Overexpressed ORF3 protein rescues the growth of ORF3null HEV in polarized human hepatocytes. (A) Immunofluorescence images showing ORF3-positive cells (green) in F2 (a subclone of HepG2/C3A cells), F2-empty (F2 cells transduced with an empty lentiviral vector), and F2-ORF3 (F2 cells transduced with an ORF3-expressing lentiviral vector) cells. Nuclei were stained with DAPI. Bars, 100 μm. (B) Immunofluorescence images showing HEV ORF2-positive cells (green) in polarized F2, F2-empty, and F2-ORF3 cells 5 and 10 days after infection with WT or ORF3null HEV (103 GE per cell). Bars, 100 μm. (C) HEV-positive areas at 5 and 10 days postinfection (dpi) were quantified using ImageJ software (NIH). Data are shown as means ± SEM from triplicate wells. n.s., not significant; ****, P < 0.0001.