FIG 4.

Simultaneous detection of HEV RNA and HA-tagged ORF1 protein in cells replicating full-length genomes. Hep293TT cells transfected with full-length B2HA or wild-type (wt) HEV RNA were fixed 5 days posttransfection and subjected to fluorescence in situ hybridization (FISH) using the HEV-specific probe V-HEV-C2 (red), followed by immunofluorescence detection of HA-tagged ORF1 protein using rabbit MAb C29F4 against HA (green) and mouse MAb 1E6 against ORF2 protein (magenta). Cell nuclei were counterstained with DAPI (blue). Nontransfected cells (nt) served as negative control. White squares indicate fields shown in higher magnification at the bottom. The empty arrowhead denotes an example of HEV RNA and ORF1 protein colocalization while the filled arrowhead denotes colocalization of HEV RNA as well as ORF1 and ORF2 proteins. The scale bar represents 20 μm.