Investigating interactions between influenza A virus and respiratory syncytial virus during coinfection

Haney, Joanne (2022) Investigating interactions between influenza A virus and respiratory syncytial virus during coinfection. PhD thesis, University of Glasgow.

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Respiratory viruses are the cause of significant disease burden and coinfections with more than one virus constitute between 10-30% of viral respiratory infections. Interactions among respiratory viruses are recognised for their importance in influencing viral dynamics, however direct virus-virus interactions are poorly understood. Influenza A virus (IAV) and respiratory syncytial virus (RSV) are important respiratory pathogens that share epidemiological characteristics, including timing of seasonal peaks of infections, and biological characteristics, including cellular tropism within the respiratory tract.

To characterise interactions between IAV and RSV during coinfection, we developed an in vitro model in A549 cells, a cell line derived from the human lung. Analysis of viral growth kinetics and viral dynamics by live cell imaging showed that, while IAV replication appears unaffected by coinfection with RSV, RSV replication is significantly decreased in coinfection. Imaging of coinfected cells stained for IAV and RSV nucleoproteins and glycoproteins show that they localise to the same regions of the plasma membrane, suggesting there may be opportunity for viral interactions during viral assembly. To further explore this hypothesis, virus particles budding from coinfected cells were examined using super-resolution confocal microscopy. Filamentous structures extended from coinfected cells, that incorporated glycoproteins from both viruses in distinct patches along the filament. The ultra-structure of these filaments, determined by cryo-electron tomography, revealed the formation of chimeric viral particles (CVPs) that contained genomes and structural features from both IAV and RSV. Additionally, coinfection by IAV and RSV generated pseudotyped RSV filaments that incorporate IAV glycoproteins. Functional assays using a sialidase showed that CVPs can facilitate entry of IAV into cells that were stripped of IAV entry receptors, demonstrating CVPs possess expanded receptor tropism.

To determine the likelihood for CVP formation in the airway epithelium, we coinfected primary differentiated human bronchial epithelial cell (hBEC) cultures at air-liquid interface. We observed that IAV and RSV infect ciliated epithelial cells and identified foci of coinfection. IAV and RSV proteins both localised at the apical surface of coinfected cells, providing opportunity for interactions to occur during viral assembly. Additionally, IAV and RSV replication kinetics and cytopathic effect in hBEC cultures reflected trends observed in the in vitro cell model, suggesting that viral interactions may be conserved between simplified and representative airway models.

Overall, this project characterises interactions between IAV and RSV during coinfection and we show that coinfection by IAV and RSV results in formation of a novel class of viral particles. By expanding viral tropism, formation of CVPs may alter viral dissemination within the respiratory tract, potentially impacting disease outcomes for a coinfected individual. Further, by defining a previously unknown source of viral interaction with implications on viral structure, we contribute more widely to the understanding of the properties of IAV and RSV, and their infection biology as a whole.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Funder's Name: Medical Research Council (MRC)
Supervisor's Name: Murcia, Professor Pablo and Swetha, Dr. Vijayakrishnan
Date of Award: 2022
Depositing User: Theses Team
Unique ID: glathesis:2022-82807
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Apr 2022 08:06
Last Modified: 01 Aug 2022 08:43
Thesis DOI: 10.5525/gla.thesis.82807

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