Reassortment of Mammalian Orthoreovirus Open Access

Hockman, Megan (Fall 2021)

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Viral evolution can facilitate the generation of viral variants that escape therapeutics, subvert vaccination, and enter the human population from other host species. Studies of viral evolution therefore provide mechanistic insight into variant emergence. One potential driver of evolution relevant for segmented viruses is reassortment. During cellular co-infection, the segments of co-infecting viruses can mix and may be co-packaged. This results in progeny bearing novel combinations of the parental genes, a potentially important source of genetic diversity. The degree to which the virus’ replication mechanism impacts its ability to undergo reassortment is an area of ongoing research. Mammalian orthoreovirus (reovirus) represents an interesting system in which to study the relationship between replication mechanism and reassortment frequency. Viral inclusion bodies, which house replication machinery and viral mRNA, are hypothesized to impose a physical barrier to the mixing of parental mRNAs. Two commonly studied serotypes of reovirus, T1L and T3D, are known to generate inclusions with filamentous and globular morphologies, respectively. A single amino acid is responsible for this difference, allowing for the generation of inclusion mutants within the same serotype and the subsequent investigation of the impact of inclusion morphology on reassortment frequency. Reassortment studies were performed using an unbiased system. We developed this system to enable quantification of reassortment in the absence of protein or nucleic acid mismatch and quantified T1L and T3D reassortment using this system. Data were then compared to a mathematical model which assumed free segment mixing. We found that T3D reassortment occurs with comparable frequency to that predicted by the model, and T1L reassortment occurs less frequently. Alteration of inclusion body morphology in each serotype did not impact reassortment efficiency. Furthermore, blocking of inclusion body merging using the microtubule depolymerizing agent nocodazole did not impact reassortment frequency. We have developed a system in which to quantify reovirus reassortment. Using this system, we have concluded that inclusion morphology is not a determinant of reassortment frequency, and that inclusion coalescence is not a requirement for genetic exchange.  

Table of Contents

Chapter I. Introduction 1

Introduction 1

References 10

Chapter II. A method for the unbiased quantification of reassortment in segmented viruses 16

Abstract 17

Introduction 17

Results 20

Discussion 24

Materials and Methods 27

Acknowledgements 35

Figures and tables 36

References 47

Chapter III. Mammalian orthoreovirus reassortment proceeds with little constraint on segment mixing 51

Abstract 52

Introduction 53

Results 55

Discussion 61

Materials and Methods 64

Acknowledgments 70

Figures and Tables 71

References 78

Chapter IV. Summary 82

Summary 82

References 91 

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