Rapid quantitation of neuraminidase inhibitor drug resistance in influenza virus quasispecies.
BACKGROUND
Emerging resistance of influenza viruses to neuraminidase inhibitors is a concern, both in surveillance of global circulating strains and in treatment of individual patients. Current methodologies to detect resistance rely on the use of cultured virus, thus taking time to complete or lacking the sensitivity to detect mutations in viral quasispecies. Methodology for rapid detection of clinically meaningful resistance is needed to assist individual patient management and to track the transmission of resistant viruses in the community.


METHODS
We have developed a pyrosequencing methodology to detect and quantitate influenza neuraminidase inhibitor resistance mutations in cultured virus and directly in clinical material. Our assays target polymorphisms associated with drug resistance in the neuraminidase genes of human influenza A H1N1 as well as human and avian H5N1 viruses. Quantitation can be achieved using viral RNA extracted directly from respiratory or tissue samples, thus eliminating the need for virus culture and allowing the assay of highly pathogenic viruses such as H5N1 without high containment laboratory facilities.


RESULTS
Antiviral-resistant quasispecies are detected and quantitated accurately when present in the total virus population at levels as low as 10%. Pyrosequencing is a real-time assay; therefore, results can be obtained within a clinically relevant timeframe and provide information capable of informing individual patient or outbreak management.


CONCLUSIONS
Pyrosequencing is ideally suited for early identification of emerging antiviral resistance in human and avian influenza infection and is a useful tool for laboratory surveillance and pandemic preparedness.
Health Protection Agency, Centre for Infection, London, UK. angie.lackenby@hpa.org.uk
Angie
A
Lackenby
Jane
J
Democratis
Marilda M
MM
Siqueira
Maria C
MC
Zambon
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001717:Birds / Q000821:virology; D002460:Cell Line; D017931:DNA Primers; D024882:Drug Resistance, Viral / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005814:Genes, Viral; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D053124:Influenza A Virus, H5N1 Subtype / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005585:Influenza in Birds / Q000821:virology; D007251:Influenza, Human / Q000821:virology; D009154:Mutation; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods*
D000998:Antiviral Agents; D017931:DNA Primers; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D009439:Neuraminidase