| 摘要 | 第5-7页 |
| Abstract | 第7-8页 |
| List of Abbrevations | 第20-22页 |
| Chapter 1 Review of literature | 第22-42页 |
| 1. General introduction | 第22-28页 |
| 1.1. Hepatitis e virus disease | 第22页 |
| 1.2. History | 第22-23页 |
| 1.3. Taxonomy and phylogeny | 第23页 |
| 1.4. Structure of HEV and genome organization | 第23-24页 |
| 1.5. Molecular morphology and physical chemical characteristics | 第24-26页 |
| 1.6. Crystalline structure of the virus | 第26页 |
| 1.7. Life cycle of HEV | 第26-27页 |
| 1.8. Animal Models | 第27-28页 |
| 1.9. Hepatitis E cell culture models | 第28页 |
| 2. HEV epidemiology | 第28-32页 |
| 2.1. Geographical distribution of HEV | 第28-29页 |
| 2.2. Human HEV discovery,characterization and epidemiology | 第29-30页 |
| 2.3. Swine HEV discovery,characterization and epidemiology | 第30页 |
| 2.4. Avian HEV discovery,characterization and epidemiology | 第30-31页 |
| 2.5. Rabbit HEV discovery,characte rization and epidemiology | 第31页 |
| 2.6. The prevalence of HEV in other animals | 第31页 |
| 2.7. Acute hepatitis E | 第31页 |
| 2.8. Chronic hepatitis E | 第31-32页 |
| 3. HEV transmission | 第32-35页 |
| 3.1. Water-borne | 第32页 |
| 3.2. Food-borne | 第32-33页 |
| 3.3. Blood-borne | 第33页 |
| 3.4. Contact with infected animals | 第33页 |
| 3.5. Vertical transmission | 第33-35页 |
| 4. Hepatitis Symptoms | 第35-36页 |
| 5. The animal model for HEV | 第36页 |
| 6. Target organs of HEV infection | 第36-37页 |
| 7. Apoptosis its molecular mechanisms and significance | 第37-40页 |
| 7.1. Caspase dependent pathway | 第37-38页 |
| 7.2. Caspase independent pathway | 第38页 |
| 7.3. Apoptosis and the role of mitochondria | 第38-39页 |
| 7.4. Viruses and viral products that induce apoptosis | 第39-40页 |
| 8. The purpose of this study and significance | 第40-42页 |
| Chapter 2 Source of Hepatitis E Virus RNA from Pig | 第42-51页 |
| 1. Introduction | 第42页 |
| 2. Materials and methods | 第42-48页 |
| 2.1. Sample | 第42页 |
| 2.2. Laboratory reagents and consumables items | 第42-43页 |
| 2.3. Instruments and equipment | 第43-44页 |
| 2.4. Extraction of RNA from tissues | 第44-45页 |
| 2.5. Histopathological observations | 第45-47页 |
| 2.6. Immunohistochemical staining | 第47-48页 |
| 3. Results | 第48-49页 |
| 3.1. Pig liver HEV PCR results | 第48-49页 |
| 3.2. Histopathological observation of livers | 第49页 |
| 3.3. Detection of ORF2 antigen in liver cells | 第49页 |
| 4. Discussions | 第49-51页 |
| Chapter 3 Swine HEV inoculation, distribution and detection of viral RNA and load inorgans tissues of Mongolian gerbils | 第51-64页 |
| 1. Introduction | 第51页 |
| 2. Materials and methods | 第51-57页 |
| 2.1. The virus preparation | 第51页 |
| 2.2. Ethics statement and experimental animal | 第51-52页 |
| 2.3. Virus inoculation into Mongolian gerbils | 第52页 |
| 2.4. Sampling | 第52页 |
| 2.5. Laboratory reagents and consumables items | 第52-53页 |
| 2.6. Main laboratory experiment apparatus | 第53页 |
| 2.7. HEV RNA detection in Virus suspension | 第53页 |
| 2.8. HEV RNA detection in feces | 第53-54页 |
| 2.9. Gerbils infection HEV viral load measurement | 第54-56页 |
| 2.10. HEV viral load test after inoculation in feces, serum and organ tissues | 第56页 |
