Figure 4.

Phyllanthus phillyreifolius extract and its major component geraniin target the early stage of ZIKV infection. (A) Schematic representation of time-of-drug addition assays used to characterize the antiviral activity of P. phillyreifolius extract (250 µg/mL) and geraniin (200 µg/mL). (B) flow cytometric analysis of GFP expression in A549 cells infected with ZIKV^GFP during 24 h at MOI of 1 under the different experimental conditions shown in (A). *** p < 0.001, n.s. = not significant. (C) A549 cells were infected with ZIKV at MOI of 1 for 1 h at 4 °C with or without P. phillyreifolius (250 µg/mL) or geraniin (200 µg/mL). Epigalocathechin gallate (EGCG) (100 µM) was used as positive control. The number of virus particles bound to cell surface was measured by RT-qPCR. (D) A549 cells were incubated 1 h with ZIKV^GFP at 4 °C and the temperature was shifted to 37 °C in absence (vehicle) or presence of 250 µg/mL of P. phillyreifolius or 200 µg/mL of geraniin which have been added at different time points post temperature shift. Q3G (200 µg/mL) was used as a positive control. ZIKV entry inhibition was calculated from the percentage of GFP-positive cells determined at 24 hpi. Data represent the means ± SD from four independent experiments performed in triplicate.