Influenza virus reassortment in the absence of segment mismatch Pubblico
Baird, Nicolle (2015)
Abstract
Influenza A virus (IAV) carries an eight-segmented genome which allows for reassortment when two different strains co-infect the same cell. Reassortment can lead to the generation of novel IAV, contributing to the development of epidemics and pandemics. A better understanding of the conditions that favor reassortment in nature would aid the development of public health measures aimed at limiting the impact of emerging IAV. It has been previously shown that reassortment is restricted by segment mismatch, including functional incompatibilities at the RNA or protein level. Due to this potent effect, additional factors that affect reassortment efficiency have been difficult to characterize. To study IAV reassortment in the absence of genetic incompatibilities, we have developed a system in which parental and progeny virus are of equal fitness, eliminating selection bias. A pair of phenotypically identical, yet genetically different viruses was generated using influenza A/Panama/2007/99 (H3N2) virus. Silent mutations introduced into each of the eight gene segments allowed our variant (var) virus to be differentiated from the wild-type virus by high resolution melt analysis. Using this system, we determined baseline frequencies of reassortment in cell culture and in-vivo. We further evaluated the effects of dosage, time and defective particles on this baseline frequency. The potential for reassortment declined quickly with increasing time intervals between two infections. Our results show that this effect is due to super-infection interference, which results from removal of sialic acids from the host cell surface by the viral neuraminidase and induction of interferon responses by the host cell. We demonstrated, using both computational and experimental methods, that semi-infectious (SI) particles promote diversification through reassortment. The introduction of defective interfering (DI) particles, by contrast, was found to potently suppress detectable reassortment, most likely due to the known interfering effects of segments carrying large deletions. These data suggest that semi-infectious, but not defective-interfering, particles may accelerate the evolution of IAV. Taken together, the work described has established a powerful new method for studying reassortment and exploited this method to reveal novel insights in to the potential for reassortment to occur within a cell and within an intact host.
Table of Contents
Chapter 1: Introduction 1
Organization and Classification of Influenza A Viruses 1
Influenza A Virus Life Cycle 2
Influenza A Virus Natural and Model Hosts 5
Human Influenza Disease 7
Influenza A Virus Epidemiology 8
Influenza A Virus Transmission 12
Influenza A Virus Evolution 13
Introduction to Thesis Project 18
Chapter 2: Influenza virus reassortment occurs with high frequency in the absence of segment mismatch 20
Abstract 21
Introduction 22
Results 25
Discussion 31
Materials and Methods 37
References 45
Figure Legend 50
Tables and Figures 53
Chapter3: Influenza virus reassortment is enhanced by semi-infectious particles but can be suppressed by defective interfering particles 62
Abstract 63
Introduction 64
Results 68
Discussion 83
Materials and Methods 89
References 103
Figure Legends 108
Tables and Figures 115
Chapter 4: Mechanisms of influenza A virus super-infection interference 130
Abstract 131
Introduction 132
Materials and Methods 134
Results 141
Figure Legends 145
Figures 148
Discussion 154
References 158
Chapter 5: Discussion 162
References 166
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