isirv Antiviral Group
Pyrosequencing is a molecular technique that can be used for the detection and quantitation of (resistance) mutations. This rapid technique can be used on cultured virus or directly on clinical material such that it can be used to inform individual patient management. It has the advantage over some methodologies which are more time consuming or lack the sensitivity to detect mutations in mixed virus populations.
Pyrosequencing is a real-time DNA sequencing technique which, via a cascade of enzymatic reactions, detects pyrophosphate (PPi) released during DNA synthesis as visible light. The light released is quantitative and enables the rapid generation of sequence information.
Pyrosequencing instruments are available in two formats: 24 well and 96 well. A limitation of the technique is that, if used alone, it can only be used to detect well-characterised mutations. Nonetheless, the technique is suitable for high throughput surveillance or drug resistance mutation screening, as demonstrated during the emergence of the oseltamivir-resistant (H275Y) seasonal influenza A(H1N1) viruses in Europe during 2007-08, and more recently during the 2009 pandemic.
Pyrosequencing protocols for antiviral resistance mutations of A(H1N1)pdm09
A protocol for screening for oseltamivir resistance describes a method for amplification and pyrosequencing of the A(H1N1)pdm09 N1 gene to detect the H275Y mutation.
Pyrosequencing protocol (provided by kind permission of the Health Protection Agency, London)
Protocols to amplify the M2 gene and NA-275 of the A(H1N1)pdm09 virus are available on the WHO website.
Film of pyrosequencing methodology
The below short film explains pyrosequencing methodology and demonstrates how the technique is undertaken (provided by kind permission of the Health Protection Agency, London)
Deyde VM, Okomo-Adhiambo M. Sheu TG, Wallis TR, Fry A, Dharan N, Klimov AI, Gubareva LV. (2008). Pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza A viruses. Antivir Res 2009; 81(1):16-24.
Duwe S, Schweiger B (2008). A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1. J Virol Methods. 2008 Nov;153(2):134-41. Epub 2008 Sep 19.
Deyde VM, Sheu TG, Trujillo AA, Okomo-Adhiambo M, Garten R, Klimov AI, Gubareva LV. (2009) Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing. Antimicrob Agents Chemother. 2010 Mar;54(3):1102-10.
Lackenby A, Democratis J, Siqueira MM, Zambon MC (20088). Rapid quantitation of neuraminidase inhibitor drug resistance in influenza virus quasispecies. Antivir Ther. 2008;13(6):809-20.