Poly a u

For infection, Vero, C6/36 cells and MoDCs were seeded in tissue culture plates 24 h prior to infection with ZIKV. Cells were washed once with pre-warmed phosphate-buffered saline solution (PBS) (Gibco) and ZIKV, diluted with culture medium without supplements, was added to reach the desired MOI. Based on preliminary experiments with two ZIKV strains in Vero and C6/36 cells, we selected a MOI of 0.1 TCID₅₀/cell to perform the African and Asian lineage strain comparison. Indeed, already at 24 h post infection at MOIs < 1 TCID₅₀/cell, we detected significant frequency of ZIKV-positive cells and high titers of live virus in supernatants. The virus was adsorbed for one hour at 37 °C or 28 °C. Next, the inoculum was discarded and the cells were washed three times with pre-warmed PBS. Fresh culture medium was added to each well and the cells were incubated at 37 °C or 28 °C, 5% CO₂ until harvesting. As controls, the culture supernatant from uninfected C6/36 cells was used for mock infection and Poly(IC) (30 µg/ml) (Sigma-Aldrich) in combination with Resiquimod (10 µM) (Sigma-Aldrich), Poly(AU) (0.1 µg/ml) (Sigma-Aldrich) and 5′ppp-dsRNA (1 µg/ml) (InvivoGen) were used as TLR ligands.

Poly I:C and poly A:U were both obtained from Sigma (St. Louis, Missouri, USA). The human agonistic anti-Fas antibody CH11 was obtained from Merck-Millipore, (Billerica, MA, USA), with murine agonistic anti-Fas antibody Jo2 obtained from BD Biosciences (San Jose, CA, USA). Staurosporine was obtained from Sigma.