Frozen tissues were thawed and homogenized before inoculation into embryonated eggs for viral culture. Briefly, 1.0 ml of phosphate-buffered saline (PBS) was added to 100-150 mg of tissue, which was homogenized, and, then centrifuged; 0.1 ml of the recovered supernatant was injected into the allantoic cavity of 11 days-old specific pathogen-free embryonated chicken eggs. The allantoic fluids were tested for haemagglutation activity with turkey red blood cells after 72 h incubation at 35uC. Samples containing virus were subjected to RT-PCR and sequencing. Three blind passages were performed on hemagglutinationnegative samples to confirm the absence of infectious virus. All steps were performed in BSL-3 containment laboratory.