REV-A Strain HLJ07I (GenBank accession No. GQ375848) was isolated from Heilongjiang Province in China in 2007. Chicken embryo fibroblasts (CEFs) were prepared as primary cultures from 10-day-old chicken embryos as previously described [16] and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum plus antibiotics. Viruses were grown in CEFs and incubated at 37uC with 5% CO 2 for 5 days. The suspension was frozen and thawed three times to disrupt cells and release virus, and then clarified by two centrifugation steps (2000 g for 15 min, and 10,000 g for 60 min). Virus present in the upper phase was precipitated with 10% (w/v) polyethylene glycol 6000 (PEG 6000) for 4 hours at 4uC. Precipitates were collected by centrifugation at 9,000 g for 30 minutes and resuspended in TNE buffer (50 mM tris-HC1, pH 7.5; 0.1 M NaC1, 10 mM EDTA). Finally, they were centrifuged through a 30% (w/v) sucrose cushion for 90 minutes at 200,000 g and resuspended in TNE buffer. The purified virus was analyzed in SDS-PAGE.