Date of Completion

12-9-2013

Embargo Period

6-7-2014

Keywords

Influenza, Interferon, Virology, Virus, Vaccine, Subpopulations

Major Advisor

Margaret J. Sekellick

Co-Major Advisor

Philip I. Marcus

Associate Advisor

Steven Geary

Associate Advisor

Charles Giardina

Field of Study

Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

This dissertation explores the complex nature of the influenza virus quasispecies. Influenza populations exist as a mixture of mostly replication-deficient biologically-active particles. These particles are measured and quantified based upon the phenotype they manifest upon entry into the host. Three subpopulations are characterized: infectious, interferon-inducing, and interferon induction-suppressing particles.

It has been observed that only 5 out of every 100 particles of influenza are infectious. A novel biological assay was developed and, for the first time, directly demonstrated that infectious particles are the progenitors of all other virus particles. From the disparity observed to exist between the numbers of physical and infectious particles, a mathematical approach is presented which quantifies the frequency of expression of individual gene segments.

Interferon-inducing particles are demonstrated to be resistant to physical and chemical inactivation when measured in avian cells –the best evidence to date that the interferon-inducing moiety is preformed within the virion. In contrast, interferon-inducing particles are inactivated at a rate consistent with inactivation of the NS gene in mammalian cells, suggesting that primary transcription is necessary for interferon-induction in these cells.

Interferon induction-suppressing particles inhibit interferon production in cells otherwise programed to induce. This activity is nonspecific, blocking interferon-induction by viruses belonging to multiple families, and is dependent upon expression of the NS1 protein. A mathematical model, based on a random (Poisson) distribution of virus particles amongst the cell monolayer, is presented that predicts the fraction of cells that will induce interferon, dependent upon the ratio of interferon-inducing and interferon induction-suppressing particles present in the virus stock. A novel method for quantifying interferon-inducing particles in preparations with high interferon induction-suppressing particle content is described. A highly potent interferon induction-suppression activity was also found to be associated with exposure to lipopolysaccharide.

The functional heterogeneity demonstrated by a single strain of influenza was quantified, utilizing interferon-induction and interferon induction-suppression as phenotypic markers of the quasispecies. The activities of 117 plaque-derived isolates were measured, and variability in these phenotypes was observed over a 1000-fold range. The genetic basis of this phenotypic variance was investigated through sequencing the NS genes of several isolates demonstrating extreme phenotypes.

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