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ILAR Journal V42(2) 2001
Animal Models of Hepatitis

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GB Virus

GB Virus B as a Model for Hepatitis C Virus
Burton Beames, Deborah Chavez, and Robert E. Lanford

Burton Beames, Ph.D., is a Research Scientist at Bayer Biological Products, Raleigh, North Carolina. Deborah Chavez, B.S., is a Senior Research Associate and Robert E. Lanford, PhD., is a Scientist in the Department of Virology and Immunology, Southwest Foundation for Biomedical Research and Southwest Regional Primate Research Center, San Antonio, Texas.

Abstract

GB viruses A and B (GBV-A and GBV-B) are members of the Flaviviridae family and are isolated from tamarins injected with serum from a human hepatitis patient. Along with a related human virus, GB virus C, or alternatively, hepatitis G virus (GBV-C/HGV), the three viruses represent the GB agents. Of the three viruses, GBV-B has been proposed as a potential surrogate model for the study of hepatitis C virus (HCV) infections of humans. GBV-B is phylogenetically most closely related to HCV and causes an acute, self-resolving hepatitis in tamarins as indicated by an increase in alanine aminotransferase and changes in liver histology. Similarities between GBV-B and HCV are found at the nucleotide sequence level with the two viruses sharing 28% amino acid homology over the lengths of their open reading frames. Short regions have even higher levels of homology that are functionally significant as shown by the ability of the GBV-B NS3 protease to cleave recombinant HCV polyprotein substrates. The shared protease substrate specificities suggest that GBV-B may be useful in testing antiviral compounds for activity against HCV. Although there are numerous similarities between GBV-B and HCV, there are important differences in that HCV frequently causes chronic infections in people, whereas GBV-B appears to cause only acute infections. The acute versus chronic course of infection may point to important differences between the two viruses that, along with the numerous similarities, will make GBV-B in tamarins a good surrogate model for HCV.

Key Words: chronic infection; GB agents; GB virus B; GBV-B; HCV; hepatitis C virus; primary hepatocytes; tamarins

Nonhuman Primate Models of Human Hepatitis Viruses

The agents recognized as causing human viral hepatitis include hepatitis viruses A (HAV1), B (HBV1), C (HCV1), D or the delta agent (HDV1), and E (HEV1). Historically, the study of human hepatitis viruses has been hindered by restricted host range and lack of permissive tissue culture systems. For decades, nonhuman primates have served to propagate these viruses and to model the liver damage from hepatitis virus infection (Purcell 1994). Hepatitis viruses A through D replicate in the chimpanzee, which has served as the major nonhuman primate model for these viruses (Purcell 1994). The macaque is the nonhuman primate model for the study of HEV replication (Bradley 1990). Because the hepatitis viruses typically do not cause major harm to the animals, evidence of hepatitis is based on biochemical changes, such as increases in the liver enzymes alanine aminotransferase (ALT1), aspartate aminotransferase, gamma-glutamyltransferase or isocitrate dehydrogenase, as well as an increase in bilirubin. Liver damage in these animals often is not as great as that seen in humans. To observe histological changes characteristic of a given viral hepatitis, samples are obtained by percutaneous needle biopsy and examined. Although the liver damage may be less, the animals often model the disease faithfully in terms of developing chronic infections with those viruses known to cause chronicity, most notably HBV and HCV.

Animal models of viral hepatitis permit studies of infectivity, pathogenesis, humoral and cellular immunity, cytokine responses, and antiviral and vaccine efficacy. Animal models are particularly valuable in that they permit analysis throughout the infection. The availability of samples from the initiation of infection provide a better assessment of the time course of the infection and insight into the early events of subclinical infection. Such studies are currently of great importance in determining the early events in HCV infection because the time of infection is rarely known for HCV in humans.

