Identifying Mechanisms of Respiratory Syncytial Virus-Induced Mucus Público

Stokes, Kate Lynn (2013)

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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



Table of Contents

List of Figures and Tables


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


References 182


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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