Total RNA was extracted from NPS specimens using QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany), and stored at 280uC. RNA was amplified using the Qiagen One Step RT-PCR Kit. A five-tube mRT-PCR was used for virus identification as previously described [43, 44] . Tube 1 targeted IAV, influenza B virus, RSV, and human metapneumovirus; tube 2, parainfluenza viruses 1 to 4; tube 3, HRV and influenza C virus; tube 4, human coronaviruses (HCoVs) 229E-HCoV, OC43-HCoV, NL63-HCoV and HKU1-HCoV; and tube 5, adenovirus and HBoV. Amplified products were analyzed in 0.5 mg/ml ethidium bromide/2% agarose gel.