Although the availability of nonhuman primate models of hepatitis diseases is of great value, the lack of suitable tissue culture systems means that some experiments that might be done in tissue culture are performed in animals. Notwithstanding the tremendous strides made in molecular analysis of the hepatitis viruses, including constructing infectious clones of hepatitis viruses A through E, development of robust fully permissive tissue culture systems has lagged. Hepatocytes frequently undergo rapid dedifferentiation upon isolation and plating, thus losing the hepatocyte-specific characteristic necessary for hepatitis virus infection and replication. The development of such tissue culture systems would reduce the need for viral hepatitis studies in nonhuman primates and help preserve these valuable animals.

GB Agent(s)

In addition to chimpanzees, smaller nonhuman primates were used in attempts to identify the human hepatitis viral agents. In the 1960s and 1970s, Deinhardt and coworkers (1967) injected marmosets and tamarins, Saguinus and Callithrix species, with sera from patients with hepatitis in an attempt to pass the human hepatitis viruses to these small (approximately 0.5 kg) New World primates. Among the sera injected into the Saguinus species, hereafter referred to as tamarins, was a sample from a 34-yr-old surgeon (initials G.B.) who presented with jaundice that persisted for 4 wk and reached a maximum total serum bilirubin level of 14.4 mg/100 mL (Deinhardt et al. 1967). Aliquots of serum taken from this patient on the third day of jaundice caused hepatitis 16 to 40 days postinjection (pi1) in four tamarins as evidenced by abnormal liver enzyme tests and biopsies. The 11th passage of serum in tamarins was designated H205 GB pass 11 and was demonstrated to be infectious in tamarins. The H205 GB pass 11 serum is the source of the GB agent(s) used for all subsequent studies (Deinhardt et al. 1975).

Questions concerning the GB agent(s) included whether they were of human or tamarin origin and whether GB might be the agent of non-A, non-B hepatitis. Transmission studies using the H205 GB pass 11 serum in tamarins demonstrated that a protective immunity was developed against rechallenge with the homologous serum, but not with an HAV inoculum (Deinhardt et al. 1975). Studies by Tabor and coworkers (1980) demonstrated that chimpanzees inoculated with the GB passage 11 serum failed to develop hepatitis but could be infected subsequently with non-A, non-B hepatitis. In the reciprocal experiment, tamarins failed to develop hepatitis when injected with non-A, non-B hepatitis-positive serum, establishing that the GB agent was separate from the major non-A, non-B hepatitis agent (subsequently identified as HCV) (Choo et al. 1989). Although repeated attempts were made to isolate and identify the GB agent through classical means, it would take the technical breakthrough of reverse transcription/polymerase chain reaction (RT/PCR1) to isolate and identify the GB agent(s).

GB Viruses A and B

In 1995, the Virus Discovery Group at Abbott Laboratories identified not one but two GB agents (Simons et al. 1995b) from the serum of a tamarin infected with an aliquot of the H205 GB pass 11 serum, using representational difference analysis (Lisitsyn et al. 1993). The viral agents, termed GB virus A (GBV-A1) and GB virus B (GBV-B1), were 9493 and 9143 nucleotides in length, respectively, and showed similarity to the Flaviviridae family. Sequence analysis of the genomes revealed single open reading frames of 2972 amino acids (GBV-A) and 2864 amino acids (GBV-B) (Muerhoff et al. 1995). Homology comparisons with flaviviruses revealed the common 5' structural protein-3' nonstructural protein organization and permitted a preliminary assignment of polyprotein domains (Figure 1).

GBV-A

The domain alignments reveal that GBV-A is distinguished from HCV and GBV-B in lacking a capsid protein domain at the amino terminal end of the polyprotein, leading to speculation that another viral or cellular protein substitutes as a capsid protein. GBV-A can be aligned with GBV-B and HCV; it is more distantly related to HCV than is GBV-B. GBV-A appears to be a common primate virus, with viral genomes having been identified in four Saguinus species as well as a member of the Callithrix species (marmoset) and Aotus species (owl monkey) (Bukh and Apgar 1997; Leary et al. 1996). Examination of 98 wild-caught New World monkeys representing 10 species revealed that 33 had GBV-A related sequences, representing five separate viral species. GBV-A causes chronic infections (Bukh and Apgar 1997) although it is believed not to cause hepatitis in tamarins by itself (Schlauder et al. 1995a).

