Pathogenesis of hepatitis C virus infection in Tupaia belangeri

Abstract

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.

FIG. 1.

Course of infection with patient serum HCR6 and RCV. (A) The results of quantitative RTD-PCR for HCV RNA and serum ALT concentrations were combined and plotted to show the course of infection in Tup.5. The bars and the ordinates on the left represent HCV RNA as genome equivalents/ml of serum. The curved line and the ordinates on the right represent serum ALT concentrations as IU/liter serum. (B) Serum HCV RNA and ALT concentrations for infection of Tup.6. (C) The graph for Tup.4. (D) The graph for Tup.8. The vertical axis for serum ALT in this graph is scaled differently from the others because of significant ALT elevation. (E) Quantification of HCV RNA in tupaia liver. HCV RNA in hepatocytes from tupaia (Tup.4, Tup.5, Tup.6, Tup.8, and Tup.15) livers was isolated 172 weeks after HCV infection and quantified by RTD-PCR. As few as 10 copies of the genome were detected, and the quantification range was between 10¹ and 10⁸ copies (26).

FIG. 2.

Micrographs of liver specimens stained with H&E. Liver tissue from HCR6-inoculated tupaias (A to D) and RCV-inoculated tupaias (E to H) was obtained at 2 and 3 years postinoculation (pi). (I and J) Liver specimens from uninfected animals age matched to each inoculated animal were also obtained. The HCV-infected tupaia livers harbored infiltrating lymphocytes (white arrowheads) and fibrosis (broken lines and black arrowheads), which indicate chronic hepatitis.

FIG. 3.

Macro- and microscopic features of tupaia liver. (A) Infection-free control tupaia (Tup.15; 92 weeks). (B) RCV-infected animal displaying liver cirrhosis (Tup.8; 84 weeks postinoculation). (C) RCV-infected animal with massive surface nodules (Tup.8; 144 weeks postinoculation). (D and G) H&E staining of the uninfected Tup.15 at 92 weeks (D) and the uninfected Tup.39 at 242 weeks (G). (E, F, H, and I) H&E and silver staining of Tup.8 at 84 weeks postinoculation (E and F) or at 144 weeks postinoculation (H and I).

FIG. 4.

Sudan IV-stained liver specimens exhibiting fatty liver degeneration. Cryosections of liver stained by Sudan IV as described in Materials and Methods show fatty liver degeneration. The left and right columns display biopsy specimens of infected animals (2 years postinoculation) and animals sacrificed at 3 years postinfection, respectively. (A and B) Uninfected controls at 2 years (Table 1 shows sample timing). (C to F) Patient serum HCR6-infected animals. (G to J) RCV-infected animals.

FIG. 5.

Results of a reinfection experiment. (A) Quantitative RTD-PCR for HCV RNA and serum ALT levels are shown. Two naive animals were inoculated with tupaia serum (using serum taken at 5 weeks postinoculation from Tup.5, originally inoculated with patient serum HCR6) containing 100 genome equivalents (GE)/ml and were monitored for 15 weeks postinoculation (Table 1). (B) Tupaia serum (taken at 10 weeks postinoculation from Tup.8, originally inoculated with RCV) that was positive for HCV RNA was passaged into two naive animals. The animals were inoculated with tupaia serum at 100 GE/animal and monitored for 15 weeks postinoculation. (C) Tupaia serum (taken at 8 weeks postinoculation from Tup.4, originally inoculated with RCV) that was positive for HCV RNA was passaged into naive animals. The animals were inoculated with serum at 100 GE/animal and monitored for 20 weeks postinoculation.