Immunofluorescence Detection of HEV Infection

S10-3 cells transfected with HEV RNA transcripts or infected with HEV were grown on slides, rinsed in PBS three times, and fixed with 80% acetone for 20 min. Cells were then rinsed in PBS 3 times followed by 1-h incubation with anti-HEV-ORF2 antibody (36 (link)). Next, cells were rinsed in PBS for another 3 times and treated with goat antimouse IgG conjugated with Alexa Fluor 488 (Invitrogen) for 1 h. After adding 4, 6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), cells were visualized under a fluorescence microscope (DMI3000B, Leica, Germany). HEV infectious titers were assessed by counting the number of ORF2-positive cell clusters (quantified as FFU).