>GBV-B

The second GB agent identified, GBV-B, is clearly distinct from HCV but phylogenetically is most closely related to HCV (Muerhoff et al. 1995; Ohba et al. 1996), with GBV-B revealing 28% amino acid similarity to HCV genotype 1 over the whole length of its open reading frame. GBV-B was shown to cause hepatitis in tamarins during transmission studies in which four tamarins were injected with a mixture of GBV-A and GBV-B (H205 GB pass 11 serum). Although all four animals developed an acute, self-limiting hepatitis, only three were positive for GBV-B, demonstrating that GBV-B alone is sufficient to cause hepatitis (Schlauder et al. 1995a). Conversely, infection with only GBV-A failed to cause hepatitis (Schlauder et al. 1995b), although coinfection with GBV-A and GBV-B is believed to lead to a more severe hepatitis (Schlauder et al. 1995a). Unlike GBV-A, GBV-B does not appear to be commonly found in tamarins or closely related nonhuman primates. All experiments with GBV-B have been performed with passages of the isolate first obtained by Deinhardt et al. (1967), and GBV-B has not been isolated from additional tamarins, with the possible exception of a tamarin hepatitis virus (Parks and Melnick 1969), which has not been confirmed as GBV-B by RT/PCR to our knowledge.

Although people at risk for non A-E hepatitis reportedly have antibodies reactive with GBV-A and GBV-B antigens (Pilot-Matias et al. 1996; Simons et al. 1995a), GBV-A and GBV-B are considered tamarin viruses (Karayiannis and Thomas 1997) based on their restricted host range. However, the lack of additional isolations of GBV-B from tamarins in the wild raises questions as to whether tamarins are the natural host for GBV-B. The question of natural host is also complicated by questions as to whether GBV-B, which appears to cause acute infections (see below), could be maintained in a population at low levels through parenteral transmission. Many questions about the origin and life cycle of GBV-B remain.

GBV-C/HGV

A third GB agent, termed GBV-C (Simons et al. 1995a), was identified in a human serum sample from a region of West Africa endemic for hepatitis using degenerate PCR primers recognizing GBV-A, GBV-B, and HCV. A second virus identified shortly afterward, termed hepatitis G virus (HGV 1) (Linnen et al. 1996), was subsequently found to be a separate isolate of GBV-C; and the two are now often termed GBV-C/HGV. Recently, a similar virus has been identified in chimpanzees (Birkenmeyer et al. 1998). Sequence analysis revealed that GBV-C/HGV also lacks a capsid gene and is more closely related to GBV-A than either GBV-B or HCV. Interest in GBV-C/HGV is great because this human virus has been found in 1 to 2% of the US general population using GBV-C/HGV specific primers and is known to cause persistent infections. Early studies identified GBV-C/HGV in patients with fulminant hepatitis (Heringlake et al. 1996; Yoshiba et al. 1995), suggesting that GBV-C/HGV might play a role in the etiology of the disease. Other reports have failed to support this association (reviewed in Karayiannis and Thomas 1997), and recent studies suggest that GBV-C/HGV RNA is a lymphotropic virus (Tucker et al. 2000). With its broad distribution in the human population, it is unlikely that GBV-C/HGV causes significant levels of severe disease. A comparison of the GB viruses to HCV is shown in Table 1.

GBV-B as a Model of HCV Infection

The close phylogenetic relation of GBV-B to HCV and the reproducible nature of the hepatitis caused by GBV-B infection of tamarins have led to suggestions of using GBV-B as a surrogate model for HCV. Molecular studies of HCV have exploded since its identification in 1989 (Choo et al. 1989). By comparison, studies on virus infectivity, replication, and response to antiviral compounds, as well as host immune responses to HCV infection, have been hindered by the expense of studying HCV infections in chimpanzees. HCV infection of chimpanzees is playing a vital role in the study of the early stages of infection, determinants of viral clearance and chronicity, humoral and cellular immune responses, and vaccine strategies. However, the chimpanzee model imposes a number of limitations including their large size, expense, and limited availability.

