Viral RNA was extracted from 140 µl of human nasal swab suspension or nasopharyngeal aspirate by QIAamp ® Viral RNA Mini Kit (QIAGEN, Germany) as described by the manufacturer or by an automated MagNA Pure LC Instrument applying the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics, Basel, Switzerland). The different gene segments were amplified by OneStep RT-PCR Kit (QIAGEN) as previously described [64] , applying a two minute elongation step for all genes. The primers for RT-PCR were segment specific but subtype universal targeting the highly conserved noncoding RNA regions at the 5'-and 3'-end of each segment [65] . PCR products were purified using the GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Germany) prior to sequencing.