Molecular analysis of acute and chronic duck hepatitis B virus (DHBV) infections in ducks.

i Declaration of originality and consent . vii Publications and presentations resulting from this thesis . viii Acknowledgements . x Abbreviations . xiv Chapter 1: Introduction . 1 1.1. Epidemiology of hepatitis B virus . 1 1.2. Hepadnaviruses . 1 1.2.1. The Hepadnaviridae family . 1 1.2.2. HBV virion structure . 2 1.2.3. HBV genome . 3 1.2.3.1. HBV genomic structure . 3 1.2.3.2. Open reading frames present on the HBV genome . 3 1.2.4. HBV replication . 4 1.2.4.1. HBV attachment and entry . 4 1.2.4.2. Transcription and replication . 5 1.2.4.3. Encapsidation, reverse transcription, budding and release . 5 1.3. HBV mode of transmission . 7 1.4. HBV infection outcomes . 8 1.4.1. Acute HBV infection . 9 1.4.1.1. Serological markers during acute HBV infection . 9 1.4.1.2. Immune responses during acute HBV infection . 9 1.4.1.3. Occult HBV infection . 10 1.4.2. Chronic HBV infection . 11 1.4.2.1. Serological markers during chronic HBV infection . 11 1.4.2.2. Immune responses during chronic HBV infection . 12 1.4.2.3. Chronic HBV-induced liver pathology . 13 1.5. HBV vaccines and treatments. 13 1.5. 1. HBV vaccines . 13 1.5. 2. HBV treatments . 14 1.5. 2.1. Immuno-modulators . 14 1.5. 2.2. Nucleoside/nucleotide analogues . 15 1.5. 2.3. Combination therapies and future potential therapies . 16 1.6. Clearance of cccDNA from infected hepatocytes . 16 1.6.1. Cytolytic hepatocyte killing . 16 1.6.2. Non-cytolytic hepatocyte curing . 17 1.7. Clonal proliferation of hepatocytes . 19 1.7. 1. Clonal proliferation of hepatocytes during acute HBV infection . 19 1.7. 2. Clonal proliferation of hepatocytes during chronic HBV infection . 20 1.8. Integration of HBV DNA into the host genome . 21 1.8. 1. Detection of integrated virus-cell junctions. 22 1.9. Animal models for the study of HBV . 24 1.10. Study aims and outline . 27 Chapter 2: Materials and Methods . 40 2.1. Animals . 40 2.2. Virus stock . 40 2.3. Procedures involving ducks . 40 2.3.1. Inoculation of ducks with DHBV . 40 2.3.2. Serum sample collection . 41 2.3.2.1. Venipuncture . 41 2.3.2.2. Blood serum extraction . 41 2.3. 3. Liver tissue collection . 41 2.3. 3.1. Liver biopsy . 41 2.3. 3.2. Autopsy . 42 2.3. 4. Tissue sample processing . 42 2.3. 4.1. Tissue fixation, embedding and sectioning . 42 2.3. 4.1.1. Ethanol:acetic acid . 42 2.3. 4.1.2. Formalin . 43 2.3. 4.1.3. 70% ethanol . 43 2.3. 4.2. Liver tissue processing . 43 2.4. Histology and immunohistochemical analysis of tissue sections . 43 2.4.1. Haematoxylin and eosin (H&E) staining. 43 2.4.2. Immunohistochemical detection of DHBsAg . 44 2.4.3. Periodic acid-Schiff diastase (PAS-D) staining of Kupffer cells . 45 2.4. 4. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) . 45 2.4. 5. Statistical analysis . 46 2.5. Serologic assays . 46 2.5. 1. DHBV DNA extraction from serum . 46 2.5. 2. Detection of DHBsAg by ELISA . 46 2.5. 3. Detection of anti-DHBc antibodies by ELISA . 47 2.5. 4. Detection of anti-DHBs antibodies by ELISA . 48 2.6. Liver DNA extraction . 48 2.6.1. DNeasy blood and tissue kit . 48 2.6.2. ChargeSwitch gDNA mini tissue kit . 49 2.6.3. Total DNA extraction . 49 2.6. 4. Low m elting t emperature a garose gel el ectrophoresis an d purification of hi gh molecular weight cellular DNA . 49 2.6. 5. Nuclear DNA extraction . 50 2.6. 6. High MW DNA extraction . 51 2.6. 6.1. High MW DNA extraction I . 51 2.6. 6.2. High MW DNA extraction II . 51 2.6. 6.3. High MW DNA extraction III . 52 2.6. 7. cccDNA extraction method . 52 2.7. Plasmid DNA extraction . 52 2.7. 1. Mini prep . 52 2.7. 2. Maxi prep .