Similarities and Differences between GBV-B and HCV

To assess the potential for using GBV-B as a surrogate for HCV, one must compare the similarities and differences between these two viruses structurally, in terms of their ability to replicate in animals or tissue culture and in terms of the disease they cause in the host. GBV-B and HCV share short regions of higher nucleotide sequence similarity including a segment of 130 nucleotides within the NS3 region of 62% homology and a 516 nucleotide segment of NS5 of 48% homology with HCV genotype 1 (Muerhoff et al. 1995). The functional significance of these short regions of high homology were highlighted when the GBV-B protease was shown to cleave at the NS4A/NS4B, NS4B/5A, and NS5A/5B junctions of an HCV polyprotein substrate (Scarselli et al. 1997). Shared substrate specificity was further demonstrated when the HCV NS3 protease was shown to cleave a GBV-B polyprotein substrate. However, some specificity does exist in that the GBV-B NS3 protease requires the GBV-B NS4 cofactor for enhanced activity (Butkiewicz et al. 2000).

Additional similarities between GBV-B and HCV can be found in the structures at the 5' and 3' ends of the genomes. HCV, like many members of the Flaviviridae, encodes an RNA 5' internal ribosome entry site (IRES1) necessary for initiation of translation several hundred nucleotides internal to the 5' end of the genome. GBV-B contains an IRES that resembles the one found in HCV (Grace et al. 1999). However, the two IRES elements differ in that the 3' limit of the GBV-B IRES is at the capsid initiator AUG (Rijnbrand et al. 2000); the 3' limit of the HCV IRES includes a portion of the capsid domain. HCV contains a distinct 3' nontranslated region RNA structure containing a poly U tract of varying length. The region is believed to play a role in polymerase recognition for the synthesis of negative strand RNA from the positive strand RNA template. While assembling an infectious clone, Bukh et al. (1999) identified an additional 259 nucleotides at the 3' end of the genomic RNA than originally reported (Simons et al. 1995b). Although the GBV-B 3' nontranslated region is structurally similar to the 3' terminus of HCV, it has a much shorter poly U tract and a region of three times greater length after the poly U tract than in HCV.

Although similarities between GBV-B and HCV are impressive, there are also some important differences including the propensity for HCV to induce chronic infections. The percentage of people developing chronic HCV after infection has been estimated to be as high as 85% (NIH 1997). However, recent chimpanzee studies showing less than 40% persistence (Bassett et al. 1998) as well as a human study showing less than 50% persistence (Barrett et al. 1999) suggest that the 85% persistence rate for humans may be an overestimate due to higher infection and viral clearance rates than previously appreciated. GBV-A and GBV-C/HGV, but apparently not GBV-B, cause chronic infections. Four of five infected tamarins cleared GBV-B within 84 days of infection; the fifth animal remained viremic until it was sacrificed at 137 days pi (Schlauder et al. 1995a). In another report, all five GBV-B-infected tamarins cleared the infection, again demonstrating that chronicity is not common with this virus (Bukh et al. 1999). Data from this laboratory, including those presented below, reveal a total of six tamarins infected with GBV-B and followed through viral clearance. Although chronic GBV-B infections of tamarins has not been reported, the number of reports in the literature is small, with fewer than 20 animals screened by RT/PCR for GBV-B through viral clearance to our knowledge.

