HDV-GT3 shows similar induction of innate immunity compared to HDV-GT1 in humanized mice despite its high infection efficiency and intrahepatic activity

Hepatitis D virus genotype 1 (HDV-GT1) is the most common genotype worldwide, while HDV-GT3 is mainly restricted to South America but is associated with a more severe outcome of the disease.

Aim: To investigate virological differences among these two genotypes and their capacity to enhance innate responses in human hepatocytes using humanized mice.

Methods: Human liver chimeric uPA/SCID/beige (USB) mice were co-infected with cell culture-derived HDV-GT1 or HDV-GT3 and HBV (GT-D). 9 weeks post inoculation (p.i.), HBV and HDV viremia, intrahepatic HDV RNA, pgRNA and RNA levels of interferon stimulated genes (ISGs) and cytokines were determined by qRT-PCR. Antigenomic HDV RNAs were detected by a specific RT-PCR-based assay and in situ hybridisation. HDAg was assessed by immunofluorescence. Results: 9 weeks p.i., mice co-infected with HBV and HDV-GT3 showed a 3.6-fold higher HDV viremia (median 2.4 × 10E8 copies/ml) and 9.3-fold higher levels of intrahepatic HDV RNA than HDV-GT1 co-infected mice. Intrahepatic analyses also revealed that the amount of antigenomic HDV RNA was 3.2-fold higher in the liver of HDV-GT3 compared to HDV-GT1 infected mice, suggesting increased virion productivity of HDV-GT3 RNAs. Also a higher number of human hepatocytes appeared HDAg-positive in mice infected with HDV-GT3. Of note, both HBV viremia and amounts of pgRNA appeared lower in mice co-infected with HDV-GT3 (median 6.1 × 10E8 copies HBV DNA/ml and median 1.9 pgRNA relative to hGAPDH) compared to those co-infected with HDV-GT1 (median 2.6 × 10E9 copies HBV DNA/ml and median 4.9 pg RNA relative to hGAPDH), indicating that HDV-GT3 can suppress HBV replication to some extent. Remarkably, both HDV genotypes induced comparable enhancement of human ISGs such as MxA (HDV-GT1: 9.0-fold; HDV-GT3: 7.9-fold induction compared to uninfected mice) and ISG15 (HDV-GT1: 13.7-fold; HDV-GT3: 12.8-fold induction) and of the cytokine CXCL10 (HDV-GT1: 5.9-fold; HDV-GT3: 3.8-fold induction).

Conclusions: Despite its higher infection efficiency, activity and HBV suppression capabilities, HDV-GT3 showed no augmented activation of hepatocyte innate responses compared to HDV-GT1 in humanized mice. These results suggest that direct virological effects may affect HBV productivity, whereas immune cell activity shall be responsible for the more severe course of disease in HDV-GT3 infected patients.