Nasal swabs placed into a transport medium (Virocult, Elitech) were transported at 4uC by special courier to the nearest National Influenza Centre (NIC) (Hospices Civils de Lyon, Lyon, or Pasteur Institute, Paris, France). Upon arrival, the swab samples were eluted into 2 ml of transport medium, processed for real-time reverse transcription (RT)-PCR analyses and inoculated onto MDCK cells for virus isolation and subsequent subtyping using a standard hemagglutination inhibition assay. For RT-PCR analyses, RNA extraction from 200 ml of specimen was performed using the QIAmp virus RNA mini kit (Qiagen) with RNA elution into a final volume of 60 ml. All real-time RT-PCR assays were performed in a final volume of 15 mL with 5 mL RNA, 0. mM of each primer, 0.2 mM probe, and 0.8 ml enzyme mix (Super-ScriptIII platinum one-step quantitative RT-PCR system, Invitrogen). Type A influenza virus RNA was detected by a real-time RT-PCR targeting the conserved matrix gene using GRAM/7Fw (59-CTTCTAACCGAGGTCGAAACGTA-39) and GRAM/ 161Rv (59-GGTGACAGGATTG GTCTTGTCTTTA-39) primers and GRAM probe/52/+ (59[Fam]-TCAGGCC CCTCAA-AGCCGAG-[BHQ-1]39) probe. The quality of the specimens was assessed by real-time RT-PCR targeting the GAPDH cellular gene [12] . Amplification was performed on a LightCycler 480 (Roche Diagnostics) (NIC, Pasteur Institute, Paris) or an ABI 7500 (Applied Biosystems) (NIC, Lyon). Cycling conditions are available upon request. Quantified synthetic RNA transcripts corresponding to the M and GAPDH genes were used as controls in parallel [13] .