1 Departments of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
2 Division of Clinical Research, New England Regional Primate Research Center, Harvard Medical School, PO Box 9102, One Pine Hill Drive, Southborough, MA 01772-9102, USA
Correspondence
Keith G. Mansfield
keith_mansfield{at}hms.harvard.edu
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ABSTRACT |
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INTRODUCTION |
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Research on HCV has not benefited from such affordable and accessible animal models. Until recently, the only model available was the HCV-infected chimpanzee. This system has several drawbacks including expense, availability, biocontainment and ethical considerations. It is also questionable whether HCV-infected chimpanzees adequately model all aspects of the human disease. Furthermore, research is impeded by the lack of appropriate infectious cell culture models for this hepatotropic pathogen (Lanford et al., 1994). A small non-human primate surrogate model of HCV infection would allow the in vivo examination of hostvirus interactions, disease pathogenesis and potential chemotherapeutic agents.
HCV is a member of the virus family Flaviviridae, composed of the flaviviruses, the pestiviruses and the hepatitis C viruses, which have similar genomic organization and replication strategies (Rice, 1996). Bovine viral diarrhoea virus (BVDV) is a pestivirus that has been developed for cell-based screening assays to assess antiviral drug activity (Meyers & Thiel, 1996
; Zitzmann et al., 1999
). The GB viruses (GBVs), based on their genetic sequences, have been categorized as flaviviruses (Muerhoff et al., 1995
). Their historical background has been reviewed (Karayiannis & McGarvey, 1995
; Robertson, 2001
). Briefly, GBV-B was isolated from New World monkeys (tamarins) after inoculation with serum from a human patient with hepatitis (Deinhardt et al., 1967
). Subsequent work has demonstrated and characterized three distinct viral agents termed GBV-A, GBV-B and GBV-C. GBV-C, previously called hepatitis G virus (HGV), has been found in 12 % of the human population (Simmons et al., 1995
; Linnen et al., 1996
). The association of GBV-B with human disease has not been firmly established (Alter et al., 1997
). All three flaviviruses are related to HCV, but GBV-B is phylogenetically most closely related to HCV, showing up to 25 % amino acid sequence identity (Muerhoff et al., 1995
; Ohba et al., 1996
). Although each exhibits different host tropisms and disease sequelae, both HCV and GBV-B have similar cellular mechanisms of processing viral gene products (Reed et al., 1998
) and both are associated with persistent infections in their respective hosts (Beames et al., 2001
).
The natural host of GBV-B is unknown but several species of New World primates including tamarins (Saguinus sp.) and owl monkeys (Aotus sp.) are susceptible to experimental inoculation (Bukh et al., 2001). Recently, susceptibility of the common marmoset (Callithrix jacchus) to GBV-B infection has been demonstrated as an alternative small-animal model (Lanford et al., 2003
; Bright et al., 2004
). In the report that follows, the successful infection of common marmosets with GBV-B and associated hepatic pathology and inflammatory changes are described. The availability of GBV-B-infected marmosets now allows investigation of both host and viral processes as they relate to the pathogenesis of HCV infection of the liver.
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METHODS |
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Inoculum.
Marmosets were inoculated intravenously with 1·0 ml of a 103 dilution of GBV-B-infectious serum. This serum was derived from a common marmoset inoculated with GB Agent Pool, Mystax 661, 8/93 kindly provided by Dr Jens Bukh (Hepatitis Viruses Section, NIH, NIAID, USA). Two animals were subject to direct transfection of the liver with recombinant (r)GBV-B RNA, as described previously (Bukh et al., 1999), with the plasmid pGGB (kindly provided by Dr Bukh). Subsequently, six animals were inoculated and three were immunosuppressed with oral FK506 (Prograf) at a dose of 0·2 mg kg1 twice daily for 4 weeks starting 1 week prior to inoculation with GBV-B-infectious serum.
Virus quantification.
