The importance of innate resistance genes in respiratory syncytial virus replication in airway epithelial cells

Apperley, Louise The importance of innate resistance genes in respiratory syncytial virus replication in airway epithelial cells. Master of Philosophy thesis, University of Liverpool.

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Abstract

Introduction: Respiratory Syncytial Virus (RSV) is a leading cause of lower respiratory symptoms in infants, the elderly and the immunosuppressed worldwide. Approximately 70% of children become infected with RSV during their first year of life, and by the age of three almost all children will have been exposed to the virus. Approximately 2-3% of RSV infected infants require hospital admission. The aims of my MPhil project were as follows: • To determine whether I could ‘knockdown’ using siRNA, the expression of 26 innate resistance genes in airway epithelial cells, previously known to limit influenza. • To determine which genes provide protection against both influenza and RSV, by infecting ‘knockdown’ cultured epithelial cells with RSV, and measuring viral replication and RSV N gene expression. • To further investigate the significance of these results by focusing on three of these genes in more detail. Methods: SiRNA was used to knockdown genes in A549 cell cultures. They were infected with RSV A2 strain two days after siRNA transfection, and a further two days later, mRNA was extracted and isolated from the cells. These samples were reverse transcribed, and RSV N gene expression analysed through real-time PCR. Further study on genes required the use of antibody staining, fluorescent-labelled RSV and confocal microscopy. Results: Initially, PCR assays were optimised and quantitative, and RSV concentration was standardised. Efficiency of siRNA reagent was proved with gene expression knocked down > 60%. Following RSV infection of these knocked down cultures, 20/26 genes were found to up-regulate RSV N gene expression after their gene expression had been reduced. Two genes (RNF168 and RFFL) caused a large increase in RSV expression after knockdown and so were investigated further as well as, UBE2G2, an associated protein, identified through an online database. Unfortunately, the impressive changes in RSV replication previously seen on knocking down RNF168 and RFFL could not be repeated, and UBE2G2 was found to have little effect on RSV replication. GPR34, which when knocked down caused morphological changes on light microscopy, was also studied further using red fluorescent-labelled RSV and antibodies. This protein was found to be located around the periphery of the cell and possibly around the nucleus, with protein knockdown causing blebbing of the cell membrane. Discussion: This work has shown that 20/26 genes previously reported to offer some protection against influenza, also provide some protection against RSV. These findings further our understanding of the pathogenesis of RSV disease, and highlight possible avenues for future therapeutic research. These might involve increasing expression of these 'innate resistance genes' in the respiratory epithelium.

Item Type:	Thesis (Master of Philosophy)
Additional Information:	Date: 2012-08 (completed)
Subjects:	?? R1 ??
Divisions:	Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	11 Jan 2013 11:58
Last Modified:	16 Dec 2022 04:37
DOI:	10.17638/00008055
Supervisors:	McNamara, Paul
URI:	https://livrepository.liverpool.ac.uk/id/eprint/8055