Fig. 4: Co-detection of HEV capsid and genome allows analysis of the dynamics of nHEV and eHEV entry.

(A) nHEV particles were immobilized on slides; capsids (magenta) were detected by immunofluorescence (IF) staining and HEV genomes (green) by RNA-FISH (version 1 kit with ORF1 probe). n = 4. Scale bar = 10 μm. (B) S10-3 cells were inoculated with nHEV (MOI = 30 GE/cell) and incubated for 2 h at 4 °C to allow binding (upper image row) followed by inoculum removal and 24 h at 37 °C for internalisation (lower image row). After fixation, cells were stained as described in (A). Line graphs on the right show the fluorescence intensities of capsids and genomes across the region of interest indicated by the white line in the images. Scale bar = 5 μm. (C) S10-3 cells were infected with nHEV (MOI = 50 GE/cell) and harvested after 3 h or 24 h followed by cell fractionation. Equal volumes from each fraction were analysed by WB against Na/K+ ATPase and tubulin. (D) Percentages of HEV genomes in each fraction in cells infected for 3 or 24 h. n = 6. (E) S10-3 cells were inoculated with nHEV (MOI = 30 GE/cell) or eHEV (MOI = 20 GE/cell) for 2 h or 6 h, respectively, followed either by fixation (0 h) or internalisation at 37 °C and fixation at indicated time points. HEV capsids and genomes were detected as described above and quantified using CellProfiler. n = 4 microscope fields for capsid and n = 6 microscope fields for RNA. (D, E) Data presented as mean ± standard derivation (SD). (F) Calculated percentages of HEV genomes colocalising with capsids (from E) out of the total number of detected genomes per cell. n = 4 microscope fields for nHEV and n = 6 microscope fields for eHEV. Statistical analysis was performed by unpaired two-tailed Student’s t test (D) or two-way ANOVA (F). ***: p < 0.001; ****: p < 0.0001; ns, non-significant. Images in (A) and (B) are maximum projections of 4 slices and 2 slices, respectively. All data are from two (A, C, D), three (E, F) or six (B) independent experiments.