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Despite their close genetic relatedness, apes and African and Asian monkeys (AAMs) differ in their susceptibility to severe bacterial and viral infections that are important causes of human disease. Such differences between humans and other primates are thought to be a result, at least in part, of interspecies differences in immune response to infection. However, because of the lack of comparative functional data across species, it remains unclear in what ways the immune systems of humans and other primates differ. Here, we report the whole-genome transcriptomic responses of ape species (human and chimpanzee) and AAMs (rhesus macaque and baboon) to bacterial and viral stimulation. We find stark differences in the responsiveness of these groups, with apes mounting a markedly stronger early transcriptional response to both viral and bacterial stimulation, altering the transcription of ~40% more genes than AAMs. Additionally, we find that genes involved in the regulation of inflammatory and interferon responses show the most divergent early transcriptional responses across primates and that this divergence is attenuated over time. Finally, we find that relative to AAMs, apes engage a much less specific immune response to different classes of pathogens during the early hours of infection, up-regulating genes typical of anti-viral and anti-bacterial responses regardless of the nature of the stimulus. Overall, these findings suggest apes exhibit increased sensitivity to bacterial and viral immune stimulation, activating a broader array of defense molecules that may be beneficial for early pathogen killing at the potential cost of increased energy expenditure and tissue damage. Copyright

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.

Background & Aims GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. Methods Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). Results GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. Conclusion The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. Lay summary New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.

Background & Aims We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice. Methods We analyzed 519 serum samples from participants in a phase 1 vaccine trial (ClinicalTrials.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n = 28) and rhesus macaques (n = 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized. Results Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human VH1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs. Conclusions In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.

Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.

Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure, and hepatocellular carcinoma. Evidence suggests that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with an RNA interference (RNAi)-based therapeutic targeting HBV transcripts, ARC-520, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients who were HBeAg-negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). HBeAg positivity is associated with greater disease risk that may be moderately reduced upon HBeAg loss. The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome but also from transcripts arising from HBV DNA integrated into the host genome, which was the dominant source in HBeAg-negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the small interfering RNAs in ARC-520, explaining the reduced response in HBeAg-negative chimpanzees and, by extension, in HBeAg-negative patients. Our results uncover a heretofore underrecognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immunosurveillance. These results could alter trial design and endpoint expectations of new therapies for chronic HBV.

Humans and nonhuman primates (NHPs) are similar in size, behavior, physiology, biochemistry, structure and function of organs, and complexity of the immune system. Research on NHPs generates complementary data that bridge translational research from small animal models to humans. NHP models of human disease offer unique opportunities to develop stem cell-based therapeutic interventions that directly address relevant and challenging translational aspects of cell transplantation therapy. These include the use of autologous induced pluripotent stem cell-derived cellular products, issues related to the immune response in autologous and allogeneic setting, pros and cons of delivery techniques in a clinical setting, as well as the safety and efficacy of candidate cell lines. The NHP model allows the assessment of complex physiological, biochemical, behavioral, and imaging end points, with direct relevance to human conditions. At the same time, the value of using primates in scientific research must be carefully evaluated and timed due to expense and the necessity for specialized equipment and highly trained personnel. Often it is more efficient and useful to perform initial proof-of-concept studies for new therapeutics in rodents and/or other species before the pivotal studies in NHPs that may eventually lead to first-in-human trials. In this report, we present how the Southwest National Primate Research Center, one of seven NIH-funded National Primate Research Centers, may help the global community in translating promising technologies to the clinical arena.

Siderophores are best known as small iron binding molecules that facilitate microbial iron transport. In our previous study we identified a siderophore-like molecule in mammalian cells and found that its biogenesis is evolutionarily conserved. A member of the short chain dehydrogenase family of reductases, 3-OH butyrate dehydrogenase (BDH2) catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. We have shown that depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of cellular iron and mitochondrial iron deficiency. These observations suggest that the mammalian siderophore is a critical regulator of cellular iron homeostasis and facilitates mitochondrial iron import. By utilizing bioinformatics, we identified an iron-responsive element (IRE; a stem-loop structure that regulates genes expression post-transcriptionally upon binding to iron regulatory proteins or IRPs) in the 3′-untranslated region (3′-UTR) of the human BDH2 (hBDH2) gene. In cultured cells as well as in patient samples we now demonstrate that the IRE confers iron-dependent regulation on hBDH2 and binds IRPs in RNA electrophoretic mobility shift assays. In addition, we show that the hBDH2 IRE associates with IRPs in cells and that abrogation of IRPs by RNAi eliminates the iron-dependent regulation of hBDH2 mRNA. The key physiologic implication is that iron-mediated post-transcriptional regulation of hBDH2 controls mitochondrial iron homeostasis in human cells. These observations provide a new and an unanticipated mechanism by which iron regulates its intracellular trafficking.

