Glass bottom 96 well plates

Vero cells were seeded at 0.5 × 10⁴ cells/well in glass bottom 96-well plates (Eppendorf) and infected with ZKV-PF13 or MR766 strains at MOI of 1 for 24 h. Alternatively, ZIKV strains were pre-incubated for 1 h at 37 °C with plant extracts in DMEM before infection (A. triplinervis and A. theiformis at a final concentration of 500 μg.mL⁻¹, EGCG at a final concentration of 50 µM). After fixation with 4% PFA for 30 min at RT, cells were stained with wheat germ agglutinin (WGA) 488 conjugate (Thermo Fisher) in PBS-BSA 0.5% over night at 4 °C. After immunostaining, ZIKV plus strand RNA from both strains was detected following the manufacturer’s protocol (ViewRNA ISH Cells Assays) using probe sets designed by Affymetrix. Alexa-Fluor 546-conjugated ZIKV probe set recognizes a region between nt position 2 and 1144 of the ZIKV genome. Nuclei were stained with NucBlue (Thermo Fisher Scientific) for 15 min at RT. Images were acquired with a Zeiss LSM 700 laser scanning confocal microscope equipped with a X63 objective. Images were processed with Zen2 (blue edition) software and analysed with the ICY software (icy.bioimageanalysis.org) using the Spot Detector plugin.

Vero cells were seeded at 1.2 × 10⁴ cells/well in glass bottom 96-well plates (Eppendorf) and infected with ZIKV HD78 or MR766 strains at MOI of 500 for 1 h at 4 °C. Alternatively, ZIKV strains were pre-incubated for 1 h at 37 °C with plant extracts in DMEM before infection (A. triplinervis and A. theiformis at a final concentration of 500 μg.mL⁻¹, EGCG at a final concentration of 50 µM). Cells were then shifted at 37 °C for 90 min and fixed with 4% PFA for 30 min at RT. FISH assays were performed as indicated above.