Duck embryo (CCL-141), chicken embryo (CRL-12203), and quail QT6 cells were obtained from the ATCC (American Type Culture Collection, Manassas, VA) and were grown at 37°C and 5% CO2. Duck embryo cells were grown in Eagle’s minimum essential medium (ATCC), while chicken embryo cells were grown in Dulbecco’s modified Eagle’s medium (ATCC); both were supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. QT6 cells were grown in Ham’s F-12K medium (ATCC) with 2 mM l -glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, supplemented with 10% tryptose phosphate broth, 5% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin.
For inoculation experiments, portions of frozen, virus-positive beak, heart, brain, cloaca, kidney, liver, and ventriculus samples were minced with scalpels, suspended in serum-free DMEM containing 25 mM HEPES (pH 7.4), homogenized with a pellet pestle or Dounce homogenizer (beak), and placed on ice for 30 min. The homogenates were clarified by centrifugation at 10,000 × g for 1 min, filtered through a 0.2-µm filter, diluted 1:60, and used to inoculate cell cultures. The cells were visually monitored for evidence of infection, and the supernatants were monitored for the presence of viral particles using quantitative real-time PCR (qRT-PCR).
For inoculation experiments, portions of frozen, virus-positive beak, heart, brain, cloaca, kidney, liver, and ventriculus samples were minced with scalpels, suspended in serum-free DMEM containing 25 mM HEPES (pH 7.4), homogenized with a pellet pestle or Dounce homogenizer (beak), and placed on ice for 30 min. The homogenates were clarified by centrifugation at 10,000 × g for 1 min, filtered through a 0.2-µm filter, diluted 1:60, and used to inoculate cell cultures. The cells were visually monitored for evidence of infection, and the supernatants were monitored for the presence of viral particles using quantitative real-time PCR (qRT-PCR).