Viral Determinants of Reliance on Multiple Infection in Influenza A Viruses 公开
Shartouny, Jessica (Summer 2022)
Published
Abstract
Viral infections involving two or more virions in a single cell have been seen to impact infection outcomes for a diverse array of virus types. We previously found that influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) virus (GFHK99) displays a particularly high reliance on multiple infection in mammalian cells tied to the PA gene segment. This dissertation sought to uncover the viral processes underlying the high reliance phenotype of GFHK99. Herein, the experimental techniques and data collected in the evaluation of GFHK99 are described. PA 26K was found to suppress endonuclease activity and viral transcription, specifically within cells infected at low multiplicity. The model arising from this research postulates that sub-optimal activity of the GFHK99 endonuclease results in inefficient priming of viral transcription, an insufficiency which can be overcome with the introduction of additional viral templates to the cell. These findings add to the burgeoning field of collective cellular interactions of viruses and have implications for continued public health surveillance of H9N2 IAVs.
Table of Contents
Chapter 1: Introduction pg. 1
Virus structure and lifecycle pg. 1
Fig. 1: The structure and proteins of an influenza A virion pg. 2
Fig. 2: Antigenic drift and antigenic shift pg. 6
IAV ecology and impact pg. 8
Interhost transmission pg. 14
Fig. 3: Barriers to IAV spread pg. 14
Reliance on multiple infection pg. 18
Fig. 4: The Lowen Lab multiple infection model pg. 19
Fig. 5: GFHK99 high reliance is linked to the PA gene segment pg. 20
Dissertation aims pg. 21
References pg. 23
Chapter 2: Beneficial effects of cellular coinfection resolve
inefficiency in influenza A virus transcription pg. 35
Introduction pg. 36
Results pg. 37
Fig 1. Replacement of the MaMN99 endonuclease region
with that of GFHK99 PA increases reliance on multiple infection. pg. 38
Fig 2. Disrupting PA-X does not alter reliance on multiple infection
phenotype of GFHK99 and MaMN99 viruses. pg. 41
Fig 3. PA 26K is associated with a higher reliance on multiple infection pg. 42
than PA 26E.
Fig 4. Levels of PA protein are not decreased by PA 26K pg. 44
Fig 5. PA 26K confers lower endonuclease activity than PA 26E pg. 46
Fig 6: Inhibition of cap-snatching increases reassortment pg. 47
Discussion pg. 47
Fig 7: Multiple infection allows a virus with an inefficient PA
endonuclease to replicate. pg. 47
Methods pg. 51
Tables pg. 57
Table 1: Primers used for chimeric PA segment Gibson Assembly
Table 2: Primers for ΔPA-X site-directed mutagenesis
Table 3: Primers used for strand-specific qPCR of MaMN99
References pg. 59
Chapter 3: Conclusions pg. 63
References pg. 69
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