Understanding the factors that drive the emergence of dominant gene constellations during influenza A virus reassortment
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2017
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Melbourne Authors
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School of Biomedical Sciences
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PhD thesis
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Open Access
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© 2017 Dr. Sanja Trifkovic
Abstract
The segmented nature of the influenza genome allows the virus to undergo reassortment (gene swapping) upon co-infection of a single cell. This process is a major contributing factor to the emergence of novel pandemic strains and in a practical context, reassortment can be utilised to produce viruses carrying the gene constellations that make them suitable for vaccine production. Despite its significance, the factors that govern the emergence of dominant gene constellations during reassortment are not well understood. Studies in this thesis have extensively examined influenza virus reassortment in the context of vaccine seed production.
Using a methodology equivalent to that used for seasonal influenza vaccine seed production, eggs have been co-infected with the A/Udorn/307/72 (Udorn) virus as a model seasonal strain and the egg-adapted, high-yielding A/Puerto Rico/8/34 (PR8) virus. Tracking of the reassortant genotypes generated throughout the reassortment process performed under the selective pressure of antibody to PR8 surface glycoproteins, identified a large variety of reassortant viruses isolated in the initial stages. However, with subsequent rounds of replication and selection, specific gene constellations began to dominate. Enhanced replicative fitness was shown to explain the emergence of certain gene constellations; however, a few had reduced replicative abilities and we demonstrated, using competitive plasmid transfection experiments, that gene co-selection can explain the dominance of these viruses. We therefore postulate that preferential co-packaging of gene segments into progeny virions is an important driver for the emergence of certain gene constellations after reassortment. As well as confirming co-selection relationship between NA and PB1 from both Udorn and PR8 viruses, a novel putative co-selection relationship was identified between the PB1 and NP genes of both viruses and an additional putative co-selection relationship between the NA, PB2 and PB1 genes of the Udorn virus. Overall, the PB1 and NP genes of the Udorn virus and the PA and M genes of the PR8 virus were found to prevail in the final viral progeny.
Seed viruses produced through classical reassortment are selected to have the seasonal haemagglutinin (HA) and neuraminidase (NA) and a high HA protein yield. From the final dominant gene constellations isolated through cloning via limiting dilution, four potential vaccine seed candidates were selected. Comparisons of viral fitness for the reassortants identified an incompatibility between the Udorn PB1 and PR8 NP that resulted in reduced replicative fitness. This incompatibility was further exacerbated by the presence of the Udorn HA. Analysis of the four vaccine seed candidates also revealed that either an increase in the HA protein density on the virion or an increase in the production of non-infectious particles were the main mechanisms contributing to the observed high HA yields.
This study of the dynamics of reassortment show it to be a largely random process initially, but selective pressures such as replicative fitness, gene co-selection and antibody pressures restrict the final viruses that dominate. Influenza virus reassortment is a complicated process and understanding the factors that govern reassortment may aid in the prediction of future outbreak isolates.
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Influenza, reassortment, packaging, co-selection, constellation