Detection of HEV Antigen and dsRNA

Detection of HEV ORF2 Ag in the infected cells was done by flow cytometry as described previously [18 (link)]. Briefly, HMOs and HMACs were challenged with the stool-derived HEV-1 and/or HEV-3 as described; the cells were fixed at day 12 post infection and permeabilized using eBioscience™ Fixation/Permeabilization Concentrate (ThermoFischer Scientific, USA) according to the manufacturer’s instruction and then stained with antibody 1E6 clone (Millipore, Billerica, MA, United States; dilution 1/1000) that targets the HEV ORF2 protein. Goat anti-mouse IgG conjugated with Alexa488 (Invitrogen, Waltham, MA, USA) was used as a secondary antibody according to the manufacturer’s instructions. For detection of ds-RNA, the cells were fixed using cold methanol, permeabilized by 0.5% Triton X100, and stained with mouse monoclonal anti-dsRNA J2 Ab (Millipore; dilution 1/200). Goat anti-mouse IgG conjugated with Alexa647 (Invitrogen) was used as a secondary antibody (dilution 1/1000), and DAPI was used for nuclei staining.