Efficacy of HDV mono-infection with genotype 1 and 3 in CRPISPR/Cas generated humanized NTCP mice compared to human liver chimeric mice

Background:

Sodium taurocholate co-transporting polypeptide (NTCP) was identified as the functional entry receptor for HBV and HDV. Previous studies showed that HDV infection through mouse NTCP is restricted by only 3 amino acids of the receptor.

Aim was to establish HDV mono-infection in immunocompetent mice expressing humanized NTCP and to compare HDV infection efficiency with that achieved in immunodeficient human liver chimeric mice.

Methods:

Murine NTCP in C57BL/6J mice was humanized by altering the residues 84 – 87 using the CRISPR/Cas technology (hNTCP mice). For infection, HDV virions (2 × 108 genome equivalent/mouse) were injected i.p. or i.v. into adult hNTCP mice (> 8 weeks old). Mice received either cell culture derived HDV genotype (GT) 1 or 3. Five to 21 days post inoculation livers were analysed by using qRT-PCR (HDV RNA, NTCP RNA), immunofluorescence staining (HDAg, NTCP protein) and in situ hybridisation (genomic and antigenomic HDV RNA, HDV mRNA). Human liver chimeric mice were generated by transplanting human hepatocytes into uPA/SCID/beige mice (USB mice) and were infected similarly.

Results:

In hNTCP mice, NTCP was expressed similarly regardless of mouse gender. 7 days after infection, i.v. administration and HDV GT3 resulted in higher intrahepatic infection levels than i.p. administration and HDV GT1 inoculation, respectively (median copies HDV RNA/ng total liver RNA in GT1 i.p.: undetectable, GT1 i.v.: 0.4, GT3 i.p.: 7.5; GT3 i.v.: 3.8). Although less than 0.1% of cells were HDAg-positive in livers of hNTCP mice inoculated i.v. with HDV GT3, the presence of genomic and antigenomic HDV RNA, as well as HDV mRNA, demonstrated intracellular viral replication. Infection kinetic studies showed detectable intrahepatic HDV RNA levels at day 5, 7, 9 and 14 post infection, with a maximum at day 7. At day 21, HDV RNA levels were below the detection limit, suggesting virus clearance or death of HDV-infected hepatocytes. Compared to hNTCP mice, human liver chimeric mice infected i.v. with the same HDV GT3 inoculum showed 36-fold higher levels of intrahepatic HDV RNA (median 137.1 copies HDV RNA/ng RNA) and higher amounts of HDAg-positive cells (0.4%).

Conclusion:

HDV mono-infection can successfully be established in hNTCP mice and HDV GT3 led to higher infection rates. However, HDV infection of human hepatocytes in USB mice appeared more efficient than in hNTCP-expressing mice, suggesting that additional restriction factors may account for the lower infection susceptibility of murine hepatocytes. In sum, hNTCP mice provide a useful model to investigate the fate of HDV mono-infection in immune competent mice and to study host-related differences.