Rabies virus replication outside the central nervous system [Elektronische Ressource] : implications for disease transmission / vorgelegt von Mirjam Preuß

Mirjam Preuss

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 60
• Poids de l'ouvrage: 9 Mo

Extrait

Aus dem Institut für Anatomie und Zellbiologie
Abteilung Molekulare Neurowissenschaften
Geschäftsführender Direktor: Prof. Dr. E. Weihe
des Fachbereichs Medizin der Philipps-Universität Marburg
in Zusammenarbeit mit dem
Department of Microbiology & Immunology
Direktor: T. L. Manser, PhD
M. J. Schnell, PhD & B. Dietzschold, DVM
der Thomas Jefferson Universität Philadelphia
RABIES VIRUS REPLICATION
OUTSIDE THE CENTRAL NERVOUS
SYSTEM
Implications for Disease Transmission
Inaugural-Dissertation
zur Erlangung des Doktorgrades der gesamten Humanbiologie
dem Fachbereich Medizin der Philipps-Universität Marburg
vorgelegt von
Mirjam Preuß aus Wertheim
Marburg, 2008Angenommen vom Fachbereich Medizin der Philipps-Universität Marburg
am: 02. Juli 2008
Gedruckt mit Genehmigung des Fachbereichs.
Dekan: Prof. Dr. M. Rothmund
Referenten: . E. Weihe; M. J. Schnell, PhD
1. Koreferent: Prof. Dr. S. BeckerTABLE OF CONTENTS
1. Abstract [English] 1
2. Abstract [German] 3
3. Introduction 5
3.1. The Rabies Disease in its Historical Context: Epidemiology and
Pathogenesis 5
3.1.1. History 5
3.1.2. Development of Vaccines 5
3.1.3. Epidemiology 6
3.1.4. Impact on Human Health 7
3.1.5. Clinical Picture of the Rabies Infection 8
3.1.6. Invariably Fatal? 9
3.1.7. Pathophysiology 9
3.2. The Rabies Virus: Structure, Genomic Organization and Cellular Cycle of
Infection 10
3.2.1. Taxonomy 10
3.2.2. Virion Structure 10
3.2.3. Cellular Cycle of Infection 12
3.2.3.1. Adsorption 12
3.2.3.2. Penetration & Uncoating 14
3.2.3.3. Intraneuronal Transport 15
3.2.3.4. Viral Protein Synthesis 16
3.2.3.5. Replication 17
3.2.3.6. Assembly & Budding 19
3.3. Rabies Virus Replication at Peripheral Sites 20
3.3.1. Muscle Tissue 20
3.3.2. Salivary Glands 22
3.3.3. Solid Organs 23
3.3.4. Other Non-Neuronal Cells 24
3.4. Bat-Derived Rabies Virus Strains 254. Problem Statement 27
5. Materials & Methods 28
5.1. Animals and in vivo Experiments 28
5.1.1. Mice Strains 28
5.1.2. Infection and Observation of Mice 28
5.1.3. Tissue Harvest 30
5.2. Cells and their Cultivation 31
5.3. Molecular Biological Methods 32
5.3.1. Restriction Digest of DNA 32
5.3.2. Agarose Gel Electrophoresis 32
5.3.3. Determination of Concentration and Purity of Nucleic Acids by
Photometry 33
5.3.4. Gel Purification of DNA Fragments 33
5.3.5. Alternative Purification of DNA Fragments from Enzymatic Reactions 33
5.3.6. Dephosphorylation of Linearized DNA by Calf Intestine Phosphatase 34
5.3.7. DNA Ligation 34
5.3.8. Non-quantitative Polymerase Chain Reaction 34
5.3.9. Transformation of Chemically Competent Bacteria with Recombinant
DNA 35
5.3.10.Small- and Large-Scale Plasmid Preparation from Bacteria 36
5.3.11.Construction of a Recombinant Rabies Virus 37
5.3.12.RNA Isolation from Cells and Murine Tissue 38
5.3.13.Quantitative Real-time Two-Step RT-PCR Assay 39
5.3.13.1.Primer and Probe Design for Quantitative Real-time PCR 40
5.3.13.2.Reverse Transcription 40
5.3.13.3.Quantitative Real-time PCR 41
5.3.13.4.Generation of Standard Curves for Absolute Quantification of Viral
RNA 41
5.4. Viruses and Virological Methods 43
5.4.1. Wildtype Rabies Virus Strains 43
5.4.2. Recombinant Rabies Viruses 445.4.3. Recovery of Recombinant Rabies Viruses 44
5.4.4. Production of Virus Stocks in vitro 46
5.4.5. Virus Titration 46
5.4.6. Concentration of Virus Stocks by Centrifugation 47
5.4.7. Virus Isolation from Tissue 47
5.5. Immunological Methods 47
5.5.1. Determination of Virus Neutralizing Antibodies 47
5.5.2. Immunohistochemistry 48
5.5.2.1. Silanization of Glass Slides 48
5.5.2.2. Tissue Sections 48
5.5.2.3. Antibodies 48
5.5.2.4. Information about the Detected Antigens 49
5.5.2.5. Immunohistochemical DAB/Nickel Staining 50
5.5.2.6. Immunohistochemical Double Fluorescence Staining 50
6. Results 51
6.1. Symptoms and Outcome of Infections in Mice after Intravenous
Inoculation with a Recombinant Silver-Haired-Bat Rabies Virus Strain in
Comparison to Intramuscular Inoculation 51
6.1.1. Concentration Dependent Survival after Intramuscular and Intravenous
Inoculation 51
6.1.2. Differences in Symptoms after Intramuscular and Intravenous
Inoculation 52
6.2. Strain Dependency of the Outcome of Intravenous Inoculation 55
6.2.1. Outcome of Intravenous Inoculation with DOG4 55
6.2.2. Strain and Infection Route Dependent Distribution of Viral Antigen in the
CNS 56
