Human liver chimeric mice as a new model of Hepatitis E Virus infection and preclinical drug evaluation

Abstract

Objective: Hepatitis E Virus (HEV) is recognized as important pathogen in developing and industrialized countries leading to acute and chronic infections with considerable morbidity and mortality. Since our knowledge about the molecular biology and pathogenicity of HEV is still rudimental, we employed humanized uPA/SCID/beige (USB) mice to establish a new small animal model of HEV infection. Methods: Homozygous USB mice repopulated with primary human hepatocytes were inoculated with either patient-derived HEV-positive serum or stool suspensions from genotype 1 or 3. Virological parameters were determined at serological and intrahepatic levels with quantitative real-time PCR as well as immunofluorescence staining. Sucrose gradients were performed with HEV from faeces and serum in order to characterize the virions. Results: HEV infection in humanized mice was successful only after intravenous (i.v.) injection of stool-derived virions (1 × 105 IU/mouse; n= 3/3 genotype 1 and genotype 3, respectively), but not after i.v. injection of serum derived virions (n= 13) obtained from 3 acutely HEV infected patients. Notably, i.v. HEV genotype 1 inoculated mice developed viremia with up to 1 × 106 IU/ml within 4 weeks and remained stable for up to 20 weeks. HEV-infected human hepatocytes could be visualized by immunofluorescence using HEV orf2 and orf3 specific antibodies. Kinetics of infection with HEV genotype 3 were slower and resulted in up to 2.5 × 104 IU/ml viremia within 8 – 11 weeks of infection. Comparable to the situation found in human samples and regardless of the genotype used, HEV concentrations in faeces were higher (1 – 2 log) than in the corresponding serum samples. Sucrose gradient analyses also revealed that HEV virions retrieved either from mouse or human serum displayed a significantly lower density than virions obtained from faeces, which indicates that serum virions are possibly associated with lipids. Notably, HEV infection could be passaged by cohousing HEV chronically infected with uninfected humanized mice, thus demonstrating the establishment of de novo HEV infection through the faecal-oral route in humanized mice. Finally, six-weeks of treatment with ribavirin (50 mg/kg/daily) led to a 2log reduction of HEV viremia and intrahepatic HEV-RNA/GAPDH as well as loss of orf2-positive hepatocytes. Conclusion: We established an efficient model of HEV infection closely resembling the human infection with respect to viral replication, viral morphogenesis and routes of infection. We could demonstrate successful infection with HEV genotype 1 and 3 and assess the strong antiviral effect of ribavirin in this model. This new infection system offers new possibilities to test the efficacy of established and new antiviral therapeutic concepts, as well as to exploit mechanisms of viral interference in human hepatocytes in vivo.