Although phylogenetically unrelated, human hepatitis viruses share an exclusive or near exclusive tropism for replication in differentiated hepatocytes. This narrow tissue tropism may contribute to the restriction of the host ranges of these viruses to relatively few host species, mostly nonhuman primates. Nonhuman primate models thus figure prominently in our current understanding of the replication and pathogenesis of these viruses, including the enterically transmitted hepatitis A virus (HAV) and hepatitis E virus (HEV), and have also played major roles in vaccine development. This review draws comparisons of HAV and HEV infection from studies conducted in nonhuman primates, and describes how such studies have contributed to our current understanding of the biology of these viruses.

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.

GBV-B induces hepatitis in tamarins and marmosets and is a surrogate model for HCV infections. Here, we cloned and characterized the antiviral activity of tamarin and marmoset interferon (IFN)alpha and IFN gamma. Potent antiviral activity was observed for tamarin and marmoset IFN alpha in primary hepatocyte cultures infected with GBV-B. The antiviral activity was greater in cultures exposed to IFN alpha prior to GBV-B infection, suggesting that either GBV-B was capable of inhibition of the antiviral activity of exogenous IFN alpha or that the preexisting endogenous IFN response to the virus reduced efficacy to exogenous IFN alpha. IFN gamma also exhibited antiviral activity in GBV-B infected hepatocytes. The transcriptional response to IFN alpha in marmoset hepatocytes was characterized using human genome microarrays. Since the GBV-B hepatocyte culture model possesses a functional innate immune response, it will provide opportunities to explore the nature of the antiviral response to a virus closely related to HCV.

To investigate chemokine expression networks in chronic hepatitis C virus (HCV) infection, we used microarray analysis to determine chemokine expression in human infection and in chimpanzees experimentally infected with HCV. The CXCR3 chemokine family was highly expressed in both human and chimpanzee infection. CXCL10 was the only CXCR3 chemokine elevated in the serum, suggesting that it may neutralize any CXCR3 chemokine gradient established between the periphery and liver by CXCL11 and CXCL9. Thus, CXCR3 chemokines may not be responsible for recruitment of T lymphocytes but may play a role in positioning these cells within the liver. The importance of the CXCR3 chemokines, in particular CXCL11, was highlighted by replicating HCV (JFH-1) to selectively upregulate its expression in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). This selective upregulation was confirmed at the transcriptional level by using the CXCL11 promoter driving the luciferase reporter gene. This synergistic increase in expression was not a result of HCV protein expression but the nonspecific innate response to double-stranded RNA (dsRNA), as both in vitro-transcribed HCV RNA and the dsRNA analogue poly(I:C) increased CXCL11 expression and promoter activity. Furthermore, we show that CXCL11 is an IRF3 (interferon regulatory factor 3) response gene whose expression is selectively enhanced by IFN-γ and TNF-α. In conclusion, the CXCR3 chemokines are the most significantly expressed chemokines in chronic hepatitis C and most likely play a role in positioning T cells in the liver. Furthermore, HCV can selectively increase CXCL11 expression in response to IFN-γ and TNF-α stimulation that may play a role in the pathogenesis of HCV-related liver disease.

Almost 800 research scientists convened in San Antonio in early October of 2008 to share recent results and new insights concerning hepatitis C virus (HCV). The 15th International Symposium on Hepatitis C Virus and Related Viruses opened with a mini-symposium recognizing progress made over the past 20 years since the discovery of HCV. This was followed by 10 keynote talks, and 61 oral and 379 poster presentations over the succeeding 4 days. Our intent is to succinctly summarize the most notable findings reported, a task made extraordinarily difficult by the volume, diversity and quality of science presented. At best, this cursory overview vividly demonstrates the robust pace of the research that is unraveling the mysteries of this unique and potentially deadly human pathogen.

