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.