Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

UNLABELLED: A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. IMPORTANCE: HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines.

Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (<200 copies/mL), the viral RNA loads in plasma were detectable intermittently during the observation period. Of note, the chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome.

A novel hepacivirus with an unusually long and intrinsically disordered NS5A protein in a wild Old World primate.

GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.

Modulation of GB virus B RNA abundance by microRNA-122: dependence on and escape from microRNA-122 restriction.

Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5'-terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance.

A comparative analysis of the substrate permissiveness of HCV and GBV-B NS3/4A proteases reveals genetic evidence for an interaction with NS4B protein during genome replication.

The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs containing HCV sequences at the C-terminus of NS4B had a pseudo-lethal replication phenotype. Replication-competent revertants contained second-site substitutions within the NS3 protease or NS4B N-terminus, providing genetic evidence for an essential interaction between NS3 and NS4B during genome replication.

Molecular evolution of GB virus B hepatitis virus during acute resolving and persistent infections in experimentally infected tamarins.

GB virus B (GBV-B) causes acute hepatitis in experimentally infected tamarins. We compared evolutionary features in acute resolving and persistent GBV-B infection. We detected no evidence of evolution in four animals with clearance during weeks 9-12, whereas three animals with clearance during weeks 13-26 had several substitutions in their polyprotein sequence. A single tamarin had long-term GBV-B viraemia; analysis of virus recovered at weeks 2, 5, 12, 20, 26, 52 and 104 demonstrated that mutations accumulated over time. Overall, the amino acid substitution rate was 3.5x10(-3) and 1.1x10(-3) substitutions per site year(-1) during weeks 1-52 and 53-104, respectively. Thus, there was a significant decrease in evolution over time, as found for hepatitis C virus. The rate of non-synonymous substitution per non-synonymous site compared with that of synonymous substitution per synonymous site decreased over time, suggesting reduction of positive selective pressure. These data demonstrate that prolonged GBV-B infection is associated with viral evolution.

Lack of adaptation of chimeric GB virus B/hepatitis C virus in the marmoset model: possible effects of bottleneck.

Approximately 3% of the world population is chronically infected with hepatitis C virus (HCV). GB virus B (GBV-B), a surrogate model for HCV, causes hepatitis in tamarins and is the virus phylogenetically most closely related to HCV. Previously we described a chimeric GBV-B containing an HCV insert from the 5' noncoding region (NCR) that was adapted for efficient replication in tamarins (Saguinus species). We have also demonstrated that wild-type (WT) GBV-B rapidly adapts for efficient replication in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that the chimeric virus failed to adapt during serial passage in marmosets. The chimeric virus was passaged four times through 24 marmosets. During passage, two marmoset phenotypes were observed: susceptible and partially resistant. Although appearing to adapt in a resistant animal during a prolonged and gradual increase in viremia, the chimeric GBV-B failed to replicate efficiently upon passage to a naïve marmoset. The resistance was specific to the chimeric virus, as the chimeric virus-resistant animals were susceptible to marmoset-adapted WT virus during rechallenge studies. Three isolates of the chimeric virus were sequenced, and 20 nucleotide changes were observed, including eight amino acid changes. Three unique changes were observed in the 5' NCR chimeric insert, an area that is highly conserved in HCV. We speculate that the failure of the chimeric virus to adapt in marmosets might be due to a bottleneck that occurs at the time of infection of resistant animals, which may lead to a loss of fitness upon serial passage.

The marmoset model of GB virus B infections: adaptation to host phenotypic variation.

Worldwide, approximately 170 million people are chronically infected with hepatitis C virus (HCV), and chronic infection frequently progresses to serious liver disease, including cirrhosis and hepatocellular carcinoma. GB virus B (GBV-B), the virus phylogenetically most closely related to HCV, causes hepatitis in tamarins. We have demonstrated the suitability of the tamarin as a host for GBV-B and as a surrogate nonhuman primate model for HCV infection, and we have initiated studies of GBV-B infection in a closely related species, the common marmoset (Callithrix jacchus). Here, we demonstrate that marmosets exhibit two phenotypes upon infection with GBV-B: the susceptible phenotype and the partially resistant phenotype. In addition, we identify changes that may correlate with adaptation of the virus to the partially resistant host. GBV-B was serially passaged five times through 14 marmosets as one lineage and two times through 6 marmosets as a second lineage. Virus adapted to the marmosets and eventually exhibited robust infections in two separate lineages, lineages 1 and 2. A third lineage was initiated with a molecular clone, and again, susceptible and partially resistant phenotypes were observed. Three isolates were fully sequenced (from lineage 1), and 21 nucleotide changes were observed, with six amino acid changes. We speculate that the marmoset partially resistant phenotype may be due to a polymorphism in the marmoset population that affects critical virus-host interactions and that wild-type GBV-B is capable of rapidly adapting to this altered host.