The mechanism of the interferon-alpha (IFN-alpha)-induced antiviral response during hepatitis C virus (HCV) therapy is n o t completely understood. In this study,we examined the transcriptional response to IFN-alpha in uninfected chimpanzees after single doses of chimpanzee, human, or human-pegylated IFN-alpha. Liver and peripheral blood mononuclear cell (PBMC) samples were used for total genome microarray analysis. Most induced genes achieved maximal response within 4 hours, began to decline by 8 hours, and were at baseline levels by 24 hours postinoculation, a time when high levels of circulating pegylated IFN-alpha were still present. The rapid downregulation of the IFN-alpha response may be involved in the transition between the observed phase I and phase II viral kinetics during IFN-alpha therapy in HCV-infected patients. The response to all three forms of IFN-alpha was similar; thus, the reasons for previous failures in antiviral treatment of chimpanzees with human IFN-alpha were not due to species specificity of IFN-alpha. The response to IFN-alpha was partially tissue-specific. A total of 1778 genes were altered in expression by twofold or more by IFN-alpha, with 538 and 950 being unique to the liver or PBMC, respectively. Analysis of the IFN-alpha and IFN-gamma responses in primary chimpanzee and human hepatocytes were compared as well. IFN-alpha and IFN-gamma induced partially overlapping sets of genes in hepatocytes. In conclusion, the response to IFN-alpha is largely tissue-specific, and the response is rapidly downregulated in vivo, which may have a significant influence on the kinetics of antiviral response.

Progress in understanding the pathogenesis of hepatitis C virus (HCV) has been slowed by the absence of tractable small animal models. Whereas GB virus B (GBV-B, an unclassified flavivirus) shares a phylogenetic relationship and several biologic attributes with HCV, including hepatotropism, it is not known to cause persistent infection, a hallmark of HCV. Here, we document persistent GBV-B infection in one of two healthy tamarins ( Saguinus oedipus ) inoculated intrahepatically with infectious synthetic RNA. High-titer viremia (10 8 to 10 9 genome equivalents per ml) and transiently elevated serum alanine transaminase activities were present from weeks 4 to 12 postinoculation in both animals. However, whereas GBV-B was eliminated from one animal by 20 weeks, the second animal remained viremic (10 3 to 10 7 genome equivalents per ml) for >2 years, with alanine transaminase levels becoming elevated again before spontaneous resolution of the infection. A liver biopsy taken late in the course of infection demonstrated hepatitis with periportal mononuclear infiltrates, hepatocellular microvesicular changes, cytoplasmic lipid droplets, and disordered mitochondrial ultrastructure, findings remarkably similar to chronic hepatitis C. GBV-B-infected hepatocytes contained numerous small vesicular membranous structures resembling those associated with expression of HCV nonstructural proteins, and sequencing of GBV-B RNA demonstrated a rate of molecular evolution comparable to that of HCV. We conclude that GBV-B is capable of establishing persistent infections in healthy tamarins, a feature that substantially enhances its value as a model for HCV. Mitochondrial structural changes and altered lipid metabolism leading to steatosis are conserved features of the pathogenesis of chronic hepatitis caused by these genetically distinct flaviviruses.

Members of the Hepadnaviridae family have been isolated from birds, rodents, and primates. A new hepadnavirus isolated from the woolly monkey, a New World primate, is phylogenetically distinct from other primate isolates. An animal model has been established for woolly monkey hepatitis B virus (WMHBV) by using spider monkeys, since woolly monkeys are endangered. In this study, a greater-than-genome length construct was prepared without amplification by using covalently closed circular DNA extracted from the liver of an infected woolly monkey. Transfection of the human liver cell line Huh7 with WMHBV DNA resulted in the production of viral transcripts, DNA replicative intermediates, and secreted virions at levels similar to those obtained with an infectious human HBV clone, demonstrating that the host range restriction of WMHBV is not at the level of genome replication. WMHBV particles from the medium of transfected cultures initiated an infection in a spider monkey similar to that obtained with virions derived from woolly monkey serum. In an attempt to adapt the virus for higher levels of replication in spider monkeys, immunosuppressed and newborn animals were inoculated. Neither procedure produced persistent infections, and the level of viral replication remained several logs lower than that observed in persistently infected woolly monkeys. These data demonstrate the production of an infectious clone for WMHBV and extend the characterization of the spider monkey animal model.

