As previously described28, RNA was directly extracted from nasal swabs or lung tissues. Reverse transcription was carried out under standard conditions with universal 12 primer. One pair of universal M gene primers was used to amplify M gene. M gene positive samples were inoculated onto monolayers of Madin–Darby canine kidney (MDCK) cells maintained in DMEM containing 1 μg/mL trypsin for 48 h at 37 °C. After three serial passages, virus isolates were identified by RT-PCR and the whole genome was amplified by eight pairs of primers and sequenced.