A viral titre reduction assay was used to determine the compounds’ effect on selected flaviviruses, including DENV2 and various ZIKV strains, as we described previously [24–26 (link)]. Briefly, viral infection was done by inoculation of approximately 2 × 105 cells with various viruses at MOI of 0.1 PFU/cell for A549 cells, or MOI of 1 PFU/cell for HPEC and HNPC cells in 24 well plates. Virus yields were quantified at 48-hour post-infection using standard viral plaque forming assay with Vero cells, as we described previously [24 (link)].
The effective concentration EC50 was determined by nonlinear regression fitting of the dose–response curve using the ORIGIN Suite 6.0 (Origin Lab, Wellesley Hills, MA) or GRAPHPAD 8.0 (San Diego, CA).
Human primary placental epithelial cells (HPECs) derived from the inner surface of the amnion were purchased from Cell Applications, Inc., and cultured according to manufacturer’s manual. Human HNPC, derived from iPSC generated using the STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) lentivirus, was purchased from EMD Millipore and cultured according to manufacturer’s manual. The antiviral efficacy experiments with HNPCs were carried out with a ZIKV MOI of 1 as described previously [24–26 (link)].
All cells were tested as free of Mycoplasma contamination.
The effective concentration EC50 was determined by nonlinear regression fitting of the dose–response curve using the ORIGIN Suite 6.0 (Origin Lab, Wellesley Hills, MA) or GRAPHPAD 8.0 (San Diego, CA).
Human primary placental epithelial cells (HPECs) derived from the inner surface of the amnion were purchased from Cell Applications, Inc., and cultured according to manufacturer’s manual. Human HNPC, derived from iPSC generated using the STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) lentivirus, was purchased from EMD Millipore and cultured according to manufacturer’s manual. The antiviral efficacy experiments with HNPCs were carried out with a ZIKV MOI of 1 as described previously [24–26 (link)].
All cells were tested as free of Mycoplasma contamination.