A novel in vitro system for simultaneous infections with hepatitis B, C, D and E viruses

Abstract

Background & aims: The liver, and more precisely hepatocytes, can be infected by several hepatotropic viruses, including HBV, HDV, HCV and HEV, with chronic infection leading to end-stage liver diseases. Since no in vitro model allowing multi-infections with the four viruses is reported, limited data are available on their interplay as well as on the potential cross-reactivity of antivirals in multi-infection cases. The aim of our study was to set up such a model.

Methods: HuH7.5-NTCP cells were cultured with 2% DMSO (dimethyl sulfoxide) for 1 week to allow partial differentiation into hepatocytes (dHuH7.5-NTCP) before infection with the different viruses and treatment with known antiviral molecules.

Results: We observed increased expression of liver specific transcripts and production of ApoB containing VLDL in dHuH7.5-NTCP cells and replication of HBV, HDV, HCV and HEV for at least 4 weeks after mono or multiple infections. We recapitulated the known antiviral effect of sofosbuvir on HCV and HEV (>90% reduction in the levels of intracellular viral RNAs, p <0.0005) and of IFN-α on HCV, HEV and HDV (80% reduction in the levels of intracellular viral RNAs, p <0.0005). Besides its already described antiviral effect on HBV and HDV, we observed that GW4064, a farnesoid X receptor (FXR) agonist, also strongly inhibited HEV replication (85 to 95% reduction in the levels of intracellular HEV RNAs, p <0.0005). Using HEV-infected HuHep mice, we confirmed the antiviral effect of vonafexor, an FXR agonist, that is currently being tested clinically against HBV/HDV.

Conclusions: We set-up the first in vitro model allowing multi-infections with hepatitis viruses that can be used for broad drug screening and highlighted FXR ligands as potential broad-acting antivirals.

Impact and implications: Hepatitis virus infections caused by HBV, HCV, HDV, and HEV represent a global health threat. Treatment options remain limited, notably due to the lack of knowledge about molecular virus-host interactions. Moreover, the interplay between these four viruses in the context of co-infections remains unknown. In this study, we report the first in vitro system that allows for mono and multi-infections with these four viruses and characterize the broad antiviral activity of farnesoid X receptor agonists, paving the way for the development of new strategies for viral cure.

Keywords: Hepatitis B Virus; Hepatitis C Virus; Hepatitis D Virus; Hepatitis E Virus; broad acting antivirals; drug screening; hepatocytes.

Graphical abstract

Fig. 1

DMSO treatment of HuH7.5-NTCP cells enables their partial differentiation into hepatocyte-like cells. (A-E) HuH7-NTCP or HuH7.5-NTCP cells were seeded and treated or not with 2% DMSO. (B) Cells were lysed, total RNAs were extracted and gene expression was assessed by RNA sequencing (GSE288204). Relative liver-specific gene expression (Z-score) of up to 250 genes (see Table S1 for details) is presented. (C, D, E) HepaRG cells were differentiated for 4 weeks (dHepaRG) and PHHs seeded for 24 h before analyses. (C) Cells were lysed and levels of the indicated proteins were assessed by western blot. Results from a representative experiment are presented. (D) 24 h before analysis, media were replaced by serum-free media. Supernatants were collected and ApoB was assessed by ELISA in each fraction of iodixanol gradients. Results from a representative experiment are presented. (E) Cells were stimulated or not with the indicated molecules for 24 h. Total RNAs were extracted, and gene expression was assessed by reverse-transcription quantitative PCR. Levels of target mRNAs were normalized to the levels of Gus-B mRNAs and the no stimulation conditions. Data are the mean of three independent experiments each performed with three biological replicates. dHepaRG, differentiated HepaRG; PHHs, primary human hepatocytes.

Fig. 2

Infections of dHuH7.5-NTCP cells by HBV, HCV, HDV and HEV and testing for broadly acting antivirals. (A) dHuH7-NTCP, dHuH7.5-NTCP, or dHepaRG cells were inoculated with HBV, HCV, HDV, or HEV (mono) or with the four viruses at the same time (multi). At the indicated dpi, cells were lysed and the levels of viral RNAs were assessed by multiplex RT-ddPCR. (B) dHuH7.5-NTCP cells were inoculated with HCV, HDV, HEV, or HBV. At the indicated dpi, cells were lysed and levels of intracellular viral proteins were analyzed by western blot and the levels of HBeAg in the cell supernatants were quantified by CLIA. (C, D) dHuH7.5-NTCP cells were inoculated with HBV, HCV, HDV, or HEV. Three dpi with HBV, HCV, HDV or 10 dpi with HEV, cells were treated or not with the indicated molecules for 10 days. Cells were lysed and the levels of intracellular viral RNAs were assessed by RT-qPCR. Data are the mean ± SD of at least three independent experiments each performed with three biological replicates. (E) HuHep mice were treated as indicated. At the indicated weeks post-infection, levels of HEV RNA were quantified by qRT-PCR in 10% (w/v) stool suspensions of the infected mice. CLIA, chemiluminescence immunoassay; dpi, days post-inoculation; dHepaRG, differentiated HepaRG; dHuH7-NTCP, DMSO-treated HuH7-NTCP; dHuH7.5-NTCP, DMSO-treated HuH7.5-NTCP; qRT-PCR, quantitative reverse-transcription PCR; RT-ddPCR, reverse-transcription droplet digital PCR. Statistical analyses were performed using a Mann-Whitney U test with the prism software.