| 2.11. ELISAs for HEV antibody and antigen detection | 第56页 |
| 2.12. Liver function test after HEV inoculation in gerbils | 第56-57页 |
| 2.13. Data analysis | 第57页 |
| 3. Results | 第57-63页 |
| 3.1. Virus suspension HEV RNA detection nested PCR results | 第57页 |
| 3.2. Preparation and identification of standards | 第57-58页 |
| 3.3. Standard curve | 第58-59页 |
| 3.4. HEV load assay in gerbils | 第59页 |
| 3.5. Number of HEV RNA positive sera in Gerbil after inoculation of HEV | 第59页 |
| 3.6. Number of HEV RNA positive feces in Gerbil after inoculation of HEV | 第59页 |
| 3.7. Number of HEV RNA positive organs tissues after HEV inoculated gerbils | 第59-60页 |
| 3.8. HEV viral load in serum,feces,kidneys and testicle after inoculation | 第60-61页 |
| 3.9. Gerbils serum HEV IgG test results | 第61-62页 |
| 3.10. Concentrations ALT after HEV inoculation | 第62页 |
| 3.11. Concentrations AST after HEV inoculation | 第62-63页 |
| 3.12. Concentrations T-BIL after HEV inoculation | 第63页 |
| 4. Discussions | 第63-64页 |
| Chapter 4 HEV antigen detection,Structural changes and up-regulation apoptotic protein inMongolian gerbil's kidney intraperitoneally infected by swine hepatitis E virus | 第64-84页 |
| 1. Introduction | 第64-65页 |
| 2. Materials and methods | 第65-73页 |
| 2.1. Specimen | 第65页 |
| 2.2 Histopathology related reagents | 第65页 |
| 2.3. Immunohistochemical staining related reagents and procedure | 第65页 |
| 2.4. The main equipment | 第65页 |
| 2.5. Immunohistochemistry | 第65页 |
| 2.6. The expression of apoptotic proteins | 第65-66页 |
| 2.7. The expression of PCNA and HSP70 | 第66页 |
| 2.8. Positive signal density ratio | 第66页 |
| 2.9. Hematoxylin and eosin staining | 第66页 |
| 2.10. Mallory's trichome staining | 第66页 |
| 2.11. Sirus red staining | 第66-67页 |
| 2.12. cDNA preparation for Quantitative real-time PCR | 第67页 |
| 2.13. qRT-PCR procedure and primer design | 第67-68页 |
| 2.14. Terminal deoxynucleotidyle transferase-mediated dUTP nick end-labeling(TUNEL) | 第68页 |
| 2.15. Transmission electron microscopy | 第68-69页 |
| 2.16. Western blotting to detect HEV ORF3 antigen in the inf-ected gerbils of tissues | 第69-73页 |
| 2.17. Statistical analysis | 第73页 |
| 3. Results | 第73-81页 |
| 3.1. Clinical symptoms and relative weight of body and kidneys | 第73-74页 |
| 3.2. Immunohistochemical staining for HEV antigen | 第74页 |
| 3.3. Light microscope observation | 第74-75页 |
| 3.4. TUNEL assays | 第75页 |
| 3.5. Immunohistochemical examination for the apoptotic proteins | 第75-79页 |
| 3.6. Electron microscope findings | 第79页 |
| 3.7. Expression of relative mRNA up-regulating proteins | 第79-80页 |
| 3.8. Apoptosis related protein expression in the kidney tissue through western blot | 第80-81页 |
| 3.9. HEV ORF3 expression through western blot | 第81页 |
| 4. Discussions | 第81-83页 |
| 5. Conclusion | 第83-84页 |
| Chapter 5 Detection of HEV antigen and up regulation of apoptotic proteins in testisMongolian gerbil inoculated with swine hepatitis E virus | 第84-97页 |
| 1. Introduction | 第84页 |
| 2. Material Methods | 第84-87页 |