Woolly monkey hepatitis B virus (WMHBV) is a new member of Hepadnaviridae that was isolated from a New World monkey and is phylogenetically distinct from the HBV family. In this study, we explored the functional significance of sequence divergence in the HBV and WMHBV genomes. Independently expressed TP and RT domains of the WMHBV reverse transcriptase (Pol) formed a complex functional for in vitro nucleotide priming, consistent with previous results from priming reactions conducted with HBV. Transcomplementation assays between HBV and WMHBV TP and RT components for in vitro priming demonstrated functional compatibility, although priming with the combination of WMHBV RT and HBV TP was reduced. Examination of cross-species protein-protein interactions revealed that WMHBV core coprecipitated with HBV TP and RT, as well as with WMHBV TP and RT. Analysis in Huh7 cells revealed that WMHBV core and Pol complemented core-negative and Pol-negative HBV mutant genomes for replication. These results highlight the conservation of function despite significant sequence divergence in these viruses.

Persistent infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) account for the majority of cases of hepatic cirrhosis and hepatocellular carcinoma (HCC) worldwide. Small, non-coding RNAs play important roles in virus-host interactions. We used high throughput sequencing to conduct an unbiased profiling of small (14-40 nts) RNAs in liver from Japanese subjects with advanced hepatitis B or C and hepatocellular carcinoma (HCC). Small RNAs derived from tRNAs, specifically 30-35 nucleotide-long 5' tRNA-halves (5' tRHs), were abundant in non-malignant liver and significantly increased in humans and chimpanzees with chronic viral hepatitis. 5' tRH abundance exceeded microRNA abundance in most infected non-cancerous tissues. In contrast, in matched cancer tissue, 5' tRH abundance was reduced, and relative abundance of individual 5' tRHs was altered. In hepatitis B-associated HCC, 5' tRH abundance correlated with expression of the tRNA-cleaving ribonuclease, angiogenin. These results demonstrate that tRHs are the most abundant small RNAs in chronically infected liver and that their abundance is altered in liver cancer.

Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-α via TLR7 signaling when cocultured with infected cells. pDCs required either close contact with infected cells or exposure to concentrated culture supernatants for IFN-α production. In isopycnic and rate-zonal gradients, pDC-activating material cosedimented with eHAV but not membrane-bound acetylcholinesterase, suggesting that eHAV, and not viral RNA exosomes, is responsible for IFN-α induction. pDC activation did not require virus replication and was associated with efficient eHAV uptake, which was facilitated by phosphatidylserine receptors on pDCs. In chimpanzees, pDCs were transiently recruited to the liver early in infection, during or shortly before maximal intrahepatic IFN-stimulated gene expression, but disappeared prior to inflammation onset. Our data reveal that, while membrane envelopment protects HAV against neutralizing antibody, it also facilitates an early but limited detection of HAV infection by pDCs.

Hepatitis C virus (HCV) poses a worldwide health problem in that the majority of individuals exposed to HCV become chronically infected and are predisposed for developing significant liver disease. DNA microarray technology provides an opportunity to survey transcription modulation in the context of an infectious disease and is a particularly attractive approach in characterizing HCV-host interactions, since the mechanisms underlying viral persistence and disease progression are not understood and are difficult to study. Here, we describe the changes in liver gene expression during the course of an acute-resolving HCV infection in a chimpanzee. Clearance of viremia in this animal occurred between weeks 6 and 8, while clearance of residual infected hepatocytes did not occur until 14 weeks postinfection. The most notable changes in gene expression occurred in numerous interferon response genes (including all three classical interferon antiviral pathways) that increased dramatically, some as early as day 2 postinfection. The data suggest a biphasic mechanism of viral clearance dependent on both the innate and adaptive immune responses and provide insight into the response of the liver to a hepatotropic viral infection.

