Fig. 5: Both nHEV and eHEV entry depend on endosomal acidification.

(A) and (B) S10-3 cells were treated with indicated concentrations of bafilomycin A (BafA1), concanamycin A (ConA) or DMSO for 30 min prior to infecting with (A) nHEV (MOI = 0.1 GE/cell) or (B) eHEV (MOI = 5 GE/cell). Drugs and virus were removed after 24 h and HEV infection was quantified by counting ORF2-positive FFUs 5 days post-infection. n = 8 (A) or 6 (B). (C) S10-3 cells were treated with 50 nM of bafilomycin A (BafA1) or vehicle control DMSO for 30 min before inoculation with nHEV (MOI = 50 GE/cell). The inoculum was removed 8 h later and replaced with fresh media containing the drug. 24 h later, cells were harvested followed by cell fractionation to extract membranes and the cytosol. Equal volumes from each fraction were separated by SDS-PAGE and probed by Western blotting against the membrane marker Na/K+ ATPase, and the cytosolic marker tubulin. (D) HEV genome copies in each fraction were quantified by RT-qPCR. Shown are percentages of HEV genomes in each fraction in cells treated with BafA1 or DMSO. Data presented as mean ± SD. n = 6 replicates. All data are from two (C, D) or three (A, B) independent experiments. Statistical analysis was performed by comparing HEV RNA in cytosolic fractions by unpaired two-tailed Student’s t test (D) or one-way ANOVA (A, B). *: p < 0.05; **: p < 0.01; ****: p < 0.0001; ns, non-significant.