Development of a primary tamarin hepatocyte culture system for GB virus-B: a surrogate model for hepatitis C virus

Abstract

GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5' exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 10(9) genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 10(7) genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.

FIG. 1

Infection profiles of GBV-B infected tamarins. (A) Progress of initial GBV-B infection in tamarins 12024 (top) and 12026 (bottom). GBV-B levels are shown in comparison to the liver damage, as measured by ALT, and rise in serum antibody titers to the GBV-B NS3 protein over the 24- to 26-week course of the experiment. (B) GBV-B levels and serum antibody titers to the NS3 protein during the course of rechallenge infection of 12024 (top) and 12026 (bottom) with a standard inoculum of GBV-B-containing 12024 tamarin serum. GBV-B TaqMan data, measured in genome equivalents per milliliter of serum, are shown as bar graphs, with line graphs representing ALT and NS3 ELISA absorbance superimposed.

FIG. 2

Persistence of GBV-B replication in primary tamarin hepatocytes. GBV-B levels were monitored for 42 days in primary hepatocytes isolated from GBV-B-infected tamarin 12036. Day 0 represents RNA isolated from hepatocytes harvested on the day of isolation from the liver.

FIG. 3

Growth curve of GBV-B in primary tamarin hepatocytes. Tamarin hepatocytes were inoculated with GBV-B-containing plasma, using an adsorption period of 6 h. Cultures were washed extensively to remove unadsorbed virus and then harvested in duplicate at various times over a 14-day period. Time zero (immediately after washing away the inoculum) represents the level of virus attached or internalized by the cells. Cell-associated (squares) or secreted (circles) GBV-B RNA was quantified by TaqMan RT-PCR and expressed as genome equivalents per microgram of cellular RNA or milliliter of culture medium, respectively.

FIG. 4

Estimation of doubling time for GBV-B in primary tamarin hepatocytes. Tamarin hepatocytes were inoculated with GBV-B-containing plasma using a shortened adsorption period (1 h). Cultures were washed extensively to remove unadsorbed virus and harvested in duplicate at various times over a 48-h period. Time (immediately after washing away the inoculum) represents the level of virus attached or internalized by the cells. Cell-associated GBV-B RNA was quantified by TaqMan RT-PCR.

FIG. 5

Immunofluorescence staining for NS3 protein. Hepatocytes grown on glass coverslips were inoculated with GBV-B 3 days postplating and harvested 3 days postinfection unless noted otherwise. GBV-B NS3 protein was detected by immunofluorescence using a rabbit anti-GST-NS3 antiserum and goat anti-rabbit IgG-fluorescein. (A) Infected hepatocytes stained with anti-NS3 (magnification, ×100); (B) uninfected hepatocytes stained with anti-NS3 (×100); (C) infected hepatocytes stained with normal rabbit (prebleed; ×100); (D) lower magnification of infected hepatocytes stained with anti-NS3 (×50); (E) hepatocytes infected with a low multiplicity and harvested 21 days p.i. (×100); (F) higher magnification of infected hepatocytes stained with anti-NS3 (×200).

FIG. 6

Specificity of rabbit anti-GST-NS3 antiserum in immunofluorescence staining for NS3 protein. Hepatocytes were inoculated with GBV-B and harvested 3 days p.i. Cells were stained for NS3 protein using rabbit anti-GST-NS3 antiserum (A). To demonstrate specificity, the antiserum was adsorbed for 16 h at 4°C with 5 μg of purified GST (B) or NS3 (C) protein prior to being used for immunofluorescence staining. Adsorption with GST had no effect on staining, while adsorption with NS3 eliminated staining.