| 2.1. Animals and experimental treatments | 第84-85页 |
| 2.2. Sampling of Serum and Tissues | 第85页 |
| 2.3. Parafin sectioning | 第85页 |
| 2.4. Histopathology examination | 第85页 |
| 2.5. Measurement testicular seminiferous tubule diameter | 第85页 |
| 2.6. Quantitative real-time PCR for apoptosis genes | 第85-86页 |
| 2.7. Immunohistochemical staining for HEV ORF2 antigen in testicular tissues | 第86页 |
| 2.8. The expression of apoptotic proteins | 第86页 |
| 2.9. Blood biochemistry and testosterone measurement | 第86页 |
| 2.10. Transmission electron microscopy(TEM)observation of testicles | 第86页 |
| 2.11. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining | 第86页 |
| 2.12. Western blotting analysis | 第86-87页 |
| 2.13 Adding of antibodies | 第87页 |
| 3. Results | 第87-94页 |
| 3.1. Gross findings and body organ weights | 第87-88页 |
| 3.2. Blood biochemistry | 第88页 |
| 3.3. Histopathological changes in testes of HEV inoculated Mongolian gerbils | 第88-89页 |
| 3.4. Histopathological changes in epididymis of HEV inoculated Mongolian gerbils | 第89页 |
| 3.5. Ultrastructural changes in testicle byTransmission electron microscopy(TEM) | 第89-90页 |
| 3.6. Immunohistochemical staining for HEV antigen in testicular tissues | 第90页 |
| 3.7. Diameter of seminiferous tubules | 第90页 |
| 3.8. TUNEL Apoptosis assay | 第90-91页 |
| 3.9. Immunohistochemical examin ation for the Fas-L and FAS | 第91-92页 |
| 3.10. Expression and up-regulation of apoptosis-regulating proteins in testes after inoculation | 第92-93页 |
| 3.11. Fas and Fas -L protein expression in the testicular tissue through western blot | 第93页 |
| 3.12. HEV ORF3 expression through western blot | 第93-94页 |
| 4. Discussions | 第94-96页 |
| 5. Conclusion | 第96-97页 |
| Chapter 6 Location and distribution HEV antigen HEV RNA in rabbit Kidney tissues | 第97-105页 |
| 1. Introduction | 第97页 |
| 2. Material and methods | 第97-99页 |
| 2.1. Main laboratory instruments and reagents | 第97页 |
| 2.2. Virus suspension | 第97-98页 |
| 2.3. Experimental animals | 第98页 |
| 2.4. Sampling | 第98页 |
| 2.5. HEV RT-PCR of rabbits | 第98页 |
| 2.6. Liver function test after HEV inoculation in rabbits | 第98页 |
| 2.7. Histology and Immunohistochemistry Stain of tissues from rabbits | 第98页 |
| 2.8. TUNEL assay | 第98页 |
| 2.9. Western blot | 第98-99页 |
| 3. Results | 第99-103页 |
| 3.1. Clinical Observation | 第99页 |
| 3.2. RT-PCR for HEV RNA in Serum and Feces from Rabbits | 第99页 |
| 3.3. RT-PCR for HEV RNA in Tissues from Rabbits | 第99-100页 |
| 3.4. AST changes in sera of rabbits | 第100-101页 |
| 3.5. ALT changes in sera of rabbits | 第101页 |
| 3.6. TBIL changes in sera of rabbits | 第101-102页 |
| 3.7. Immunohistochemistry Stain for HEV antigen in tissues from rabbits | 第102页 |
| 3.8. Histopathological observation of kidneys of rabbits | 第102页 |
| 3.9 TUNEL | 第102-103页 |
| 3.10. Western Blotting | 第103页 |
| 4. Discussion | 第103-104页 |
| 5. Conclusion | 第104-105页 |
| Conclusions | 第105-106页 |
| Innovations | 第106-107页 |
| Plates | 第107-121页 |
| References | 第121-136页 |
| Acknowledgement | 第136-138页 |
| Author CV | 第138-139页 |
