Contribution of viral morphology to influenza virus fitness Public
Seladi-Schulman, Jillian Marie (2014)
Published
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
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