Quantification of GBV-B was performed using previously described protocols (Beames et al., 2000). One-step RT-PCR amplification utilized the following primer pair and probe: forward, 5'-AACGAGCAAAGCGCAAAGTC-3'; reverse, 5'-CATCATGGATACCAGCAATTTTGT-3'; and probe, 5'-6FamAGCGCGATGCTCGGCCTCGTATamra-3' (6Fam, 6-carboxyfluorescein; Tamra, 6-carboxytetramethylrhodamine). The primer pairs and fluorescence reporter probe for the sequence analysis were synthesized commercially (Applied BioSystems). RT-PCR was employed for quantifying GBV-B serum titres (TaqMan system, Applied BioSystems, Sequence Detection System). A reference standard of GBV-positive serum was calculated to contain 1x107 GBV genome equivalents (g.e.) ml1 in a head-to-head comparison with an rGBV RNA transcript standard (Bukh et al., 1999
).
Histology.
In initial experiments, eight animals were inoculated intravenously with serially passaged GBV-B serum. Two of these animals were euthanized and necropsies were performed at 4 weeks after infection. One animal was euthanized at 7 weeks and the remainder were followed to 22 weeks. Hepatic biopsies were obtained under general anaesthesia with ultrasonic guidance and samples divided for histological evaluation, RNA isolation and determination of lymphocyte subsets. Sections of liver were stained with haematoxylin and eosin (H&E) and examined for morphological evidence of hepatic inflammation. To examine the immunophenotype of cells within the liver, tissues were also stained for CD3, CD8, CD20 and HLA-DR (Dako). Tissue sections (5 µm) were immunostained using an avidinbiotinhorseradish peroxidase complex (ABC) technique with diaminobenzene chromogen as described previously (Mansfield et al., 1995; Wykrzykowska et al., 1996
). Lymphocytes were also isolated by mechanical disruption and Ficoll separation from hepatic biopsies and stained for CD3, CD8, CD4, CD20 and HLA-DR (Dako). Lymphocyte subsets were determined by fluorescence-activated cell sorting (FACS) analysis (Genain & Hauser, 1996
).
Peripheral blood mononuclear cell (PBMC) analysis.
Whole blood was collected at weekly intervals from two marmosets inoculated with infectious GBV-B serum as described above. PBMCs were isolated from whole blood by density-gradient centrifugation. Briefly, after centrifugation of 2 ml whole blood in EDTA anticoagulant at 4500 r.p.m. using a Sorvall SH3000 rotor, the plasma was removed and the cell pellet (1 ml) suspended in 3 ml RPMI and layered onto 3 ml lymphocyte separation medium, followed by centrifugation at 4500 r.p.m. The PBMC interface (0·7 ml) was collected with a needle and syringe, diluted twofold in PBS and centrifuged at 4500 r.p.m. The cell pellets (50 µl) were diluted into 1·5 ml PBS, followed by centrifugation at 4500 r.p.m. In one case, PBMCs were stained for CD3+ and isolated by FACS analysis as described above. The final cell pellets were suspended in 100 µl PBS and stored at 80 °C. Total cell RNA was isolated and titres of GBV-B RNA were quantified in both the plasma and PBMC extracts by the procedures described above. At 7 weeks post-inoculation (p.i.), animals were euthanized for the recovery of primary hepatocytes used for in vitro assays.
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RESULTS |
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DISCUSSION |
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In comparison with the tamarin model, the marmoset is equally susceptible to GBV-B infection. In a study presented by Bright et al. (2004), GBV-B-infectious serum from tamarins led to increased infectivity upon serial passage in the marmoset model, from a rate of infection with tamarin serum of 65 % (13/20 marmosets) to a rate of 100 % (9/9 marmosets) with marmoset-derived material. The onset of viraemia peaked at 3 weeks p.i. and attained serum titres of approximately 1091010 g.e. ml1 before clearing virus by 8 weeks p.i. The onset of viraemia in the study reported here occurred as early as 12 weeks p.i. followed by increasing titres to 7 weeks p.i. averaging 108 g.e. ml1. In a report by Lanford et al. (2001)
, viraemia was detected in tamarins as early as 12 weeks p.i., with sustained viral titres of 107 g.e. ml1 for 12 weeks until clearance of virus from the serum. In a study comparing the viraemia in GBV-B-infected marmosets and tamarins, the kinetics of infection were similar (Lanford et al., 2003
). In this report, persistent GBV-B infection of immunologically normal marmosets was documented for periods of up to 24 weeks p.i.
