FIG 4.

ZIKV production and spread at suboptimal temperature. (A) C6/36 cells with established ZIKV infection were split and incubated at 20°C or 28°C for 5 days. (B) C6/36 cells infected at an MOI of 0.1 were split at 36 h postinfection (p.i.) and incubated at 20°C and 28°C for 5 days. Supernatants were collected and replaced daily, and virus titers were determined. Data shown for persistent ZIKV infection are means ± SEM of results from two biological replicates, each consisting of three technical replicates (n = 6). Data shown for an MOI of 0.1 are means ± SEM of results from three independent experiments. Dashed lines represent when the cells were split. (C and D) The percentage of ZIKV-positive cells was assessed using flow cytometry. (C) Cells were infected with ZIKV (MOI 0.1) for 2 h at 28°C and then split and incubated at 20°C or 28°C. (D) Cells infected with ZIKV were kept at 28°C for 36 h and then split and incubated at 20°C or 28°C. ZIKV-positive cells were measured after splitting (2 or 36 h p.i.) and 2, 4, and 6 days p.i. Data shown are means ± SEM of results from seven independent experiments. Statistical differences were assessed by a two-way ANOVA with Bonferroni’s correction, where cells at 20°C were compared to cells at 28°C at every time point. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.