FIG. 1.

Plaque morphology and growth analysis of parental E5 virus and its E5-104 derivative in Vero cells and growth in neuroblastoma cells of human or murine origin. (A) Plaques of E5 and E5-104 on Vero cell monolayers were visualized using immunoperoxidase staining on day 4 postinfection. The mean plaque diameter of 11 randomly selected E5 plaques was 2.2 mm. Plaques of E5-104 (mean diameter of 0.18 ± 0.02 mm; n = 9) are indicated by arrows. (B) Growth of the wt LGT, E5, and E5-104 viruses in Vero and neuroblastoma cell cultures at temperatures ranging from 32°C to 39°C. Confluent cell monolayers in 24-well plates were infected with parental or mutant virus at an MOI of 0.01 and incubated at the indicated temperature for 4 days. Virus titers in cell culture medium were determined by plaque assay in Vero cells incubated at 32°C. The limit of detection was 0.7 log₁₀ PFU/ml. Bars represent the standard error of three to four separate experiments. (C) Replication kinetics of wt LGT, E5, and E5-104 viruses in Vero cells or in Neuro-2A or SH-SY5Y neuroblastoma cells following incubation at 32°C or 37°C. Virus was inoculated at an MOI of 0.01, and the virus titer in cell culture medium was determined in Vero cells cultivated at 32°C.