The chimpanzee (Pan troglodytes) is the only experimental animal susceptible to infection with hepatitis C virus (HCV). The chimpanzee model of HCV infection was instrumental in the initial studies on non-A, non-B hepatitis, including observations on the clinical course of infection, determination of the physical properties of the virus, and eventual cloning of the HCV nucleic acid. This review focuses on more recent aspects of the use of the chimpanzee in HCV research. The chimpanzee model has been critical for the analysis of early events in HCV infection because it represents a population for which samples are available from the time of exposure and all exposed animals are examined. For this reason, the chimpanzee represents a truly nonselected population. In contrast, human cohorts are often selected for disease status or antibody reactivity and typically include individuals that have been infected for decades. The chimpanzee model is essential to an improved understanding of the factors involved in viral clearance, analysis of the immune response to infection, and the development of vaccines. The development of infectious cDNA clones of HCV was dependent on the use of chimpanzees, and they will continue to be needed in the use of reverse genetics to evaluate critical sequences for viral replication. In addition, chimpanzees have been used in conjunction with DNA microarray technology to probe the entire spectrum of changes in liver gene expression during the course of HCV infection. The chimpanzee will continue to provide a critical aspect to the understanding of HCV disease and the development of therapeutic modalities.

We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.

Elevated iron levels have been associated with raised serum alanine transaminase (ALT) levels in hepatitis C virus (HCV)-infected humans. However, it is not clear if HCV infection causes increased iron accumulation by the liver or if the severity of HCV infection is actually worsened by higher iron levels in the host. To better understand the relationship between iron and persistent HCV infections, we examined the effect of excess dietary iron on disease severity in HCV-infected chimpanzees. Iron was supplemented in the diets of four HCV-infected and two uninfected chimpanzees for 29 weeks to achieve iron loading. Iron loading was confirmed by increases in serum iron levels, percentages of transferrin saturation, ferritin levels, elevations in hepatic iron concentration (HIC), and by histological examination. The majority of HCV-infected chimpanzees had higher iron levels before iron feeding than the uninfected animals. Although various degrees of iron loading occurred in all chimpanzees, HCV-infected animals exhibited increased loading in comparison with uninfected animals. The effects of iron loading on HCV disease expression was determined by comparing disease parameters during an extended baseline period before iron loading with the period during iron loading and immediately following iron loading. Iron loading did not influence the viral load, but did exacerbate liver injury in HCV-infected chimpanzees, as evidenced by elevated ALT and histological changes. Because all chimpanzees on high iron diets experienced iron loading, but pathological effects were only observed in HCV-infected chimpanzees, HCV infection appears to increase the susceptibility of the liver to injury following iron loading. These results confirm and extend previous observations made in human populations and serve to further validate the chimpanzee model of chronic hepatitis C.

Hepatitis B virus (HBV) infections are a major worldwide health problem with chronic infections leading to cirrhosis and liver cancer. Viruses related to human HBV have been isolated from birds and rodents, but despite efforts to find hepadnaviruses that infect species intermediate in evolution between rodents and humans, none have been described. We recently isolated a hepadnavirus from a woolly monkey ( Lagothrix lagotricha ) that was suffering from fulminant hepatitis. Phylogenetic analysis of the nucleotide sequences of the core and surface genes indicated that the virus was distinct from the human HBV family, and because it is basal (ancestral) to the human monophyletic group, it probably represents a progenitor of the human viruses. This virus was designated woolly monkey hepatitis B virus (WMHBV). Analysis of woolly monkey colonies at five zoos indicated that WMHBV infections occurred in most of the animals at the Louisville zoo but not at four other zoos in the United States. The host range of WMHBV was examined by inoculation of one chimpanzee and two black-handed spider monkeys ( Ateles geoffroyi ), the closest nonendangered relative of the woolly monkey. The data suggest that spider monkeys are susceptible to infection with WMHBV and that minimal replication was observed in a chimpanzee. Thus, we have isolated a hepadnavirus with a host intermediate between humans and rodents and establishes a new animal model for evaluation of antiviral therapies for treating HBV chronic infections.