A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC)) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC) genome (within the 3'NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC) RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

Chimeric GB virus B genomes containing hepatitis C virus p7 are infectious in vivo.

BACKGROUND/AIMS: The development of new therapies for hepatitis C virus (HCV) infection has been hampered by the lack of a small animal model. GB virus B (GBV-B), which infects new world monkeys, has been proposed as a surrogate system for HCV replication. Despite their short genetic distance, however, difficulties exist when extrapolating results from GBV-B to the HCV system. One way of addressing this is the creation of chimeric GBV-B containing HCV elements. METHODS: Construction and analysis of GBV-B chimeras in which the p13 ion channel was replaced by its HCV counterpart, p7. RESULTS: Replacing all, or part of, the GBV-B p13 protein with HCV p7 resulted in viable chimeras which replicated at wild-type levels in marmosets following intra-hepatic RNA injection. Serum from one animal injected with chimeric RNA was infectious in three naïve recipients, indicating that chimeras formed fully infectious virions. Amantadine, which blocks the ion channel activity of both HCV and GBV-B proteins in vitro, also inhibited GBV-B replication in primary hepatocytes. CONCLUSIONS: These viruses highlight the potential for chimeric GBV-B in the development of HCV-specific therapies and will provide a means of developing HCV p7 as a therapeutic target.

A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B.

GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of beta-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.

Restricted quasispecies variation following infection with the GB virus B.

The extent of genetic variability following acute infection of tamarins with GB virus B (GBV-B) is not known. In this study we attempted to define the quasispecies variation of GBV-B 17 days post-infection, by PCR amplification of GBV-B RNA extracted from serum and liver. Cloning followed by sequencing revealed a small number of changes in the three regions studied, namely the 5' untranslated region, E2 and NS3. Moreover, there was no region of high amino acid variability in E2, akin to hypervariable region 1 of hepatitis C virus. This was further confirmed by analysing sequences from two additional animals obtained at a similar time point post-infection. Nevertheless, it was apparent that different variants with one or two amino acid substitutions in the region studied had been selected when comparing the sequences from the three animals. This restricted sequence variation of GBV-B during acute hepatitis may explain the infrequent progression of the infection to a chronic stage.

Immunity against the GBV-B hepatitis virus in tamarins can prevent productive infection following rechallenge and is long-lived.

GB virus-B (GBV-B) is the virus most closely related to hepatitis C virus (HCV). Thus, we have used GBV-B infection of tamarins, which develop acute hepatitis following experimental infection, as a surrogate model to study protective immunity. As challenge virus, we first produced a GBV-B pool from an infected tamarin, which was not infected with the related GBV-A viruses. Its infectivity titer was 10(6.6) tamarin 50% infectious doses per ml. Next, two tamarins that were convalescent from recombinant GBV-B infection were re-challenged. In the original infection viremia persisted for 8 and 12 weeks, respectively, and both animals developed moderately severe hepatitis. Each tamarin was re-challenged four times with 10(4.3) tamarin 50% infectious doses of the GBV-B challenge virus. In one animal, each re-challenge produced 1-2 weeks of viremia; hepatitis was observed following the first re-challenge. In the other animal, however, only the first re-challenge produced viremia, lasting 1 week. During the primary infection, peak GBV-B titers were about 10(8) genome equivalents/ml in both animals; following re-challenges, peak titers ranged from 10(3) to 10(6) genome equivalents/ml. Analysis of the polyprotein sequence of viruses recovered from both animals following the first re-challenge demonstrated that these did not represent immune escape variants since mutations were not detected. Neutralization studies suggested that the immunity was not humoral in nature. We also demonstrated that the immunity was long-lived: 1 year after the fourth challenge, the animal with sterilizing immunity had low titer viremia for only 1 week following an additional challenge.

Efficient regulation of viral replication by siRNA in a non-human primate surrogate model for hepatitis C.

RNA interference (RNAi) represents a new technology which could offer potential applications for the therapeutics of human diseases. RNAi-mediated therapy has recently been shown to be effective toward infectious diseases in in vitro and rodent models, however, it remains unclear whether RNAi therapy with systemic application could be effective in primates. In this study, we examined if RNAi therapy could be effective toward infectious diseases by using a non-human primate surrogate model for hepatitis C. Administration into marmosets of cationic liposome-encapsulated siRNA (CL-siRNA) for GB virus B (GBV-B), which is most closely related to hepatitis C virus, repressed GBV-B replication in a dose-dependent manner. Especially, 5 mg/kg of the CL-siRNA completely inhibited the viral replication. Since the serum interferons (IFNs) were induced by CL-siRNA in vivo, inhibition of viral regulation by anti-GBV-B CL-siRNA may include an antiviral effect of IFN. However, contribution of induced IFN may be partial, since the control CL-siRNA which induced a stronger IFN response than GBV-B CL-siRNA could only delay the viral replication. Our results suggest the feasibility of systemic administration of CL-siRNA as an antiviral strategy.