The apparent lack of GBV-B chronic infections may represent important differences in the manner in which GBV-B is presented and responded to by the immune system. Acute, self-resolving infections would produce progeny virus that had less time to mutate within the host. Because all transmission studies have been performed with virus derived from the original Deinhardt isolation (Deinhardt et al. 1967), comparisons of independently derived GBV-B isolates are not possible. However, sequence analysis of GBV-B isolates after passage through animals has revealed remarkable genetic stability. Microheterogeneity within the GBV-B polyprotein coding region, as a measure of the total number of positions with variability, was 0.8% at the nucleotide level and 1.3% at the amino acid level, with the highest level of heterogeneity, 2.6%, found in the E2 (envelope) region (Bukh et al. 1999). By comparison, the E2 protein of HCV is hypervariable, giving rise to quasispecies among individual genomes (Bukh et al. 1995). The low level of variation in the GBV-B genomes presumably facilitated the generation of an infectious GBV-B molecular clone that initiated a hepatitis indistinguishable from GB viral infection upon intrahepatic injection of synthetic RNA copies (Bukh et al. 1999) (S. Lemon, University of Texas Medical Branch at Galveston, personal communication, 2000).

Another potential difference between GBV-B and HCV is the apparent protective immunity developed by tamarins after infection with GBV-B. Tamarins that had cleared GBV-B infections were protected against GBV-B rechallenge (Schlauder et al. 1995a), in agreement with earlier work demonstrating that tamarins were protected from rechallenge with the GB pass 11 serum containing both GBV-A and GBV-B (Deinhardt et al. 1975). The protection afforded by previous infection with HCV is controversial, partly because its genetic variability from a single source may be so great as to form a quasispecies (Farci et al. 1997). The advent of infectious HCV molecular clones permits the generation of defined challenge inocula. Chimpanzees that have cleared an HCV infection can then be rechallenged with a homologous challenge inoculum of limited variability to determine the protection afforded to chimpanzees after clearance of the initial HCV infection.

Infection Profiles

Recently, studies were initiated in our laboratory to investigate GBV-B infection and replication in tamarins and tamarin cell culture for comparison with HCV infections in chimpanzees. Studies were initiated by infecting a cotton top tamarin (Saguinus oedipus) with an aliquot of GB serum, which originated from the H205 GB pass 11 serum, obtained from the American Type Culture Collection. The GB inoculum was positive for both GBV-A and GBV-B RNA as assessed by RT/PCR, and this first animal (tamarin 12024) had only GBV-B RNA present in it serum. An aliquot of GBV-B-containing serum from this tamarin became the virus stock for additional studies.

To define the magnitude of the initial GBV-B infection better, a quantitative, real-time 5' exonuclease (TaqMan) assay for GBV-B RNA was developed using a primer/probe set hybridizing to the capsid region of GBV-B as the target. Excellent sensitivity was seen with the assay detecting GBV-B RNA in the 10 to 100 copy range. The GBV-B infection profile of tamarin 12024 is shown in Figure 2 (Beames et al. 2000). By the week 2 pi time point, replication was robust with GBV-B RNA detected at 4.8 × 10 8 genome equivalents (ge1)/mL of serum. The GBV-B RNA titer remained above 1 × 107 ge/mL through 12 wk pi before dropping to 3.4 × 102 ge/mL at 14 wk and to undetectable level by 16 wk pi (Figure 2). Biochemical evidence of hepatitis, as measured by an increase in serum ALT level, closely coincided with high-level viremia. ALT reached a maximum value of 328 IU/L at 10 wk pi, and then dropped rapidly in parallel with the decrease in serum GBV-B titers. The tamarin recovered with no obvious side effects.

A second tamarin (12026) was inoculated with a day 16 pi serum from the first animal (12024) containing approximately 1 × 105 ge. GBV-B replicated to 3.1x109 ge/mL at 2 wk pi in tamarin 12026 and remained high through 10 wk pi. The ALT pattern resembled that of the first tamarin with a peak value of 160 on week 12 pi before rapidly decreasing. Each animal was rechallenged with approximately 1 × 105 ge of GBV-B at 52 wk and 38 wk for tamarins 12024 and 12026, respectively. Both animals showed transient GBV-B viremia at week 1 pi that disappeared by week 2 pi, indicative of rapid clearance of GBV-B and demonstrating that the animals were protected from rechallenge.

