The development of vaccination and novel therapy for hepatitis C virus (HCV) has been hampered by the lack of suitable small-animal models. GB virus B (GBV-B), closely related to HCV, causes viral hepatitis in common marmosets (Callithrix jacchue jacchus) and might represent an attractive surrogate model for HCV infection. However, differences exist between GBV-B and HCV in spite of a short genetic distance between the two viruses. Here we report common marmosets infected with two HCV/GBV-B chimeras containing HCV structural genes coding for either whole core and envelope proteins (CE1E2p7) or full envelope proteins (E1E2p7) substituted for the counterpart elements of GBV-B. Naïve animals intrahepatically injected with chimeric RNA transcripts or intravenously injected with sera from primary infected animals produced high levels of circulating infectious chimeric viruses and they developed chronic infection. Tacrolimus-treated marmosets inoculated with a CE1E2p7 chimera had higher viral loads and long-term persistent infection. A moderate elevation of serum aspartate aminotransferase (AST) levels was observed in parallel with viral replication. Chimeras recovered from liver samples revealed 1/958 adaptive viral mutations. Histopathological changes typical of viral hepatitis were observed in liver tissues from all types of HCV chimeras-infected marmosets. HCV core and E2 proteins were detected in liver tissues from infected animals by immunohistochemical staining. Fluctuations of chimeric virus replication in marmosets with spontaneous and sporadic viral clearance might be related to specific antibody and T-cell response to HCV proteins in vivo. Replication of CE1E2p7 chimera was observed in primary hepatocyte cultures by immunofluorescent staining in vitro. Conclusion: Infectious HCV chimeras causing chronic hepatitis in marmosets might constitute a small primate model suitable for evaluation of virus-cell interaction, vaccination, and antiviral therapy against HCV infection. (Hepatology 2014;59:789–802)
Hepatitis C virus (HCV) has a host range restricted to humans and chimpanzees and lacks suitable primate models,[1-3] limiting progress in developing HCV vaccines and antiviral drugs. The chimpanzee is a rare and expensive experimental animal whose scientific utilization raises an ethical dilemma. Marmosets (Callithrix jacchue jacchus) as well as tamarins (Saguinus oedipus) are small New World monkeys susceptible to GB virus B (GBV-B), a hepatotropic flaviviridae phylogenetically close to HCV that rarely infects small primates.[4, 5] GBV-B has been considered a surrogate for HCV in infected marmosets or tamarins.[6-9] Despite the short genetic distance between the two viruses, there are differences in structural proteins which affect the immune response. It has been hypothesized that chimeric HCV/GBV-B viruses might mimic HCV infection in small primates. Only chimeras integrating short pieces of HCV 5′-NCR, p7, or HVR1 sequences were previously constructed to investigate gene functions in vivo.[10-13] Such constructs, however, are unable to answer questions related to virus-host interaction, viral pathogenesis, and immune response.
The core, envelope glycoproteins E1E2, and p7 structural proteins are major components of HCV. We hypothesized that if HCV/GBV-B chimeras containing whole HCV structural protein genes (CE1E2p7) or intact envelope protein genes (E1E2p7) were infectious to marmosets, a major model to study the functions of HCV structural proteins in vivo would become available. Here the infectivity and pathogenicity of several chimeric constructs were investigated in marmosets.
This study describes the construction of two HCV/GBV-B chimeric viruses that contained full-length HCV genes (1,854-2,427 bp) encoding either the whole structural core and envelopes or all envelope proteins. Both the CE1E2p7 and E1E2p7 chimeras appear infectious to common marmosets and caused viral hepatitis. Several lines of evidence supported this conclusion.
First, substantial levels of viremia were found in two HCV chimera-infected marmosets and GBV-B controls during the initial 16 weeks postintrahepatic injection. In one of two animals (M15-P0) primarily infected with CE1E2p7 chimera, high levels of viremia persisted up to 44 weeks (Fig. 2A). Passaged infection with HCV chimeras presented viremia patterns similar to previously described GBV-B infections.[9, 13] An initial peak of viremia observed at 30 days (4-5 weeks) was followed in some animals by a second peak at 8-10 weeks, depending on the infectious dose.[9, 12, 13] This pattern resembles the fluctuating viremia observed in acute human HCV infection. These HCV chimeras also appear infectious since, after primary infection, circulating chimeric viruses were able to infect naïve marmosets, although the viremia pattern was slightly different, possibly related to the infectious dose and the different route of infection (intravenous versus intrahepatic). An initial high viral load (>105 copies/mL) was detected 1-5 weeks postinoculation, and was cleared between weeks 4-7, and then remained detectable at a lower level up to 29th weeks postinfection (Fig. 2A, P1). In addition, both intrahepatic (M16) and intravenous (M17) injections in FK506-immunosuppressed animals developed consistently high viral load infections (Fig. 2A). In situations of both primary and passaged infections, the ALT level appeared to be a poor indicator of liver damage, as previously described, while AST levels were moderately elevated in most infected marmosets. The presence of circulating HCV chimeras was shown by the simultaneous detection of GBV-B and HCV genome fragments. The viremia from the two chimeras was further authenticated by RT nested-PCR specific of regions from either the HCV core, E1, or adjacent regions of HCV and GBV-B gene sequences. As shown in Fig. 2B, two qRT-PCR systems were significantly correlated between HCV and GBV-B target amplification.
