The development and application of pyrosequencing assays to determine antiviral susceptibility of Influenza A to neuraminidase inhibitors.

Gatica-Wilcox, Bree 2014. The development and application of pyrosequencing assays to determine antiviral susceptibility of Influenza A to neuraminidase inhibitors. MPhil Thesis, Cardiff University. Item availability restricted.

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Abstract

Influenza A can result in complicated disease and death in high risk individuals, and can be treated with the neuraminidase inhibitors oseltamivir and zanamivir. During the 2007-2008 season a rise in oseltamivir resistance in H1N1 viruses was observed worldwide. This resistance was caused by a point mutation in the neuraminidase gene segment at position 274 causing an amino acid change from histidine to tyrosine. Other point mutations have also been observed conferring resistance to neuraminidase inhibitors in H1N1 and H3N2 viruses, such as D151, E119V, R292K and N294S. Assays for the detection of resistance to the neuraminidase inhibitors have been well documented, the most novel being pyrosequencing. The aim of this study was to optimise pyrosequencing assays for routine use in a diagnostic laboratory. Once optimisation and validation were achieved using reference strains of influenza A, clinical validation was performed on influenza A isolates collected during the 2008-2009 season. Optimisation and clinical validation of a SNP pyrosequencing assay for the detection of the H274Y mutation in H1N1pdm09 isolates was also performed on isolates collected during the 2009-2010 and 2010-2011 seasons. Routine diagnostic assays were optimised for the H274Y mutation in pre 2009 H1N1 viruses and H1N1pdm09 viruses, and for the E119V mutation in H3N2 viruses. Only SQA assays were able to be optimised for the D151, R292K and N294S mutations in H3N2 viruses. Clinical validation showed that all seasonal H1N1 viruses isolated before April 2009 possessed the H274Y mutation at nearly 100%. The E119V mutation was detected in nearly 50% of H3N2 samples tested at varying levels from 1% to 31%. The D151, R292K and N294S mutations were not detected in any of the samples and the majority of the H1N1pdm09 samples contained a low level (1-10%) of H274Y mutation in the viral quasi-species.

Item Type:	Thesis (MPhil)
Status:	Unpublished
Schools:	Medicine
Subjects:	Q Science > QR Microbiology > QR180 Immunology R Medicine > R Medicine (General)
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Oct 2023 10:43
URI:	https://orca.cardiff.ac.uk/id/eprint/64917

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