RNA extraction, cDNA synthesis, and real-time reverse transcription-PCR (RT-PCR) were used as described (15) . Virus isolation methods have also been described (16) but in general involved supernatants from a 1,000-μL sample homogenized by vortex and centrifuged at 2,500-3,000 rpm into 9-to 10-day-old specifi c pathogen-free eggs. Those positive in the hemagglutination assay were tested by hemagglutination-inhibition test with reference antiserum (A/chicken/West Java/Hamd/2006).