A second line of evidence was obtained by investigating the chimeric RNA in the liver tissue. Chimeric GBV-B and HCV RNA was detected in the M1-E1E2p7-P0 and M17-CE1E2p7-P1-FK506 animals. Furthermore, the sequence of the nearly-complete genome (nt 22-9604) was observed, including the entire ORF of M17-CE1E2p7-P1-FK506 was 99.89% homologous to the initial construct. This demonstrates a practically identical sequence identity between the injected and replicating viral RNA (Table 1). Only three mutations from the consensus sequence were observed in 50%-100% sequenced clones and seven mutations in 2-4 of eight clones are not unexpected after 25 weeks of HCV CE1E2p7 chimera replication.
Third, the histopathological evidence suggests viral hepatitis was induced. Typical but relatively discreet changes were observed in liver tissues, although clinical symptoms were not clearly recorded. Pathological examination of liver from all HCV chimera-infected marmosets revealed lymphocyte infiltration, severe ground glass degeneration, cholestasis, eosinophilic cells, fibrous expansion, hepatic edema, and cell disarray, and ultrastructural changes including abnormal mitochondrial, lipid droplets and increased numbers of lysosomes (Fig. 3, Table 2). Furthermore, the immunohistochemical staining indicated the presence of viral protein in pathologically modified liver tissues, suggesting that the infection with either of the two HCV chimeras was associated with production of HCV proteins in vivo (Fig. 4).
Lastly, indirect evidence was shown in the immunological parameters. Modest antibody response to HCV core and E2 proteins was detected in the HCV chimera-infected animals. A strong HCV-specific T-cell response from three representative marmosets were identified by ELISpot from a panel of 15 predicted HCV T-cell epitope peptides, of which 10 peptides triggered T-cell responses (Fig. 5). Among the reactive peptides, six were identified previously from the HCV database, and four are unique to this report (Supporting Table 5).
Previous studies with GBV-B chimeras integrating short sequences (<212 bp) of HCV 5′-NCR, p7 or HVR1 resulted in acute resolving infection in marmosets and tamarins mostly within 20 weeks,[8, 10-13] while GBV-B persistent infections were associated with viral molecular evolution.[20, 21] However, in this study HCV CE1E2p7 and E1E2p7 chimeric viruses remained persistently detectable up to 23-29 weeks and in one animal up to 44 weeks, suggesting that chronic infection had developed in these marmosets. A long-term HCV chimera replication with high viral load was more firmly established with oral administration of anti-T-cell drug FK506, as described previously. Few high-frequency substitutions (T2466A/I674K in 67%, C642T/P66L and T1051C/S202P in both 21%) within multiple clones of HCV chimeras were detected from M15-P0 (Table 1), but it remains to be seen whether adaptive mutations for high viral load are involved in viral persistence in vivo.
Fluctuations of low-level HCV RNA has been described often in humans. Compared with HCV clearance or persistence in humans,[24-26] sporadic spontaneous viral clearance of HCV chimeras might be associated with cellular immune response to viral proteins. Virus-specific T-cell response against viral epitopes was described in HCV infection.[27-29] In this study, three representative animals infected with HCV chimeras presented a strong T-cell response to HCV antigens. These data suggest that the HCV chimera-infected small primate model might prove useful in evaluating cellular immune response in HCV infection. Neutralizing antibodies were reported as contributing to the early control of viral replication, but might not be sufficient for long-term protective immunity to HCV infection. Antibodies to HCV chimera core and E2 antigens were found to be weak or undetectable in the study. Whether or not HCV antibodies played a role in the clearance of viremia in HCV chimera-infected marmosets, or whether spontaneous clearance or persistence of chimeras in marmosets mimicked the outcome of human HCV infection associated with IL28B genetic variation in human, requires further investigation.
The data presented in this report raise new questions regarding the mechanisms of interaction between HCV and marmoset host in terms of receptor, tropism, and replication. HCV is restricted to infecting humans and chimpanzees and rarely infects marmosets.[4, 5] Some important results indicated that the envelope glycoproteins E1 and E2 interacted with multiple host cell surface receptors.[32-34] If those human receptors are specific for HCV entry into the host cells, marmosets susceptible to HCV CE1E2p7 and E1E2p7 chimeric viruses might carry similar viral receptors. In this study, HCV CE1E2p7 chimera could infect marmosets, as well as primary marmoset hepatocyte culture (Figs. 2, 6), but could not infect human Huh7.5.1 cells (data not shown). Similarly, HCV JFH-1 could infect Huh 7.5.1 cells but not marmoset liver cells (Fig. 4A). Our data suggest that the interaction between cell receptors and viral envelope proteins may not tightly restrict HCV and GBV-B to infect humans or marmosets.
In conclusion, marmosets infected with replicating HCV chimeras carrying HCV whole structural proteins could provide a novel small primate model for investigation of a virus-host interaction, vaccination, and antiviral therapy in HCV infection.
We thank Dr. Jens Bukh (NIH, Bethesda, MA) for kindly providing the plasmid pGBB. We thank Dr. Ping Zhao (Department of Microbiology, Second Military Medical University, Shanghai, China) for kindly providing mAbs specific to HCV E2. We thank Dr. Qingling Zhang for histopathological analysis, Mr. Mourong Liu, Ms. Xianghui Wu, and Mr. Junling Zeng for assistance in animal surgery and blood sample collection. We thank Dr. Peter Gowland (BTS SRC Berne, Berne, Switzerland) for editing the article.