title: Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing.
abstract: The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta Georgia 30333, USA.
authors.forename: Varough M
authors.initials: VM
authors.lastname: Deyde
authors.forename: Tiffany G
authors.initials: TG
authors.lastname: Sheu
authors.forename: A Angelica
authors.initials: AA
authors.lastname: Trujillo
authors.forename: Margaret
authors.initials: M
authors.lastname: Okomo-Adhiambo
authors.forename: Rebecca
authors.initials: R
authors.lastname: Garten
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Klimov
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Gubareva
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics*; D005819:Genetic Markers; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000235:genetics; D007251:Influenza, Human / Q000821:virology; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics; D053139:Oseltamivir / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D014763:Viral Matrix Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D000086082:Viroporin Proteins
chemical_list: D000998:Antiviral Agents; D005819:Genetic Markers; D009439:Neuraminidase; D053139:Oseltamivir; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C487630:NA protein, influenza A virus