Therapeutic in vitro and in vivo approach of influenza virus infection by simultaneous reduction of virus titre and cytokine expression through inhibition of virus-induced {NF-_k63B [NF-kappa-B] and Raf-MEK-ERK activation [Elektronische Ressource] / by Pinto, Ana Ruth Jorge Portugal Machado

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Therapeutic in vitro and in vivo approach of influenza virus
infection by simultaneous reduction of virus titre and cytokine
expression through inhibition of virus-induced NF- B and Raf-
MEK-ERK activation
Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfilment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen
by
Pinto, Ana Ruth Jorge Portugal Machado
of
Lisbon, Portugal
Giessen 2008From the Institute of Medical Virology
Head: Prof. Dr. Wolfram Gerlich
of the Justus Liebig University Giessen
FIRST SUPERVISOR AND COMMITTEE MEMBER: Prof. Dr. Stephan Pleschka
SECOND SUPERVISOR AND COMMITTEE MEMBER:Prof. Dr. Stephan Ludwig
COMMITTEE MEMBERS: Prof. Dr. Heinz-Juergen Thiel (Chairman)
Prof. Dr. Juergen Lohmeyer
Date of Doctoral Defense: 9 December 2008I. Table of Contents
I. Table of contents
I. TABLE OF CONTENTS............................................................................................I
II. LIST OF FIGURES................................................................................................. V
III. LIST OF TABLES ................................................................................................ VI
IV. ABBREVIATIONS .............................................................................................. VII
V. SUMMARY ........................................................................................................... XI
VI. ZUSAMMENFASSUNG...................................................................................... XII
1. INTRODUCTION.....................................................................................................1
1.1. Influenza viruses ................................................................................................1
1.1.1. Different types of influenza viruses ................................................................1
1.1.2. Influenza A virus ............................................................................................2
1.1.2.1. Morphology and genome structure of influenza A virus...............2
1.1.2.2. Propagation and genome replication of influenza A virus............5
1.1.3. Antigenic variation of influenza virus infection .............................................10
1.1.4. Avian influenza viruses ..........................................11
1.1.4.1. History ...................................................................................................11
1.1.4.2. Current situation (epidemics and pandemics) ..............12
1.1.5. Clinical symptoms of influenza virus infection..............................................13
1.2. Mechanisms of intracellular signal transduction and influenza A viruses .15
1.2.1. The MAPK pathway (Raf/MEK/ERK signalling cascade).............................16
1.2.1.2. Role of Raf/MEK/ERK signalling cascade in influenza A virus infection 19
1.2.2. The NF- B pathway.....................................................................................21
1.2.2.1. Role of NF- B signalling cascade in influenza A virus infection ............24
1.3. Immune response and cytokine interplay......................................................25
1.4 Aims....................................................................................................................32
2. MATERIALS AND METHODS..............................................................................34
2.1. Materials............................................................................................................34
2.1.1. Instruments..........34
2.1.2. Reagents and general materials ..................................................................35
2.1.3. Monoclonal and polyclonal antibodies.......37
2.1.4. Materials for cell culture ...............................................................................37
2.1.5. Materials for mice experiments ..................38
2.1.6. Kits...............................................................................................................38
2.1.7. Virus strains and cell lines ...............................................................38
2.1.8. Inhibitors and solvent.................................38
II. Table of Contents
2.1.9. Media...........................................................................................................39
2.1.10. Buffers and solutions ..................40
2.1.11. Gels and other media.................................................................................42
2.2. Methods.............................................................................................................44
2.2.1. Working with cell cultures ............................................................................44
2.2.1.1. Maintenance of cell culture....................................................................44
2.2.1.2. Storage of cell cultures........................44
2.2.1.3. Infection of cells........................................................................45
2.2.2. Preparation of cell lysates for Western blot analysis...........45
2.2.3. Cell viability (cytotoxicity) analysis ...............................................................46
2.2.3.1. MTT-assay ...................................................................46
2.2.3.2. WST-1-assay.......................................47
2.2.3.3. Trypan Blue dye exclusion ...........................................47
2.2.4. Raising virus stocks ............................................................47
2.2.5. Analysis of infectious virus titres by immunohistochemistry.........................48
2.2.6. Haemagglutination (HA) Assay....................................................................49
2.2.6.1. Preparation of red blood cells (RBCs) from chicken blood.......49
2.2.6.2. HA assay...............................................................................................49
2.2.6.3. HI assay .....................50
2.2.7. Confocal Laser Scanning Microscopy and Immunofluorescence Assay (IFA)
...............................................................................................................................50
2.2.8. Western blotting (Semi-dry)..........51
2.2.8.1. Measurement of relative protein concentration (Bio-Rad protein assay)
...........................................................................................................................51
2.2.8.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
...........................................................................................................................51
2.2.8.3. Transfer to membrane in a "Semi-dry" electroblotter.............................52
2.2.8.4. Immunodetection of proteins .................................................................52
2.2.8.5. Enhanced Chemiluminescence (ECL) reaction.....................................53
2.2.8.6. Quantification of protein bands.....................................53
3. RESULTS..............................................................................................................57
3.1. Viability of A549 cells upon treatment with specific inhibitors ....................57
3.2. Virus infection induces the NF-B signal cascade in A549 cells and Bay 11-
7082 can inhibit this activation as well as decrease virus titres.........................58
3.3. Virus infection induces the Raf/MEK/ERK signal cascade in A549 cells and
U0126 can inhibit this activation as well as decrease virus titres ......................61
3.4. Bay 11-7082 and U0126 can decrease influenza A virus-induced cytokine
production in A549 cells.........................................................................................64
3.5. FPV and PR8-induced nuclear RNP export is efficiently blocked by Bay 11-
7082 and U0126 in A549 cells.................................................................................66
Primary mice alveolar epithelial cells (AECs).......................................................69
III. Table of Contents
3.6. Viability of mice primary alveolar epithelial cells upon treatment with
specific inhibitors....................................................................................................69
3.7. Viability of mice primary alveolar epithelial cells upon treatment with
specific inhibitors during the course of infection ................................................70
3.8. Both Bay 11-7082 nd U0126 can decrease virus titres in mice primary AECs
..................................................................................................................................72
3.9. Bay 11-7082 and U0126 can decrease influenza A virus-induced cytokine
production in mice primary AECs..........................................................................74
3.10. FPV and PR8 induced nuclear RNP export is efficiently blocked by Bay 11-
7082 and U0126 in AECs.........................................................................................76
3.11. Cell viability in A549 cells with combination treatment (Bay and U0126). 79
3.12. Virus titres treated with combination treatment (Bay and U0126). ............80
3.13. Cell viability in AECs with combination treatment (Bay and U0126). ........81
3.14. C57BL/6 mice........................................................