6.2.3. Long-term Infection with DOG4 after Intravenous Inoculation 59
6.3. Presence of Rabies Virus at the Inoculation Site 61
6.4. Primary and Secondary Infection of Peripheral Organs 64
6.4.1. Time Dependent Progress of Viral CNS Infection after Intramuscular and
Intravenous Inoculation of Mice with rSB 64
6.4.2. Presence of rSB at Peripheral Sites after Intramuscular and Intravenous
Inoculation 666.4.3. Inoculum Size Dependent Presence of rSB in the CNS and at Peripheral
Sites after Intravenous Inoculation 70
6.4.4. Virus Isolation from Peripheral Tissue 72
6.5. Identification of rSB Target Cells in Peripheral Organs on the Example of
the Heart 74
6.6. Identification of the Main Viral Routes from the Periphery to the CNS after
Intravenous or Intramuscular Inoculation 77
7. Discussion 92
7.1. The Rationale for the Selection of DOG4 and rSB as Model Rabies Virus
Strains 92
7.2. The Relevance of Intravenous Inoculation for Naturally Occurring Rabies
Virus Infections 93
7.3. Usefulness of the TaqMan® Technique for the Absolute Quantification of
Viral RNA 94
7.4. Implications of the Long-term Persistence of DOG4 in the CNS of Healthy
Mice 97
7.5. The Impact of Persistent Rabies Virus Viremia 100
7.6. Rabies Virus at the Inoculation Site 102
7.7. Further Insight into the Independency of Symptoms from the Localization
of Rabies Virus within the CNS 104
7.7.1. Rabies Virus Migration Pattern after Intramuscular Inoculation 105
7.7.2. Direct Invasion of the CNS by rSB after Intravenous Inoculation through
Secretory Circumventricular Organs 107
7.7.3. Independence of Motor Dysfunctions from Viral Burden in Function-
Related CNS Areas 110
7.8. Immunologic Determinants of Symptoms and Outcome in Murine Rabies
Virus Infection 111
7.9. New Findings on the Primary and Secondary Infection of Peripheral
Organs - an Explanatory Model for the Route of Rabies Virus Infection
after Organ and Tissue Transplantation 115
7.9.1. Innervation Patterns of Organs Relevant for Rabies Virus Infection 116
7.9.2. Quantity of Ganglion Cells and their Accessibility to Rabies Virus from the
Vascular System Matter 119
7.9.3. Autonomic Ganglion Cells: not the Best Nursery for Rabies Virus
Progeny 1207.9.4. Closed Gateways: no Transition of Rabies Virus from Peripheral Organs
into the CNS on Nerval Pathways 121
7.10. Concluding Remarks and Outlook 123
8. Abbreviations 125
9. Bibliography 127
10. Appendix 146
10.1. Curriculum Vitae: Mirjam Preuß (July 2, 2008) 146
10.2. List of Academic Teachers 147
10.3. Acknowledgments 1481. Abstract [English]
1.
Rabies is a fatal disease in mammals which is transmitted by the neurotropic Rabies
virus (RV). Most often, classical RV infections originate from muscle tissue after a bite
through an infected canine and ascend to the central nervous system (CNS) via peripheral
nerves. In contrast, transfer of non-classical RV by bat bites or scratches, the most common
cause for human rabies in North America and also an emerging disease in Europe, most likely
introduces RV in rather small amounts superficially into a new host. In both cases, classical
and non-classical RV can have access to lymph and/or blood. The impact and effects of the
hematogenously and lymphatically distributed share of the viral inoculum is unclear.
Taking this into account combined with recent RV infections through unrecognized RV
infected organ transplantations the questions arose whether RV is able to infect peripheral
organs primarily via a vascular route or only by centrifugal spread via neuronal pathways
from the CNS and if this postulated route is strain dependent. Subsequently it was thought to
be elucidated, whether RV is able to replicate in organs and if its target cells for direct
invasion of organs are different from those it reaches after centrifugal spread from the CNS.
With regard to the transmission of RV by tissue transplants it was also investigated whether
RV originating from organs is more likely to ascend into the CNS by neuronal pathways or on
alternative routes.
In order to answer these questions, mice were infected either with a dog-derived
classical RV (DOG4) or a bat-derived non-classical RV (rSB) as representatives for the two
RV strains with the largest impact in naturally occurring human rabies, and monitored for
weight loss and disease symptoms. To maximize the hematogenous dissemination of the
inoculum, mice were infected intravenously (i.v.) and compared to mice inoculated
intramuscularly (i.m.). A TaqMan® probe based quantitative reverse-transcription polymerase
chain reaction (qRT-PCR) assay was developed to quantify strain-specifically negat