The clinical course of hepatitis C virus (HCV) infections in a chimpanzee cohort was examined to better characterize the outcome of this valuable animal model. Results of a cross-sectional study revealed that a low percentage (39%) of HCV-inoculated chimpanzees were viremic based on reverse transcription (RT-PCR) analysis. A correlation was observed between viremia and the presence of anti-HCV antibodies. The pattern of antibodies was dissimilar among viremic chimpanzees and chimpanzees that cleared the virus. Viremic chimpanzees had a higher prevalence of antibody reactivity to NS3, NS4, and NS5. Since an unexpectedly low percentage of chimpanzees were persistently infected with HCV, a longitudinal analysis of the virological profile of a small panel of HCV-infected chimpanzees was performed to determine the kinetics of viral clearance and loss of antibody. This study also revealed that a low percentage (33%) of HCV-inoculated chimpanzees were persistently viremic. Analysis of serial bleeds from six HCV-infected animals revealed four different clinical profiles. Viral clearance with either gradual or rapid loss of anti-HCV antibody was observed in four animals within 5 months postinoculation. A chronic-carrier profile characterized by persistent HCV RNA and anti-HCV antibody was observed in two animals. One of these chimpanzees was RT-PCR positive, antibody negative for 5 years and thus represented a silent carrier. If extrapolated to the human population, these data would imply that a significant percentage of unrecognized HCV infections may occur and that silent carriers may represent potentially infectious blood donors.

Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy.

Hepatitis B virus (HBV) capsid proteins, termed core proteins, with two- to four-amino-acid insertions were assessed for capsid formation, RNA encapsidation, and the ability to support reverse transcription of the pregenome by the polymerase molecule. Velocity sedimentation analysis of insect cell-expressed recombinant core proteins revealed that only two of the nine insertion mutant proteins formed capsids with the tight banding patterns of wild-type capsids. The remaining mutant core proteins were spread over the gradients, suggesting aggregate formation, or at the top of the gradients, suggesting lack of stable capsid formation. The mutant capsid proteins were coexpressed in Huh7 cells with an HBV genome lacking a functional core gene to test for trans complementation of HBV replication. Three of the mutant core proteins formed capsids containing HBV RNA, but only two of these contained reverse-transcribed HBV DNA. While the core protein has shown resiliency in capsid formation following insertion of foreign residues into the major B-cell epitope, several of the small insertions severely reduced the efficiency of capsid formation and inhibited capsid function.

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virus-specific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses ('eHAV') resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between 'enveloped' and 'non-enveloped' viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate., Author Summary While viruses that target the liver often cause lengthy infections with considerable morbidity, there is limited understanding of how they evade host responses. We have studied hepatitis A virus (HAV), an important cause of acute hepatitis in humans. Although HAV infection typically results in hepatic inflammation, there is no disease in the liver during the first weeks of infection despite robust virus replication. This suggests that HAV either fails to stimulate or efficiently evades recognition by host innate immune sensors. Our prior work showed HAV disrupts RIG-I/MDA5 signaling by targeting MAVS, an essential adaptor protein, for degradation by 3ABC, a precursor of the only HAV protease, 3Cpro. Here, we show here that a distinct viral processing intermediate, the 3CD protease-polymerase, disrupts TLR3 signaling by degrading its adaptor protein, TRIF. HAV has evolved a novel strategy to target two different host adaptor proteins with a single protease, using its 3Dpol RNA polymerase to modify the substrate specificity of its 3Cpro protease when fused to it in the 3CD precursor, thus allowing it to target non-canonical 3Cpro recognition sequences in TRIF. This remarkable example of viral adaptation allows the virus to target two different host adaptor proteins with a single viral protease.

Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1–2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3–4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10–20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus–host interactions within the liver.

Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzar's solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1'-dioctadecyl-3,3,3,3'-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration.(ABSTRACT TRUNCATED AT 250 WORDS)

An ELISA was developed for measuring serum antibodies against the arenavirus lymphocytic choriomeningitis virus (LCMV) and a closely related isolate termed callitrichid hepatitis virus (CHV). The ELISA was used to test sera from healthy adults and from hepatitis patients. In Birmingham, Alabama, the seropositivity rate for healthy black women was 5.1% (7/138), and the rate for patients with all types of hepatitis or cirrhosis was 4.3% (2/46). In San Antonio, Texas, the seropositivity rate among a clinical series of patients with non-A, non-B hepatitis was 0 (0/20), and the rate among persons rejected from blood donation because of high serum alanine aminotransferase levels was 2.4% (2/82). These results indicate that infection with LCMV or CHV is common in Birmingham but that infection is not associated with hepatitis.

