Fig. 4: HBV infection of primary porcine hepatocytes with partially humanized porcine NTCP.

PPH were isolated using a 2-step collagenase method and seeded on a 24-well plate. Cells were allowed to attach for 24 h, transduced with adenoviral vectors expressing hNTCP or phNTCP (157-167), and subsequently differentiated in DMSO 2% for 48 h. A Cells were stained with fluorescently labeled Myrcludex B (Myr₅₆₅) and analyzed via fluorescence microscopy, showing binding to the NTCP variants expressed on the cell surface (scale bars: 100 µm). B–D In parallel, cells were inoculated with HBV (MOI 1000 vp/cell). B Cell culture supernatants were collected at days 4 and 7 post-infection and analyzed for HBeAg. C 7 days post-infection, southern blot analysis was performed utilizing a modified Hirt extraction procedure. Analyzed samples include (1) PPH transduced with Ad-hNTCP and inoculated with HBV, (2) PPH transduced with Ad-hNTCP only, (3) PPH transduced with Ad-phNTCP (157-167) and inoculated with HBV, (4) PPH transduced with Ad-phNTCP (157-167) only, (5) naive PPH inoculated with HBV, (6) naive PPH without HBV, (c) HBV infected HepG2-NTCP as a positive control. D 7 days post-infection, cells were lysed, total DNA was isolated from the lysate and analyzed for cccDNA via PCR. Values are displayed in relation to prion protein gene. B, D Mean values +/− standard deviation of at least three experiments are given. Statistical analysis was performed by (B) one-way ANOVA with Dunnett’s multiple comparison correction or (D) Kruskal–Wallis test with Dunn’s multiple comparison correction. **p < 0,01, ****p < 0,0001. For (B), the dotted line indicates the cut-off between non-reactive and reactive.