Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States Ha T. Nguyen a,c,d , Alma A. Trujillo a , Tiffany G. Sheu a,d , Marnie Levine a,d , Vasiliy P. Mishin a , Michael Shaw a , Edwin W. Ades b , Alexander I. Klimov a , Alicia M. Fry a , Larisa V. Gubareva a,⇑ a Influenza Division, Atlanta, GA, USA b Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA c Atlanta Research and Education Foundation, Atlanta, GA, USA d Battelle Memorial Institute, Atlanta, GA, USA article info Article history: Received 14 October 2011 Revised 13 January 2012 Accepted 17 January 2012 Available online 7 February 2012 Keywords: Pandemic H1N1 Neuraminidase inhibitor Pyrosequencing Neuraminidase inhibition assay H275Y I223K I223R abstract During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir treatment, were accepted for testing. Respiratory specimens were tested for the presence of the oseltamivir resistance-conferring H275Y substitution in the neuraminidase (NA) by pyrosequencing. Virus isolates propagated in MDCK cells were tested in phenotypic NA inhibition (NI) assays using licensed NA inhibitors (NAIs), zanamivir and oseltamivir, and investigational NAIs, per- amivir and laninamivir. Conventional sequencing and plaque purification were conducted on a subset of viruses. Pyrosequencing data were obtained for 87 specimens collected from 58 of the 72 (81%) patients. Of all patients, 27 (38%) had at least one specimen in which H275Y was detected. Analysis of sequential samples from nine patients revealed intra-treatment emergence of H275Y variant and a shift from wild- type-to-H275Y in quasispecies during oseltamivir therapy. A shift in the H275Y proportion was observed as a result of virus propagation in MDCK cells. Overall, the NI method was less sensitive than pyrose- quencing in detecting the presence of H275Y variants in virus isolates. Using the NI method, isolates con- taining H275Y variant at P 50% exhibited resistance to oseltamivir and peramivir, but retained full susceptibility to zanamivir. H275Y viruses recovered from two patients had an additional substitution I223K or I223R that conferred a 38–52- and 33–97-fold enhancement in oseltamivir- and peramivir- resistance, respectively. These viruses also showed decreased susceptibility to zanamivir and laninamivir. These data suggest that pyrosequencing is a powerful tool for timely detection of NAI resistant viruses and that NI assays are needed for comprehensive testing to detect novel resistance substitutions. Ó 2012 Elsevier B.V. All rights reserved. 1. Introduction The emergence of neuraminidase (NA) inhibitor (NAI) resistance in influenza viruses has both clinical and public health relevance. Circulation of oseltamivir-resistant viruses in the community limits antiviral treatment options, and the development of oseltamivir resistance while on therapy requires prompt identification of resistance to optimize clinical management. Resistance to NAIs can be detected by functional (a substitute for a phenotypic assay which is unavailable for this class of drugs) and genotypic assays. Functional assays detect susceptibility to specific NAIs and report a 50% inhibitory concentration (IC 50 ) for each drug that is inter- preted based on laboratory standards. Genotypic assays detect molecular markers that are associated with drug resistance. Cur- rently, genotypic assays for detection of H275Y substitution, a mar- ker associated with oseltamivir resistance in the NA of 2009 pandemic influenza A(H1N1) viruses (H1N1pdm09), are available at a number of commercial, clinical, and public health laboratories in the US. The availability of functional assays is currently limited to the Collaborating Centers of the World Health Organization and some academic and public health laboratories. During the 2009 pandemic, all but very few H1N1pdm09 viruses were resistant to adamantanes and susceptible to the licensed NAIs, oseltamivir and zanamivir (Bautista et al., 2010; Gubareva et al., 2010). However, prior to the emergence of H1N1pdm09 viruses, oseltamivir-resistant seasonal influenza A(H1N1) viruses circulated in high prevalence in the community (Dharan et al., 2009; Meijer 0166-3542/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2012.01.006 ⇑ Corresponding author. Address: Influenza Division, Centers for Disease Control and Prevention, Mail Stop G-16, 1600 Clifton Road, Atlanta, GA 30333, USA. Tel.: +1 404 639 5181; fax: +1 404 639 2350. E-mail address: LGubareva@cdc.gov (L.V. Gubareva). Antiviral Research 93 (2012) 381–386 Contents lists available at SciVerse ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral