Contribution of viral morphology to influenza virus fitness Pubblico

Abstract

Influenza viruses come in two shapes - spheres and filaments. Laboratory strains that have been grown extensively in embryonated chicken eggs or in MDCK cells are made up of predominantly spherical and ovoid virions. Filament-producing strains are generally found in low-passage or clinical isolates. Previous work has shown that the filamentous morphology can be gradually lost upon continued passage in embryonated chicken eggs in favor of a predominantly spherical morphology. The fact that filaments are maintained in nature but not in the laboratory suggests the hypothesis that filament-producing viruses have a selective advantage within the infected host that is not necessary for growth in the laboratory. Through serial passage of two filament-producing viruses - A/Netherlands/602/2009 (H1N1) [NL602] and A/Georgia/M5081/2012 (H1N1) [M5081] - in embryonated chicken eggs and MDCK cells, we found that a conversion to a predominantly spherical morphology is not necessary for improved growth in laboratory substrates. However, we did identify two individual point mutations in the M1 matrix protein within the egg-passaged NL602 virus that conferred a spherical morphology as well as a growth advantage in eggs. Additionally, we observed that a filamentous morphology was selected for through passaging of a spherical laboratory strain - A/Puerto Rico/8/1934 (H1N1) [PR8] - in guinea pigs. Through sequencing of the guinea pig passage twelve virus pool, we identified several point mutations within the M1 matrix protein that conferred a filament-producing morphology when introduced individually. The selection for filament production through passaging in an animal host indicated that filament formation in vivo is in some way advantageous to the virus. Using a panel of in vitro assays, we compared the functional properties of PR8wt and two of the filament-producing M1 single mutants identified through guinea pig passage. Results indicated that filament-producing viruses have a higher neuraminidase activity than their spherical counterparts. No differences were observed in red blood cell binding avidity, hemagglutination inhibition, plaque reduction, or thermostability. Taken together, we have demonstrated that while filament formation can be lost upon passage in laboratory substrates, this trait is selected for within an animal host, and the observed selective advantage may relate to the increased neuraminidase activity of filament-producing viruses.

Table of Contents

Chapter 1: Introduction 1

Influenza Virus Classification and Organization 1

IAV Disease and Pandemics 2

Overview of the Influenza Virus Life Cycle 5

Influenza Virus Transmission 6

Influenza Virus Morphology 10

The M1 Matrix Protein 11

Additional Factors that Influence Influenza Virus Morphology 13

Project Background 15

Figure Legends 19

Figures 20

Chapter 2: Spherical Influenza Viruses Have a Fitness Advantage in Embryonated Eggs, while Filament-Producing Strains Are Selected In Vivo 22

Abstract 23

Introduction 24

Materials and Methods 26

Results 30

Discussion 37

References 41

Figure Legends 46

Figures 51

Chapter 3: Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1) virus have a higher neuraminidase activity than the spherical wild-type 63

Abstract 64

Introduction 65

Materials and Methods 67

Results 73

Discussion 80

References 84

Figure Legends 89

Figures 92

Chapter 4: Supplementary Data - Paired Spherical and Filamentous Viruses 98

Introduction 98

Material and Methods 98

Results 101

Conclusions 103

Figure Legends 104

Figures 106

Chapter 5: Discussion and Future Directions 111

Introduction of artificial mutations previously shown to affect viral morphology results in general attenuation 111

An exclusively spherical morphology is not necessary for growth in laboratory substrates, while filament formation is selected through passaging in vivo 112

Filament-forming rPR8 M1 mutant viruses have a higher neuraminidase activity compared to rPR8wt virus 114

Possible mechanisms promoting increased neuraminidase activity observed in filament-forming strains 115

Possible benefits of increased neuraminidase activity to viral fitness 118

Overall summary and conclusions 121

Figure Legends 121

Figures 123

References 127

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Virology Biology, Microbiology
Parola chiave	morphology influenza
Committee Chair / Thesis Advisor	Lowen, Anice Carmen, Emory University
Committee Members	Moore, Martin L, Emory University Speck, Sam, Emory University Plemper, Richard Karl, Emory University Steinhauer, David, Emory University

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