Development of a Primary Tamarin Hepatocyte Culture System for GB Virus-B: a Surrogate Model for Hepatitis C Virus

Hepatitis C virus (HCV) is a major worldwide health problem, with an estimated 2% of the population chronically infected with this virus. Chronic HCV infection can cause significant liver disease and cirrhosis of the liver and, in some patients, lead eventually to liver cancer. The current animal model for the study of HCV is the chimpanzee. However, this model system suffers from the limited availability of chimpanzees and the high cost associated with conducting studies on large nonhuman primates. A smaller, less expensive model system would be desirable. One alternative model is the hepatitis GB virus-B (GBV-B) in Saguinus species (tamarins). The GB agents, GBV-A, -B, and -C, are members of Flaviviridae (21); GBV-B is the virus most closely related to HCV (22). GBV-A and GBV-B were isolated from tamarins inoculated with a blood sample from a surgeon (GB) suffering from acute hepatitis (10). Although GBV-A and GBV-B were isolated from a tamarin inoculated with human serum believed to contain a human hepatitis virus, GBV-A and GBV-B are considered tamarin viruses. While GBV-A has been isolated from a number of tamarins (5), GBV-B has been isolated only once, and its origins are unclear. A third GB agent, GBV-C (27), also known as hepatitis G virus (19), was isolated from human serum samples in attempts to isolate new hepatitis viruses; however its association with hepatitis is tenuous (1). GBV-A causes no recognized disease in tamarins, while GBV-B causes an acute, self-limited hepatitis, as evidenced by a reproducible rise in the serum level of alanine transaminase (ALT), an indication of liver damage. HCV infection in humans frequently results in persistent infection with associated disease sequelae, while GBV-B-infected tamarins appear to clear the viral infection with no long-lasting ill effects. However, relatively few animals have been followed long term by reverse transcription-PCR (RT-PCR) since the virus was identified in 1995 (28), and so the potential for a low percentage of chronic infections exists. The acute nature of GBV-B infections in tamarins distinguishes this hepatitis from HCV infections in humans, although the viruses share many characteristics. GBV-B is the virus most closely related to HCV genetically (22). The polyproteins possess approximately 25 to 30% homology at the amino acid level (21), while the 5′ and 3′ untranslated regions are more distinct (6, 21, 24). The functional similarities between the viruses were demonstrated by the correct processing of the HCV polyprotein by the GBV-B NS3 protease and the creation of functional chimeric NS3 proteins between HCV and GBV-B (7, 25). The studies support the premise that antiviral compounds with activity against GBV-B might show similar activities against HCV. In this report, we describe the development of a tissue culture system for GBV-B that utilizes primary cultures of tamarin hepatocytes. Our studies were initiated by the development of a quantitative, real-time 5′ exonuclease PCR (TaqMan) assay for GBV-B and the characterization of the infection profile of GBV-B in the absence of GBV-A in two tamarins. GBV-B replication in culture was documented for 42 days using hepatocytes from an infected tamarin. Replication could also be demonstrated by detection of the NS3 protein in infected cells by immunofluorescence. In vitro infection of normal hepatocytes was highly efficient and was accompanied by high levels of replication and secretion of infectious virions. This system should provide a much-needed surrogate tissue culture system for HCV.