The humoral immune response in these animals was investigated using an enzyme-linked immunosorbent assay (ELISA1) to detect antibodies to a portion of the NS3 domain expressed as a glutathione S-transferase fusion protein. Tamarin 12024 seroconverted for anti-NS3 antibody on week 8 pi with peak ELISA values observed on week 12 pi (Figure 2). Tamarin 12026 showed a similar pattern of immune response to NS3 with seroconversion by week 10 pi. In both cases, the anti-NS3 response decreased rapidly, as was reported for an NS3/NS4 protein used to detect antibodies to GBV-B (Schlauder et al. 1995a).

The magnitude of GBV-B replication may influence the outcome of the infection. Our data demonstrate a rapid increase in GBV-B titer up to 1 × 109 ge/mL. Such high-level replication exposes the tamarin immune system to large amounts of GBV-B antigens that may effectively prime the immune response to clear the virus quickly, thus providing little chance to establish chronic infection through immune evasion strategies. Recent studies of the tamarin immune response to GBV-B infection have focused on the antibody response to the capsid and nonstructural proteins. It will be necessary for future studies to investigate the response to the envelope proteins to determine whether a neutralizing antibody response is involved in viral clearance and protection from rechallenge. This model could be valuable for HCV vaccine development. It will also be necessary to investigate the cellular immune response to GBV-B infection to determine its role in virus clearance. Although differences in the course of infection of these two viruses may be thought of as a weakness in using GBV-B as a model for HCV, they may also indicate important differences that influence acute versus chronic infections.

Tissue Culture Systems for Hepatitis Viruses

A much-needed accompaniment to tamarin transmission studies is a tissue culture system permissive for GBV-B infection and replication. Hepatitis viruses are notoriously difficult to grow in cultured cells. HAV isolates have been adapted to growth in continuous cell culture (Lemon and Robertson 1993), and HDV and HEV replicate in primary chimpanzee or macaque hepatocytes, respectively (Sureau et al. 1991; Tam et al. 1997). HBV replicates after transfection of the genome into hepatocyte cell lines. However, primary cells or continuous cell lines reproducibly susceptible to infection with HBV or HCV have been more elusive. Although the blocks to infection or replication may be at many different levels, often it is thought to be due to rapid dedifferentiation of hepatocytes upon isolation and plating. Difficulties in establishing a tissue culture system for HCV include a shortage of high-quality human and chimpanzee hepatocytes as well as and difficulty in devising media formulations to maintain the differentiated state.

Efforts in our laboratory to develop hepatocyte tissue culture systems have focused on obtaining high-quality hepatocytes and maintaining them in a serum-free medium supplemented with growth factors and hormones (Lanford et al. 1989). Promising results have been obtained with baboon and macaque hepatocytes in terms of maintaining differentiated hepatocytes and in using hepatocytes that have been frozen and revived to extend their utility greatly. Baboon hepatocytes can be maintained in this medium in a highly differentiated state for up to 100 days. The baboon hepatocytes have been used extensively in our research on apolipoprotein(a) synthesis and lipoprotein biogenesis (Lanford et al. 1996; Rainwater and Lanford 1989; White and Lanford 1994a; White et al. 1993, 1994b, 1997, 1999). Experience with the baboon hepatocyte system has been applied to culturing hepatocytes from other nonhuman primate species for the study of human hepatitis viruses.

Macaque Primary Hepatocyte Cultures

Our experience with HEV replication in primary rhesus macaque hepatocytes has provided insight into development of a tissue culture system for GBV-B. Hepatocytes from naive macaques were isolated by standard methods (Lanford and Estlack 1998) and then used fresh or upon reviving after frozen storage. The hepatocytes were exposed to infectious HEV serum and assayed at various times after infection using a strand-specific RT/PCR assay (Lanford and Chavez 1998) to distinguish true replication from residual inoculum. Negative-strand HEV RNA was routinely detected in HEV-infected hepatocytes, demonstrating HEV replication (Tam et al. 1997).