Immunosuppressive treatment of marmosets prior to inoculation of GBV-B resulted in higher viral load and more severe liver pathology. These data suggest that the virus may have established a more productive infection in the liver during immunosuppressive treatment, leading to a more vigorous immune response once the suppression diminished. Although both an FK506-treated and control animal showed evidence of chronic infection for 24 weeks, a larger cohort of animals, treated in a similar fashion, must be monitored for a period of >6 months to determine the full outcome of immunosuppressive therapy. Immunosuppressive therapy on GBV-B-infected tamarins resulted in some persistent infections (Lanford et al., 2003). These results coupled with those presented here imply that immune modulation of either New World primate model will allow us to investigate factors that contribute to the establishment of the persistent carrier state.
The common marmoset is an accepted model to study inflammatory diseases such as demyelination associated with multiple sclerosis in humans (Genain & Hauser, 1996; Villoslada et al., 2001
; Genain et al., 1996
). The origins for utilizing this New World primate as an alternative in vitro model for hepatitis research have been described as well (Stephensen et al., 1991
). Based on the susceptibility of the marmoset to infection with GBV-B, we believe it should be useful as a small primate model to investigate the mechanism of hepatic damage and inflammatory changes during the course of viral infection. The presence of lymphoid nodules was particularly interesting, as this is often cited as a defining change in chronic HCV infection in man. The increase in alkaline phosphatase coincided with a prominent portal inflammatory cell infiltration. This suggests ongoing damage to the biliary epithelium, which is a feature of chronic HCV in man as well. The finding of increased numbers of MHC class I-restricted CD8+ lymphocytes, indicative of cytotoxic T cells, is in accordance with previous studies showing CD8+ cells predominant in the liver during either chronic HBV or HCV infections (Fiore et al., 1997
). The contribution of class I-restricted CD8+ T cells to liver injury during virus clearance has been demonstrated in other animal models of viral hepatitis (Fiore et al., 1997
). However, HCV infections persist despite an immune response, and the role of CD8+ cells and whether HCV is able to escape immune elimination are issues of pathogenesis that need to be studied further (Cerny & Chisari, 1999
; Ferrari et al., 1999
). In this study, MHC class I expression in association with the observed increase in CD8+ cells was not examined. An increase in the CD4+ T lymphocytes was not detected; whether this reflects a dysfunction of the immune response requires further experimentation.
Extrahepatic sites of replication are characteristic for some members of the Flaviviridae, specifically BVDV (Liebler-Tenorio et al., 1997). BVDV can replicate in PBMCs and antigen expression can be localized to several organs including brain tissues (Bielefeldt-Ohmann et al., 1987
; Gruber et al., 1993
). Extrahepatic manifestations of disease have not been well characterized in animal models for HCV (Gruber et al., 1993
). Several papers suggest that HCV and HGV (GBV-C) may be lymphotropic (Fogeda et al., 1999
; Afonso et al., 1999
; Rodriguez-Inigo et al., 2000
) and a recent paper describes the identification of HCV genomic material in brain tissue from humans (Radkowski et al., 2002
).
In this study, the data suggest that lymphocytes may be an extrahepatic site of GBV-B replication in the common marmoset. Although there was variation in detecting GBV-B in PBMCs from infected animals, the quantified levels of GBV-B in RNA extracted from PBMCs were 12 logs greater than could be accounted for by the dilution factor of contaminating serum carried through the PMBC isolation procedure (1 : 7200). It is possible that GBV-B adhered to the erythrocytes or to the platelets in the PMBC preparation. However, if this were the case, we would expect detectable levels of GBV-B in the PMBCs of cj96-01, which were prepared in parallel with cj500-00 samples. GBV-B was not detected in the PBMCs of cj96-01 and in a control experiment we did not detect GBV-B in normal PBMCs seeded with infectious virus. Furthermore, a recent report (Ducoulombier et al., 2004) describes the absence of HCV compartmentalized to the CD4+ and CD8+ fractions of PMBCs isolated from chronic carriers. This attests to our finding of increased GBV-B titres in the non-CD3+ fraction of PBMCs isolated from infected marmosets.
As discussed previously, the availability of the marmoset for research purposes provides an affordable, assessable animal resource allowing experimental studies utilizing the surrogate GBV-B system. Our data show characteristic immunophenotypic and morphological features following GBV-B inoculation of marmosets that have been observed during chronic HCV infections in man. This work and that reported by others further supports the use of New World primates infected with GBV-B as a surrogate model for the study of HCV pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 13 February 2004;
accepted 20 May 2004.