FIG 8.

G₀/G₁-phase arrest of SNB-19 cells does not promote ZIKV replication. (A) Flow cytometry analysis of SNB-19 cells cultured for 24 h in 10, 1, and 0.1% serum media, respectively. Samples were stained with propidium iodide (PI) and analyzed by flow cytometry. (B and C) SNB-19 cells were infected for (B) 24 h or (C) 36 h with ZIKV^PR (MOI of 0.5) or ZIKV^M (MOI of 0.5) or 48 h with DENV (MOI of 0.5). (B) Western blot images of intracellular viral protein expression in serum-starved SNB-19 cells 24 h postinfection. Following culture for 24 h in standard (10% FBS) or reduced (1% or 0.1% FBS) serum medium, cells were infected by direct addition of virus culture to the existing culture medium. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the 10% FBS control, representing the mean ± SD from three biological replicates. (C) Infectivity titers of culture supernatants as measured by FFU (ZIKV^M and ZIKV^PR) or PFU (DENV) on Vero cells. Error bars are the mean ± SD from three biological replicates. (D) Western blot images of intracellular viral protein expression in SNB-19 cells 24 h postinfection. Following a 2-h infection in standard serum medium (10% FBS) with ZIKV^PR (MOI of 2.0), ZIKV^M (MOI of 2.0), or DENV (MOI of 2.0), the medium was changed to standard serum medium (10% FBS) or reduced serum medium (1% or 0.1% FBS) for 24 h prior to collection. Viral protein was detected by anti-ZIKV NS1 or anti-DENV NS3 for each respective sample. Viral protein band intensities were normalized to the 10% FBS control, representing the mean ± SD from three biological replicates. In panels B to D, * indicates P ≤ 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001, and **** indicates P ≤ 0.0001 (one-way ANOVA).