Influenza virus reassortment in the absence of segment mismatch Open Access

Baird, Nicolle (2015)

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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

Table of Contents Abstract Table of Contents List of Figures and Tables 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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