Figure 1.

West Nile virus (WNV) infects human monocyte‐derived dendritic cells (DCs). (a) DCs were infected with multiplicity of infection (MOI) = 5 WNV Kunjin isolate (WNV_KUN). RNA was collected at the indicated time‐points (4–44 h) post‐infection, and the cDNA assessed for expression of the ENV gene by quantitative polymerase chain reaction (qPCR). Shown is the expression of the WNV ENV gene relative to the host ACTB gene, with the data normalized to the values at the 4‐h time‐point. The data are representative of two independent experiments. (b) DCs were left uninfected (Uninf), or infected with MOI = 1 or MOI = 5 WNV_KUN for 44 h. Symbols represent individual donors (n = 22). Three samples were infected at MOI = 50 for 24 h. The expression of the WNV ENV gene relative to the host ACTB gene is shown. Calculations were made using the 2^–ΔCt method, as described in the Methods section, and a Ct of 40 for the WNV ENV gene was used in the calculations for the uninfected samples, as no signal for viral RNA was detected. The significance of the data was evaluated using a Friedman test followed by Dunn's multiple comparison test (comparing each stimulated value to the corresponding unstimulated value). **P < 0·01; ****P < 0·0001. (c,d) The presence of intracellular viral proteins envelope (Env) protein and non‐structural (NS‐1) protein in DCs (infected at the indicated MOIs) was determined by flow cytometry after 24 or 44 h. These data are representative of three to six similar experiments, and the results of all experiments varying MOIs and time post‐infection are compiled. (e) DCs were infected with WNV at MOI = 50, and the virus washed away after 6 h or left in for 44 h. RNA was collected at the indicated time‐points post‐infection, and the cDNA assessed for expression of the ENV gene by qPCR. Shown is the expression of the WNV ENV gene relative to the host ACTB gene, with the data normalized to the signal at the 6‐h time‐point. The data are representative of two independent experiments.