Chronic hepatitis associated with GB virus B persistence in a tamarin after intrahepatic inoculation of synthetic viral RNA

Abstract

Progress in understanding the pathogenesis of hepatitis C virus (HCV) has been slowed by the absence of tractable small animal models. Whereas GB virus B (GBV-B, an unclassified flavivirus) shares a phylogenetic relationship and several biologic attributes with HCV, including hepatotropism, it is not known to cause persistent infection, a hallmark of HCV. Here, we document persistent GBV-B infection in one of two healthy tamarins (Saguinus oedipus) inoculated intrahepatically with infectious synthetic RNA. High-titer viremia (108 to 109 genome equivalents per ml) and transiently elevated serum alanine transaminase activities were present from weeks 4 to 12 postinoculation in both animals. However, whereas GBV-B was eliminated from one animal by 20 weeks, the second animal remained viremic (103 to 107 genome equivalents per ml) for >2 years, with alanine transaminase levels becoming elevated again before spontaneous resolution of the infection. A liver biopsy taken late in the course of infection demonstrated hepatitis with periportal mononuclear infiltrates, hepatocellular microvesicular changes, cytoplasmic lipid droplets, and disordered mitochondrial ultrastructure, findings remarkably similar to chronic hepatitis C. GBV-B-infected hepatocytes contained numerous small vesicular membranous structures resembling those associated with expression of HCV nonstructural proteins, and sequencing of GBV-B RNA demonstrated a rate of molecular evolution comparable to that of HCV. We conclude that GBV-B is capable of establishing persistent infections in healthy tamarins, a feature that substantially enhances its value as a model for HCV. Mitochondrial structural changes and altered lipid metabolism leading to steatosis are conserved features of the pathogenesis of chronic hepatitis caused by these genetically distinct flaviviruses.

Fig. 1.

(A) Alignments of the HCV and GBV-B genomes, showing putative protein assignments in the GBV-B polyprotein coding sequence and the similarities in GBV-B and HCV genome structure. (B) Overlapping segments of the GBV-B genome that were amplified by RT-PCR and were assembled into the full-length clone pGBV-B/2.

Fig. 2.

Profiles of acute GBV-B infections in tamarins: T12024, which was inoculated intravenously with GBV-B-positive tamarin serum, and T12023, which was infected by intrahepatic inoculation of synthetic pGBV-B/2 RNA. •, viremia measured by quantitative RT-PCR with a sensitivity of detection of ≈10³ ge/ml (except at weeks 143 and 147, when testing of a larger volume of serum improved sensitivity to ≈50 ge/ml). ○, serum ALT (IU); (▵), anti-NS3 (ELISA OD at 405 nm).

Fig. 3.

Persistent GBV-B infection in tamarin T12025, which was infected by intrahepatic inoculation of synthetic RNA (see the Fig. 2 legend). The arrow marks the timing of the liver biopsy. The dashed line represents preinfection serum ALT activity.

Fig. 4.

Hematoxylin/eosin-stained sections of normal tamarin liver (A) and liver from tamarin T12025 (B–E), 107 weeks after intrahepatic inoculation of RNA. See text for findings. Original magnification: A, B, and D, ×50; C, ×100; E, ×200.

Fig. 5.

Immunohistochemical staining for GBV-B NS3 (A and B) and GBV-B NS5B (C and D) antigens in normal tamarin liver (A and C) and liver taken from T12025 (B and D), 107 weeks after RNA inoculation. Arrows indicate cells containing abundant cytoplasmic viral antigen.

Fig. 6.

Transmission electron microscopy of normal tamarin liver (A) and persistently infected tamarin liver from T12025 (B–D). , mitochondria, with ill-defined cristae in the infected tissue. →, lipid droplets with electron-dense surface deposits. ▸, clusters of small smooth-surface vesicles. N, nucleus. (Bar, 1 μm.)

Fig. 7.

Mutations present in virus circulating in T12025 between weeks 40 and 48 postinoculation of RNA. Those occurring by week 10 were also observed in acutely infected tamrins. Solid arrows indicate nonsilent mutations in the ORF or nucleotide substitutions in the noncoding regions of the viral genome. Lightly shaded arrows indicate mutations that were present at weeks 40–42 but reverted to wild-type sequence by weeks 44–48.