV DNA --- p.55 / Chapter 2.6 --- Assembly of HBV Full-genome Sequence --- p.58 / Chapter 2.7 --- Phylogenetic Analysis --- p.59 / Chapter 2.7.1 --- Construction of phylogenetic tree --- p.59 / Chapter 2.7.2 --- Genotype and subgenotype determination --- p.60 / Chapter 2.8 --- HBV Mutations --- p.62 / Chapter 2.9 --- Info-gain program --- p.64 / Chapter 2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Results --- p.67 / Chapter 3.1 --- Patient Information --- p.68 / Chapter 3.2 --- Phylogenetic Analysis --- p.69 / Chapter 3.3 --- HBV genotypes/subgenotypes --- p.76 / Chapter 3.4 --- “Hot-spo´tح HBV Mutants --- p.79 / Chapter 3.5 --- HBV Mutation Associated with Liver Fibrosis --- p.82 / Chapter 3.5.1 --- Mutant selection --- p.82 / Chapter 3.5.2 --- Clinical significance of novel mutants --- p.84 / Chapter Chapter Four: --- Discussion --- p.88 / Chapter 4.1 --- Full-genome Sequencing Strategy --- p.89 / Chapter 4.2 --- HBV genotypes/subgenotypes Distribution and Disease Activity --- p.90 / Chapter 4.2.1 --- HBV genotypes/subgenotypes distribution --- p.90 / Chapter 4.2.2 --- Clinical significance of genotypes/subgenotypes --- p.91 / Chapter 4.3 --- HBV Hotspot Mutants and Disease Activity --- p.93 / Chapter 4.4 --- HBV Novel Mutants --- p.96 / Chapter 4.5 --- Limitation of the Study and Future Work --- p.97 / Chapter 4.5.1 --- Limitation --- p.97 / Chapter 4.5.2 --- Future Direction --- p.98 / Chapter Chapter Five: --- Conclusions --- p.99 / References --- p.102
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Study of mutations on hepatitis B virus promoters and construction of a replication-competent hepatitis B virus clone.January 2006 (has links)
Chan Ka Ping Sophie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 140-144). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.viii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter 1 --- Introduction / Chapter 1.1 --- Pathogenesis of HBV Infection --- p.1 / Chapter 1.2 --- Classification and Structure --- p.2 / Chapter 1.3 --- HBV Genome --- p.4 / Chapter 1.4 --- Replication Cycle --- p.7 / Chapter 1.5 --- HBV Genotypes and Nomenclature --- p.9 / Chapter 1.5.1 --- Asian prevalent genotypes --- p.9 / Chapter 1.5.2 --- Numbering system --- p.9 / Chapter 1.6 --- Identification of Markers in HBV Genome for HCC Development --- p.11 / Chapter 1.7 --- Project Objective --- p.13 / Chapter 1.8 --- Promoters of HBV --- p.14 / Chapter 1.8.1 --- Pre-S1 promoter --- p.14 / Chapter 1.8.2 --- X promoter and enhancer I --- p.14 / Chapter 1.8.3 --- Core promoter and enhancer II --- p.15 / Chapter 1.8.4 --- Pair of mutations at BCP --- p.17 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of pGL3-promoter Plasmids --- p.18 / Chapter 2.1.1 --- Templates selection --- p.18 / Chapter 2.1.2 --- Amplification of promoters --- p.19 / Chapter 2.1.3 --- Cloning into pGL3-basic vector --- p.21 / Chapter 2.1.4 --- Screening and plasmid preparation --- p.21 / Chapter 2.2 --- Construction of Mutant Promoter Clones --- p.23 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.23 / Chapter 2.2.2 --- pPreS 1 /2712C mutant clone --- p.24 / Chapter 2.3 --- Cloning of Full-length HBV Genomes --- p.26 / Chapter 2.3.1 --- Replication-competent HBV clone --- p.26 / Chapter 2.3.2 --- Amplification of full-length HBV genome --- p.28 / Chapter 2.3.3 --- Cloning into pUC19 vector --- p.28 / Chapter 2.3.4 --- Screening for insert and sequence confirmation --- p.29 / Chapter 