Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5'UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5' end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.

Equine Mesenchymal Stromal Cells from Different Sources Efficiently Differentiate into Hepatocyte-Like Cells.

Adult equine hepatocytes have proven challenging to culture long term in vitro as they rapidly lose their morphology and functionality, thus limiting studies on liver function and response to disease. In this study, we describe for the first time the differentiation of equine mesenchymal stromal cells (MSC) from a variety of sources into functional hepatocyte-like cells (HLC). First, we differentiated equine umbilical cord blood (UCB)-derived MSC into HLC and found that these cells exhibited a distinct polygonal morphology, stored glycogen as visualized by periodic acid Schiff's reagent staining, and were positive for albumin and other hepatocyte-specific genes. Second, we demonstrated that UCB-HLC could be revived following cryopreservation and retained their phenotype for at least 10 days. Third, we differentiated three sets of MSC from bone marrow (BM), adipose tissue (AT), and peripheral blood (PB), matched within the same horse. We achieved a 100% differentiation success rate with BM, 0% with AT, and 66% with PB. An additional set of nine PB-MSC samples resulted in an overall success rate of 42% (n = 12), and age or gender did not seem to have an effect on the success of hepatic differentiation from that source. In a final set of experiments, we evaluated the use of these HLC as tools in different fields of biomedical research like virology, to study viral growth, and toxicology, to study chemicals with hepatic toxicity. Equine HLC were found susceptible for infection with the equine herpesviruses type 1 (EHV-1), -2, and -5, and exhibited a more sensitive dose-dependent response to arsenic toxicity than the commonly used human hepatocellular cell line HepG2. Taken together, these data indicate that equine MSC can be efficiently differentiated into HLC and these equine HLC could be a useful tool for in vitro studies.

AIC649 Induces a Bi-Phasic Treatment Response in the Woodchuck Model of Chronic Hepatitis B.

AIC649 has been shown to directly address the antigen presenting cell arm of the host immune defense leading to a regulated cytokine release and activation of T cell responses. In the present study we analyzed the antiviral efficacy of AIC649 as well as its potential to induce functional cure in animal models for chronic hepatitis B. Hepatitis B virus transgenic mice and chronically woodchuck hepatitis virus (WHV) infected woodchucks were treated with AIC649, respectively. In the mouse system AIC649 decreased the hepatitis B virus titer as effective as the "gold standard", Tenofovir. Interestingly, AIC649-treated chronically WHV infected woodchucks displayed a bi-phasic pattern of response: The marker for functional cure--hepatitis surface antigen--first increased but subsequently decreased even after cessation of treatment to significantly reduced levels. We hypothesize that the observed bi-phasic response pattern to AIC649 treatment reflects a physiologically "concerted", reconstituted immune response against WHV and therefore may indicate a potential for inducing functional cure in HBV-infected patients.

Sustained efficacy and seroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuck model of chronic hepatitis B.

BACKGROUND & AIMS: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). METHODS: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n = 7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. RESULTS: GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8(+) T cell, NK cell, B cell and interferon response transcriptional signatures. CONCLUSIONS: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients.

Capture of syncytin-Mar1, a fusogenic endogenous retroviral envelope gene involved in placentation in the Rodentia squirrel-related clade.

Syncytin genes are fusogenic envelope protein (env) genes of retroviral origin that have been captured for a function in placentation. Within rodents, two such genes have previously been identified in the mouse-related clade, allowing a demonstration of their essential role via knockout mice. Here, we searched for similar genes in a second major clade of the Rodentia order, the squirrel-related clade, taking advantage of the complete sequencing of the ground squirrel Ictidomys tridecemlineatus genome. In silico search for env genes with full coding capacity identified several candidate genes with one displaying placenta-specific expression, as revealed by quantitative reverse transcription-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with recognizable hallmarks of an integrated provirus. Cloning of the gene in an expression vector for ex vivo cell-cell fusion and pseudotype assays demonstrated fusogenicity on a large panel of mammalian cells. In situ hybridization on placenta sections showed specific expression in domains where trophoblast cells fuse into a syncytiotrophoblast at the fetomaternal interface, consistent with a role in syncytium formation. Finally, we show that the gene is conserved among the tribe Marmotini, thus dating its capture back to about at least 25 million years ago, with evidence for purifying selection and conservation of fusogenic activity. This gene that we named syncytin-Mar1 is distinct from all seven Syncytin genes identified to date in eutherian mammals and is likely to be a major effector of placentation in its related clade. Importance: Syncytin genes are fusogenic envelope genes of retroviral origin, ancestrally captured for a function in placentation. Within rodents, two such genes had been previously identified in the mouse-related clade. Here, in the squirrel-related rodent clade, we identified the envelope gene of an endogenous retrovirus with all the features of a Syncytin: it is specifically expressed in the placenta of the woodchuck Marmota monax, at the level of cells fusing into a syncytium; it can trigger cell-cell and virus-cell fusion ex vivo; and it has been conserved for >25 million years of evolution, suggesting an essential role in its host physiology. Remarkably, syncytin-Mar1 is unrelated to all other Syncytin genes identified thus far in mammals (primates, muroids, carnivores, and ruminants). These results extend the range of retroviral envelope gene "domestication" in mammals and show that these events occurred independently, on multiple occasions during evolution to improve placental development in a process of convergent evolution.