Dramatic progress is being made toward the development of less-toxic and simpler alternatives to the current standard-of-care therapy for chronic hepatitis C, which involves a combination of pegylated interferon (peg-IFN) and ribavirin (RBV). Several accessible viral targets have been identified and licensure of the most advanced clinical compounds can be anticipated within the next several years. However, the highly replicative nature of HCV infection, coupled with error-prone viral RNA synthesis and considerable genome diversity, pose extraordinary challenges to drug development. Peg-IFN is likely to remain a mainstay of therapy for the foreseeable future, or until such time that multiple direct-acting antiviral (STAT-C) inhibitors are available and shown to provide a sufficiently high barrier to resistance when used in combination.

During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks ( Marmota monax ) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102: 1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from ∼35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (∼1 mg) containing, in total, ∼3 × 10 7 liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, ∼30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.

Three human yolk sac carcinoma cell lines were characterized for the expression of several markers. Each of the cell lines expressed alpha-fetoprotein, without detectable levels of chorionic gonadotropin, and the level of alpha-fetoprotein expression increased dramatically when the cultures were held without passage for extended periods. The secretion of a number of plasma proteins was documented by metabolic labeling, immunoprecipitation, and gel analysis. The major plasma proteins detected were alpha-1-antitrypsin, alpha-fetoprotein, transthyretin, beta-2 microglobulin, and plasminogen, with lower levels of transferrin and complement C4 released. Apolipoproteins B, E, and A1 were secreted in high levels as well and were found in the form of lipoprotein particles. Time course experiments on the synthesis of apolipoproteins E and A1 indicated that, as with alpha-fetoprotein, the level of synthesis increased substantially when the cultures were held without passage. The results indicate that these yolk sac carcinoma cells display a protein expression profile similar to that observed for the human yolk sac, and the possibility that the cells may have the potential to differentiate is discussed.

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.

Polyclonal antibodies were used to assay human embryonal carcinoma (EC), differentiating EC, yolk sac carcinoma, and teratoma cells for expression of viral early antigen (T-Ag) after infection with simian virus 40 (SV40). Cells of four EC lines were induced to differentiate by cultivation at low density or by exposure to retinoic acid or dimethyl sulfoxide. After infection with SV40, T-Ag was expressed by 1%, or less, of the cells (presumed to be differentiated derivatives) in only some EC cultures whereas the antigen was synthesized by a significant percentage of the yolk sac carcinoma, teratoma, and differentiating EC cells. Also, viral late proteins were produced by EC cells infected with human adenovirus type 7 (Ad7), and SV40 T-Ag was expressed by EC cells after infection with PARA, which is an Ad7-SV40 hybrid virus containing the SV40 T-Ag sequence controlled by Ad7 late regulatory sequences. Thus, T-Ag is not synthesized by the parental EC cells infected with SV40, but it is expressed in cultures of infected differentiated derivatives. The EC cells produce T-Ag, however, when expression of the viral protein is controlled by the Ad7 regulatory sequences in PARA particles. These results demonstrate that expression of T-Ag after infection with SV40 is an indicator of EC cell differentiation and also raise the possibility that, as in mouse EC cells infected with the virus, the SV40 regulatory sequences controlling T-Ag synthesis are not active in human EC cells.

Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.

Recent studies in humans and chimpanzees suggest that immunity can be induced to diminish the incidence of chronic hepatitis C virus (HCV) infection. However, the immunity that promotes viral recovery is poorly understood, and whether the breadth of this adaptive immunity is sufficient to overcome the substantial intergenotype antigenic diversity represents a final obstacle to demonstrating the feasibility of vaccine development. Here we demonstrate that recovery from a genotype 1 HCV infection protects chimpanzees against infection with representatives of other genotypes that exhibit up to 30% divergence at the amino acid level, including challenges with genotype 4, a mixture of genotypes 2 and 3, and a complex inoculum containing genotypes 1, 2, 3, and 4. In each instance, the level and duration of viremia were markedly reduced in comparison to the primary infection in the same animal. The data indicate that epitopes conserved between genotypes must play an essential role in immunity. The inocula used in the rechallenge studies induced typical primary infection profiles in naïve chimpanzees. Rechallenge infections were associated with rapid increases in the intrahepatic transcripts of interferon-stimulated genes, even in animals exhibiting apparent sterilizing immunity. Protective immunity was often associated with an early increase in gamma interferon transcripts in the liver and increases in intrahepatic transcripts of Mig, a T-cell chemokine that is a gamma interferon response gene. These studies are the first to show that cross-genotype immunity can be induced to HCV, demonstrating the feasibility of developing a vaccine protective against all HCV strains.