2.3.5 --- Excision of full-length HBV from plasmid --- p.29 / Chapter 2.4 --- Re-construction into a 1.3-fold HBV Clone --- p.32 / Chapter 2.4.1 --- Cloning of HBV fragment nucleotide 979-2617 (nt 979-2617) --- p.32 / Chapter 2.4.2 --- Screening for insert and sequence confirmation --- p.33 / Chapter 2.4.3 --- Cloning of HBV fragment (nt 905-2000) --- p.33 / Chapter 2.4.4 --- Construction of a 1.3-fold HBV genotype Cs clone --- p.34 / Chapter 2.5 --- Cell Culture --- p.37 / Chapter 2.5.1 --- Cell culture maintenance --- p.37 / Chapter 2.5.2 --- Transient transfection of promoter clones --- p.37 / Chapter 2.5.3 --- Transient transfection of HBV genomes --- p.38 / Chapter 2.6 --- Dual-Luciferase® Reporter Assay System --- p.40 / Chapter 2.6.1 --- Principle of the assay --- p.40 / Chapter 2.6.2 --- Cell harvest --- p.43 / Chapter 2.6.3 --- Luciferase assay --- p.43 / Chapter 2.7 --- Data Analysis --- p.44 / Chapter 2.8 --- Extraction of HBV DNA from Intracellular Cores --- p.45 / Chapter 2.8.1 --- Harvest of intracellular cores --- p.45 / Chapter 2.8.2 --- Phenol/chloroform extraction --- p.45 / Chapter 2.9 --- Southern Blotting --- p.47 / Chapter 2.9.1 --- Transfer of DNA to membrane --- p.47 / Chapter 2.9.2 --- Preparation of probes --- p.47 / Chapter 2.9.3 --- Hybridization with radiolabeled probes --- p.48 / Chapter 2.10 --- Detection of HBeAg and HBsAg --- p.50 / Chapter 2.10.1 --- HBsAg assays --- p.50 / Chapter 2.10.2 --- HBeAg assays --- p.51 / Chapter 2.11 --- SEAP Reporter Gene Assay --- p.52 / Chapter 3 --- Results / Chapter 3.1 --- Templates Selected --- p.53 / Chapter 3.2 --- Results of Luciferase Assays --- p.58 / Chapter 3.2.1. --- BCP mutation of genotype A as control --- p.58 / Chapter 3.2.2. --- Effect of C1165T mutation on Xpro/enhI activity of HBV genotype B --- p.60 / Chapter 3.2.3. --- Effect ofT2712C mutation on pre-S1 promoter activity of HBV Genotype B --- p.60 / Chapter 3.2.4. --- Effect of G1613A mutation on core pro/enhII activity of HBV Genotype Cs --- p.64 / Chapter 3.2.5. --- G1613A and BCP mutation --- p.67 / Chapter 3.3 --- Full-length HBV Genome Clones --- p.70 / Chapter 3.3.1. --- Construction of replication-competent full-length HBV genome clones --- p.70 / Chapter 3.3.2. --- Drawbacks of the system --- p.78 / Chapter 3.4 --- Construction of a Replication-competent 1.3-fold HBV Clone --- p.82 / Chapter 3.4.1. --- Construction of the HBV (nt 979-2617) clone --- p.82 / Chapter 3.4.2. --- Construction of the HBV (nt 905-2000) clone --- p.86 / Chapter 3.4.3. --- Construction of 1.3-fold genotype Cs HB V clone --- p.89 / Chapter 3.4.4. --- Test for replication competency --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- BCP Mutation as Control of the Luciferase Assay --- p.94 / Chapter 4.2 --- Promoter Activities Not Altered by T2712C and C1165T --- p.96 / Chapter 4.3 --- Mutation G1613A of Core pro/enhll --- p.98 / Chapter 4.3.1 --- Mutation resides in negative regulatory element of core promoter --- p.98 / Chapter 4.3.2 --- NRE and NRE-binding protein --- p.98 / Chapter 4.3.3 --- Relationship with BCP mutation --- p.101 / Chapter 4.4 --- HBV Constructs --- p.103 / Chapter 4.4.1 --- Rationale in re-construction of 1.3-fold HB V clone --- p.103 / Chapter 4.4.2 --- Replication competency --- p.104 / Chapter 4.5 --- Conclusion --- p.106 / Chapter 4.6 --- Future Work --- p.107 / Appendix --- p.108 / References --- p.140
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