Identifying Mechanisms of Respiratory Syncytial Virus-Induced Mucus Público
Stokes, Kate Lynn (2013)
Abstract
Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis in infants. Viral infection of the bronchiolar epithelium results in destruction of epithelial cells and mucus production, leading to plugs that block airways and cause breathing difficulty. Disease severity has been correlated with RSV genotype; however, the impact of strain differences remains unclear.
We hypothesized that the pathogenesis of RSV disease is strain specific and used low-passage clinical isolates to examine differences in the BALB/cJ mouse model. Infection with isolate A2001/2-20 (2-20) induced higher lung IL-13 levels, airway mucin expression, and airway dysfuction than the genetically related isolate A2001/3-12 (3-12). It was previously shown that the fusion (F) protein of a mucus-inducing strain, line 19, is a factor in RSV mucin induction. We hypothesized that the F protein of 2-20 plays a role in RSV-induced mucin expression. We generated a chimeric RSV harboring the F gene of 2-20 in the genetic background of the non-mucogenic A2 strain. A2-2-20F infection resulted in early airway necrotic cell debris and lung mucin production. We also observed more neutrophil infiltration in the lungs of A2-2-20F-infected mice compared to mock- and A2-infected mice. This data indicates that F is a factor in RSV 2-20-induced mucus expression.
Neutrophils are abundant in the lungs of infants with severe RSV disease. Thus, we hypothesized that they play a role in 2-20 pathogenesis. Anti-Ly6G antibody was used to deplete neutrophils in RSV-infected mice. We observed lower lung mucin expression, less TNF-a, and less IL-13-expressing CD4+ T cells in neutrophil-depleted, RSV-infected mice compared to controls. Our findings demonstrated a novel role of neutrophils in virus-induced mucin response.
This body of data suggests that viral strain differences play a role in RSV severity. Sequence differences within F resulted in increased fusion activity and subsequent epithelial damage, followed by a robust neutrophil response that mediated lung mucin expression. Elucidating factors in RSV-associated airway pathology may have implications for treatment of severe RSV disease.
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Figures and Tables
CHAPTER 1: INTRODUCTION 1
Introduction 1
1.1 RSV Classification 1
1.2 RSV Virus Structure 2
1.3 RSV Antigenic Diversity
7
1.4 RSV Reverse Genetics System 9
1.5 RSV Pathogenesis in Infants
11
1.6 RSV Infection Models 13
1.7 RSV Infection in BALB/c Mice 16
1.8 Infection of the Airway Epithelium
18
1.9 Mucus Regulation and Production 20
1.10 Neutrophils In Respiratory Infection 21
1.11 T Cells in RSV Infection 25
1.12 RSV and Asthma 27
1.13 RSV Vaccines 28
1.14 Current Study 30
References 32
CHAPTER 2: Differential Pathogenesis of Respiratory Syncytial Virus Clinical Isolates in BALB/c Mice 66
Abstract 68
Introduction 69
Materials and Methods 70
Results 78
Discussion 98
Acknowledgements 102
References 103
CHAPTER 3: The Respiratory Syncytial Virus Fusion Protein and Neutrophils Mediate the Airway Mucin Response to Pathogenic RSV infection 117
Abstract 119
Introduction 120
Materials and Methods 122
Results 130
Discussion 147
Acknowledgements 154
References 155
CHAPTER 4: DISCUSSION 167
References 182
LIST OF FIGURES AND TABLES
Chapter 1 :
Figure 1: Schematic of RSV genome 3
Figure 2: RSV reverse genetics system
10
Chapter 2 :
Figure 1: Differential weight loss patterns with RSV
clinical isolates 79
Figure 2: Weight loss after infection with UV-inactivated
RSV 2-20 80
Figure 3: IL-13 levels in RSV-infected mice 81
Figure 4: Gob-5 Western blotting
82
Figure 5: In vitro growth of RSV strains A2, 2-20, and 3-12 85
Figure 6: Viral load of RSV strains A2, 2-20, and 3-12
86
Figure 7: RSV antigen in bronchiolar epithelium 88
Figure 8: Early histopathologic lesions of RSV 2-20 and 3-12 infection 90
Figure 9: RSV 2-20 induced high levels of pulmonary mucin expression 92
Figure 10: RSV 2-20 induced mucin expression is IL-13-dependent 93
Figure 11: Lung dysfunction caused by RSV 2-20 95
Figure 12: RSV A2 and 3-12 induced higher levels of IFN-g producing CD8+ T cells than 2-20 and line 19 96
Figure 13: IFN-g producing CD8+T cells in 2-20 infected BALB/cJ and IL-13 KO mice 97
Chapter 3 :
Figure 1: In vitro growth and in vivo viral load of RSV strains A2, 2-20, and A2-2-20F 131
Figure 2: RSV A2-2-20F infection resulted in early lung lesions in BALB/cJ mice 133
Figure 3: 2-20 F is a mucogenic factor in RSV infection
135
Figure 4: 2-20 F is more fusogenic than A2 F when co-expressed with RSV G protein 137
Figure 5: A2-2-20F causes neutrophil infiltration in the lungs of BALB/cJ mice 139
Figure 6: Anti-Ly6G treatment results in depletion of neutrophils in the blood and lungs of RSV-infected mice 141
Figure 7: 1A8 administration does not affect macrophage numbers in mock- or RSV-infected mice 142
Figure 8: Viral load after neutrophil depletion 143
Figure 9: Neutrophil depletion decreased mucin production 144
Figure 10: Neutrophil depletion results in decreased TNF-a levels during RSV infection 145
Figure 11: Neutrophil depletion reduces IL-13 producing CD4+ T cells in lungs of RSV-infected BALB/cJ mice 147
Figure 12: Side-view ribbon representation of the prefusion RSV F protein trimer (PDB 4JHW), colored by monomer 151
Chapter 4 :
Table 1: Comparison of RSV clinical isolates 171
Table 2: Comparison of chimeric and parent RSV strains 172
Figure 1: Sequence alignment of A2 and 2-20 F proteins 174
Figure 2: Thermostability of RSV Strains 177
Figure 3: Overview of the T cell events in viral clearance and disease 181
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