2.6 Virus isolation and virus replication in cell
cultures
Samples which were detected N-DRV positive were processed for virus
isolation using LMH cell, according to the conventional pathogen
separation method. We extract the spleen tissues of ducklings and
hatching eggs samples, homogenize them by centrifugation, and then
freeze and thaw the extracted supernatant three times. The supernatant
was filtered through a filter driven by a 0.22 μm syringe, and the
filtered supernatant was seeded into cell monolayers. Then, the culture
was incubated at 37 ℃, 5% CO2, and the giant cell or
pattern cell lesions were examined daily. When we observed more than
75% CPE, we collected the virus culture and sub-cultured it until
stable CPE could be harvested (Folch et al., 1957). Finally, the cell
culture was frozen and thawed three times, and the rest of the study was
saved.