GBV-B Replication in in Vivo Infected Hepatocytes

Recent studies in this laboratory have focused on developing a tamarin hepatocyte tissue culture system. Initial studies demonstrated that hepatocytes isolated from a GBV-B infected tamarin could be maintained for up to 42 days post isolation with no signs of cytopathic effect (Beames et al. 2000). TaqMan RT/PCR analysis of the in vivo-infected hepatocytes revealed the cells contained 2.6 × 107 ge/m g RNA at day 2 after plating. Cell-associated GBV-B RNA was detected throughout the experiment, dropping by approximately two logs at day 42. Medium collected at this time point contained 3.1 ´ 105 ge/mL after 18 media changes, indicating that the cells continued to secrete virus for prolonged periods after plating. The GBV-B NS3 protein was visualized in in vivo infected hepatocytes by immunofluorescence (IF1) using a rabbit antiglutathione-S-transferase (anti-GST1)-NS3 serum generated against the antigen used for the NS3 ELISA in Figure 2. Strong reactivity was detected with the anti-GST-NS3 rabbit serum demonstrating high-level GBV-B antigen expression, compared with HCV proteins in chimpanzee hepatocytes that are more difficult to detect by IF (unpublished data).

GBV-B Infection of Naive Hepatocytes

The utility of tamarin hepatocytes was greatly increased with the demonstration that GBV-B could infect naive tamarin primary hepatocytes in culture (Beames et al. 2000). Hepatocytes isolated from a naive tamarin replicated the virus after exposure to GBV-B-containing serum as demonstrated by TaqMan analysis of cells and media from the cultures (Figure 3). Cell-associated virus increased from 6.3 ´ 104 ge/m g cellular RNA on day 0 after inoculation and extensive washing to remove residual inoculum, to 2.4 ´ 106 ge/m g cellular RNA at day 1. The 40-fold increase represents the difference between virus internalized or bound to the cells during the 6-hr exposure on day 0 and robust replication of GBV-B detected at day 1. Media-associated virus increased from no virus in the fresh medium added to the cells on day 0, to a maximum of 5.9 ´ 106 ge/mL at day 3. Cell-associated and media-derived virus levels remained high throughout the 14-day experiment. Staining of hepatocytes infected in vitro with GBV-B by IF using the rabbit anti-GST-NS3 antiserum revealed a strong NS3 signal, indicating high-level GBV-B nonstructural protein expression.

Screening of Antiviral Compounds

Experiments have been initiated to use tamarin hepatocytes in tissue culture to test potential antiviral compounds for activity against GBV-B. The close phylogenetic relatedness of GBV-B and HCV as well as the activity of the GBV-B NS3 protease in cleaving the HCV substrates suggests that GBV-B in tamarin hepatocytes may be useful in direct testing of antiviral compounds. Two antiviral compounds in clinical use for chronic HCV infection, interferon a -2b and ribavirin, are being tested in this system. Treatment with interferon resulted in an approximately 2-log decline in cell-associated viral RNA and secreted virus (unpublished data). Treatment with ribavirin, a guanosine analog, yielded a 3-log inhibition of GBV-B RNA replication. Preliminary experiments suggest that ribavirin may be incorporated into the viral genome leading to missense or nonsense mutations (unpublished data), supporting the contention that GBV-B replication in tamarin hepatocyte cultures may prove useful in investigating the mechanism of antiviral compounds. The data suggest that the GBV-B/primary hepatocyte system can be effectively used to evaluate antivirals. The utility of this system as a surrogate for HCV will increase dramatically with the creation of viable GBV-B/HCV chimeric viruses.