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.

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex–single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.

The recent isolation of a nonhuman primate hepadnavirus from woolly monkeys prompted an examination of other primates for potentially new hepadnaviruses. A serological analysis of 30 captive gibbons revealed that 47% were positive for at least one marker of ongoing or previous infection with a hepatitis B virus (HBV). The amino acid sequences of the core and surface genes of human and gibbon virus isolates were very similar. Phylogenetic analysis indicated that the gibbon isolates lie within the human HBV family, indicating that these HBV isolates most likely stem from infection of gibbons from a human source.

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.

The relationship of viral persistence, the immune response to hepatitis C virus (HCV) envelope proteins, and envelope sequence variability was examined in chimpanzees. Antibody reactivity to the HCV envelope proteins E1 or E2 was detected by enzyme-linked immunosorbent assay (ELISA) in more than 90% of a human serum panel. Although the ELISAs appeared to be sensitive indicators of HCV infection in human serum panels, the results of a cross-sectional study revealed that a low percentage of HCV-inoculated chimpanzees had detectable antibody to E1 (22%) and E2 (15%). Viral clearance, which was recognized in 28 (61%) of the chimpanzees, was not associated with an antibody response to E1 or E2. On the contrary, antibody to E2 was observed only in viremic chimpanzees. A longitudinal study of animals that cleared the viral infection or became chronically infected confirmed the low level of antibody to E1, E2, and the HVR-1. In 10 chronically infected animals, the sequence variation in the E2 hypervariable region (HVR-1) was minimal and did not coincide with antibody to E2 or to the HVR-1. In addition, low nucleotide and amino acid sequence variation was observed in the E1 and E2 regions from two chronically infected chimpanzees. These results suggest that mechanisms in addition to the emergence of HVR-1 antibody escape variants are involved in maintaining viral persistence. The significance of antibodies to E1 and E2 in the chimpanzee animal model is discussed.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. We recently expressed human hepatitis B virus (HBV) reverse transcriptase (pol) in insect cells by using the recombinant baculovirus system. The purified protein is active in nucleotide priming and reverse transcription reactions. In this report, we demonstrate that the tyrosine residue at amino acid number 63 within the TP (terminal protein) domain of the polymerase is the site of covalent linkage of the first nucleotide of minus-strand DNA. Analysis of pol polypeptides with mutations in the TP and RT (reverse transcriptase) domains indicated that both domains were required for in vitro nucleotide priming activity. Polymerase proteins with mutations in the TP and RT domains were not capable of complementing each other in the nucleotide priming reaction, suggesting that transcomplementation between full-length polypeptides was not possible. However, when the TP and RT domains were expressed as separate polypeptides, they formed a highly stable complex that was active in nucleotide priming and reverse transcription. The presence of an epsilon stem-loop dramatically increased the nucleotide priming activity in transcomplementation assays, even though full-length pol displayed similar activities in the absence and presence of epsilon. These data raise the possibility that in the transcomplementation assay, epsilon may play a role in the formation of a functional complex between TP and RT, rather than being required only as the template for nucleotide priming. The results indicate that using the baculovirus system, it is possible to dissect the protein-protein and protein-RNA interactions required for HBV genome replication.

Historically, hepatitis B virus (HBV) has been considered species specific and unable to infect baboons. Based on this premise, two patients with HBV end-stage liver disease underwent baboon liver xenotransplantation. To study whether baboons are susceptible to HBV infection, four baboons (two receiving immunosuppressive therapy) were inoculated with HBV. Animals were followed for 6 months: clinical examinations and biochemical studies were normal, hepatitis B surface antigen and hepatitis B core antigen staining of biopsies was negative, and HBV serology remained negative. HBV polymerase chain reaction was transiently positive in one animal, which most likely reflects the initial inoculation. This pilot study corroborates historical evidence and beliefs that baboons are resistant to HBV.