Figure 3.

Production and Morphology of SCV-CHIK in SCS Cells

(A) Immunoblot analysis confirmed expression of the HA-tagged D13 (assembly protein; 56 kDa) and FLAG-tagged CP77 (host-range protein; 77 kDa) in the SCS line using anti-HA and anti-FLAG antibodies, respectively. (B) Monolayers of CHO-D13L, CHO-CP77, and the SCS line were infected with VACV or SCV-CHIK (MOI, 0.001 PFU/cell). After 48 hr, the cells were fixed and viral plaques were examined by immunofluorescent staining using polyclonal anti-VACV antibody. Representative plaque images (scale bars, 200 μm) and single infected cells (inserts; scale bars, 10 μm) are shown. Bar chart shows mean plaque size (micrometers ± SD; n = 16–25) for the two viruses in the corresponding three cell lines. Statistics by Kolmogorov-Smirnov test. (C) SCS monolayers were infected with SCV-CHIK and VACV (MOI, 0.01 PFU/cell) and at the indicated times, virus titers were determined by plaque assay. Data are represented as the fold increase in the viral titer over the original inoculum (PFU ± SD; n = 4). (D) SCS cells, passaged 6 or 30 times, were infected with SCV-CHIK (MOI, 0.01 PFU/cell), and after 48 hr, titers were determined (n = 4 replicates). (E) Electron microscopy examination of virus morphology in SCV-non-permissive BHK-21 and SCV-permissive SCS cells infected with SCV-CHIK (MOI, 2 PFUs/cell). SCV-CHIK production was blocked at the viroplasma stage in BHK-21 cells, whereas virion assembly and maturation were observed in the SCS line. V, viroplasma; IV, immature virions; MV, mature virions.