Figure 2. Growth properties and genetic stability of caspase-cleavage mutant viruses.

(A) Multi-cycle replication of mutant viruses in MDCK cells. MDCK cells were infected with 10⁻³ MOI of each virus and the supernatants were harvested every 24 hpi. (B) Single-cycle replication of mutant viruses. MDCK cells were infected with 10 MOI of each virus and the supernatants were harvested every 2 hpi. Viral titers of the supernatants were determined by plaque assay. Data are the mean of three independent experiments. (C) Plaque-forming ability of mutant viruses. Viral plaque assay with WT and mutant viruses were performed on MDCK cells and the plaques were stained at five days after the assay. The representative images of crystal violet-stained plaques were shown. (D) Viral yields of mutant viruses in embryonated chicken eggs. Each egg was inoculated with 100 PFU of each virus and incubated at various temperatures. Four days later, the alantoic fluids were harvested for viral titrations. Data are the mean of at least 5 eggs. (E) Cleavage of mutant NP and NS1 in chicken embryonic fibroblasts (CEF). Primary CEF were infected with 1 MOI of WT virus or DM-C and incubated at various temperatures. The cells were harvested at different time points and the cell lysates were subjected to anti-NP and NS1 western blots. (F) Chromatograms showing nucleotide sequences encoding caspase cleavage motifs in NP and NS1 of plaque purified DM-C after ten passages in eggs, MDCK cells, and A549 cells.