GBV-B as a pleiotropic virus: distribution of GBV-B in extrahepatic tissues in vivo.

GB virus B (GBV-B) infection of New World monkeys is considered to be a useful surrogate model for hepatitis C virus (HCV) infection. GBV-B replicates in the liver and induces acute resolving hepatitis but little is known whether the other organs could be permissive for the virus. We investigated the viral tropism of GBV-B in tamarins in the acute stage of viral infection and found that the viral genomic RNA could be detected in a variety of tissues. Notably, a GBV-B-infected tamarin with marked acute viremia scarcely showed a sign of hepatitis, due to preferential infection in lymphoid tissues such as lymph nodes and spleen. These results indicate that GBV-B as well as HCV is a pleiotropic virus in vivo.

A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo.

Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane.

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks approximately 35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.

Amantadine inhibits the function of an ion channel encoded by GB virus B, but fails to inhibit virus replication.

A chemically synthesized peptide representing the C-terminal subunit (p13-C) of the p13 protein of GB virus B (GBV-B), the most closely related virus to hepatitis C virus (HCV) showed ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to potassium ions than to chloride ions. Amantadine but not hexamethylene amiloride (HMA) inhibited the ion channel function of p13-C in the lipid membranes. However, neither agent was able to inhibit the replication and secretion of GBV-B from virus-infected cultured marmoset hepatocytes, which were harvested from a marmoset that was infected in vivo or inhibit replication after in vitro infection of naive hepatocytes. These data suggest that the GBV-B ion channel, contrary to the data derived from the lipid membranes, is either resistant to amantadine or that virus replication and secretion are independent of ion channel function. As the p7 protein of HCV also has ion channel activity that is apparently resistant to amantadine in vivo, the former possibility is most likely. Ion channels are likely to have an important role in the life cycle of many viruses and compounds that block these channels may prove to be useful antiviral agents.

Functional analyses of GB virus B p13 protein: development of a recombinant GB virus B hepatitis virus with a p7 protein.

GB virus B (GBV-B), which infects tamarins, is the virus most closely related to hepatitis C virus (HCV). HCV has a protein (p7) that is believed to form an ion channel. It is critical for viability. In vitro studies suggest that GBV-B has an analogous but larger protein (p13). We found that substitutions of the -1 and/or -3 residues of the putative cleavage sites (amino acid 613/614 and 732/733) abolished processing in vitro and rendered an infectious GBV-B clone nonviable in tamarins. Internal cleavage was predicted at two sites (amino acid 669/670 and 681/682), and in vitro analysis indicated processing at both sites, suggesting that p13 is processed into two components (p6 and p7). Mutants with substitution at amino acid 669 or 681 were viable in vivo, but the recovered viruses had changes at amino acid 669 and 681, respectively, which restored cleavage. A mutant lacking amino acid 614-681 (p6 plus part of p7) was nonviable. However, a mutant lacking amino acid 614-669 (p6) produced high titer viremia and acute resolving hepatitis; viruses recovered from both animals lacked the deleted sequence and had no other mutations. Thus, p6 was dispensable but p7 was essential for infectivity. The availability of a recombinant GBV-B virus containing a p7 protein with similarities to the HCV p7 will enhance the relevance of this model and will be of importance for identifying compounds that inhibit p7 function as additional therapeutic agents.

Acute GB virus B infection of marmosets is accompanied by mutations in the NS5A protein.

GBV-B, a member of the Flaviviridae family of viruses, is the virus most closely related to HCV, and GBV-B infection in tamarin monkeys might represent a valuable surrogate animal model of HCV infection. In the current study, GBV-B was successfully transmitted to two marmosets (Callithrix jaccus). The infection resulted in viremia of 14- and 17-week duration, respectively, and was accompanied by elevation of isocitrate dehydrogenase activity. These data confirm that marmosets might represent an attractive model for GBV-B infection. The sequence of GBV-B NS5A, which was previously reported to have one of the highest mutation rates during infection in tamarins, was determined for viruses recovered from the inoculum and from marmoset blood samples obtained at weeks 1, 8, and 14 post inoculation in one marmoset and at weeks 2, 8, and 17 post inoculation in the other marmoset. In both animals, we detected four substitutions (R1945K, K2052G, F2196L, and G2268E), in the virus recovered immediately before viral clearance. Interestingly, two of these mutations (F2196L and G2268E) were described recently for viruses recovered from persistently infected tamarins. Appearance of these mutations presumably reflects a mechanism of immune escape rather than adaptation of the virus to a new host.