Potential of GBV-B Model: Conclusions and Future Directions

Although the major differences between GBV-B and HCV are mentioned above, the compelling value of GBV-B as a model for HCV warrants restatement. The relatedness of the two viruses as well as the similar tropism for the liver supports the use of GBV-B as a model for HCV. The ability of the GBV-B NS3 protease to cleave HCV polyprotein substrates suggests that protease inhibitors with activity against GBV-B may have activity against HCV. The ability to infect primary hepatocytes isolated from naive tamarins provides the basis of a neutralization assay. Test antisera could be incubated with a GBV-B inoculum to determine whether a given antiserum contains neutralizing antibodies. Neutralizing antibodies could be used to map epitopes important for infection. Such an approach might identify similar HCV envelope epitopes involved in cell attachment or internalization. Since their identification in 1995, rapid progress has been made on the analysis of the GB agents, including determination that GBV-B is the causative agent of GB-associated hepatitis in tamarins. The GBV-B genome has been cloned and sequenced, and infectious synthetic genomes have been generated. The model of GBV-B hepatitis in tamarins has been refined further by including measures of virus replication and the humoral immune response in the analysis.

Although progress has been impressive, there are still many questions concerning GBV-B and its relation to HCV. On a practical note, it will be necessary to explore the immune response to GBV-B more fully, especially in terms of epitopes of the envelope proteins that are likely to be important in protection against rechallenge. The in vitro infection system may permit development of a neutralization assay to study the humoral immune response to the envelope proteins. Efforts should be directed toward immortalizing tamarin hepatocytes to develop continuous cell lines that are fully permissive for GBV-B infection and replication. Efforts are underway to establish chronic infections of GBV-B in immunosuppressed tamarins and to determine whether such infections would better model chronic HCV infection.

Other questions that remain concern the origins of GBV-B. To date, it has been isolated one time from an animal injected with the serum from the patient GB.

Acknowledgment

This week was supported by National Institute of Health grant P51 R13986 to the Southwest Regional Primate Research Center.

1Abbreviations used in this article: ALT, alanine aminotransferase; anti-GST, antiglutathione-S-transferase; ELISA, enzyme-linked immunosorbent assay; GBV-A, GB virus A, GBV-B, GB virus B;GBV-C/HGV, GB virus C or hepatitis G virus; ge, genome equivalents; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus, HDV, hepatitis D virus; HEV, hepatitis E virus; IF, immunofluorescence; IRES, internal ribosome entry site; pi, post infection; RT/PCR, reverse transcription/polymerase chain reaction.

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Figure 1 Hepatitis C virus (HCV) comparison with tamarin GB viruses (GBVs). The coding regions of HCV, GBV-B, and GBV-A are shown as rectangles for comparison purposes. The structural proteins, capsid (C) and envelope proteins 1 and 2 (E1 and E2), are located in the amino portion of the polyprotein. Downstream are located the nonstructural (NS) protein domains representing a metalloprotease (NS2), a serine protease and helicase (NS3), a cofactor for the NS3 protease (NS4), a phosphoprotein (NS5A), and the RNA-dependent RNA polymerase (NS5B). Approximate limits of the GBV-B domains are based on published data (Bukh et al. 1999; Muerhoff et al. 1995).

Figure 2 GB virus B (GBV-B) infection profile. The course of GBV-B infection (left panel) or rechallenge (right panel) in tamarin 12024 was assayed by monitoring viral RNA levels, alanine aminotransferase (ALT), and anti-NS3 antibody levels in the serum. The bars represent GBV-B RNA in genome equivalents (ge)/mL, ALT is indicated by the circles (IU/L), and anti-NS3 antibody response is indicated as squares (absorbance at 405 nm).

Figure 3 GB virus B (GBV-B) in vitro infection of tamarin hepatocytes. A plot of TaqMan RT/PCR titers over the course of a 14-day in vitro GBV-B infection is shown. The squares represent cell-associated viral RNA in (ge)/m g cellular RNA, and circles represent medium-derived virus in ge/mL of medium.

Table 1Comparison of the GB viruses with hepatitis C virus





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