Glucosidase inhibition enhances presentation of de-N-glycosylated hepatitis B virus epitopes by major histocompatibility complex class I in vitro and in woodchucks.

UNLABELLED: In this report, the possibility of pharmacologically altering the hepatitis B virus (HBV) epitopes presented by major histocompatibility complex class I on infected cells is demonstrated. The HBV middle envelope glycoprotein (MHBs) maturation appears to require calnexin-mediated folding. This interaction is dependent on glucosidases in the endoplasmic reticulum. Prevention of HBV envelope protein maturation in cultured cells through use of glucosidase inhibitors, such as 6-O-butanoyl castanospermine and N-nonyl deoxynorjirimycin, resulted in MHBs degradation by proteasomes. The de-N-glycosylation associated with polypeptide degradation was predicted to result in conversion of asparagine residues into aspartic acid residues. This prediction was confirmed by showing that peptides corresponding to the N-glycosylation sequons of MHBs, but with aspartic acid replacing asparagine, (1) can prime human cytotoxic T lymphocytes that recognize HBV-producing cells and (2) that the presentation of these envelope motifs by major histocompatibility complex class I is enhanced by incubation with glucosidase inhibitors. Moreover, although peripheral blood mononuclear cells isolated from woodchucks chronically infected with woodchuck hepatitis virus and vaccinated with woodchuck hepatitis virus surface antigen could be induced to recognize the natural MHBs asparagine-containing peptides, only cells isolated from animals treated with glucosidase inhibitor recognized the aspartic acid-containing peptides. CONCLUSION: These data suggest that pharmacological intervention with glucosidase inhibitors can alter the MHBs epitopes presented. This editing of the amino acid sequence of the polypeptide results in a new epitope, or "editope", with possible medical significance.

Semliki forest virus expressing interleukin-12 induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma.

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with >or=1.5-log(10) reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.

Rapid immunity to vaccination with woodchuck hepatitis virus surface antigen using cationic liposome-DNA complexes as adjuvant.

Complexes of cationic liposomes and non-coding DNA (CLDC) have shown promise as vaccine adjuvant. Using the woodchuck animal model of hepatitis B virus (HBV) infection, the immunogenic effects of CLDC were evaluated following vaccination with three doses of woodchuck hepatitis virus surface antigen (WHsAg) adjuvanted with either CLDC or conventional alum and administered intramuscularly (im) or subcutaneously (sc). IM vaccination with WHsAg and CLDC elicited antibodies earlier, in more woodchucks, and with higher titers than WHsAg and alum. After two vaccine doses, antibody titers were higher following im than sc administration. Woodchucks administered two vaccine doses sc received the third vaccine dose im, and antibody responses reached titers comparable to those elicited by im administration. Following the second vaccine dose, im vaccination with WHsAg and CLDC induced T cell responses to WHsAg and selected WHs peptides and expression of the leukocyte surface marker CD8 and of the Th1 cytokines interferon-gamma and tumor necrosis factor alpha in woodchucks. T cell responses and CD8/cytokine expression were diminished in woodchucks from the other groups suggesting that this vaccine regimen induced a skew toward Th1 immune responses. The present study in woodchucks demonstrates that CLDC-adjuvanted WHsAg vaccine administered im resulted in a more rapid induction of humoral and cellular immune responses compared to conventional, alum-adjuvanted WHsAg vaccine. While less rapid, the immune responses following sc administration can prime the im immune responses. This adjuvant activity of CLDC over alum may be beneficial for therapeutic vaccination in chronic HBV infection.

Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular carcinoma contributes to the malignant phenotype.

Alterations in microRNA (miRNA) expression in both human and animal models have been linked to many forms of cancer. Such miRNAs, which act directly as repressors of gene expression, have been found to frequently reside in fragile sites and genomic regions associated with cancer. This study describes a miRNA signature for human primary hepatitis B virus-positive human hepatocellular carcinoma. Moreover, two known oncomiRs--miRNAs with known roles in cancer--the miR-17-92 polycistron and miR-21, exhibited increased expression in 100% of primary human and woodchuck hepatocellular carcinomas surveyed. To determine the importance of these miRNAs in tumorigenesis, an in vitro antisense oligonucleotide knockdown model was evaluated for its ability to reverse the malignant phenotype. Both in human and woodchuck HCC cell lines, separate treatments with antisense oligonucleotides specific for either the miR-17-92 polycistron (all six members) or miR-21 caused a 50% reduction in both hepatocyte proliferation and anchorage-independent growth. The combination of assays presented here supports a role for these miRNAs in the maintenance of the malignant transformation of hepatocytes.

Natural iminosugar derivatives of 1-deoxynojirimycin inhibit glycosylation of hepatitis viral envelope proteins.

A silkworm (Bombyx mori L.) extract known to contain naturally occurring iminosugars, including 1-deoxynojirimycin (1-DNJ) derived from the mulberry tree (Morus alba L.), was evaluated in surrogate HCV and HBV in vitro assays. Antiviral activity of the silkworm extract and one of its purified constituents, 1-DNJ, was demonstrated against bovine viral diarrhea virus (BVDV) and GB virus-B (GBV-B), both members of the Flaviviridae family, and against woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV), both members of the Hepadnaviridae family of viruses. The silkworm extract exhibited a 1,300 fold greater antiviral effect against BVDV in comparison to purified 1-DNJ. Glycoprotein processing of BVDV envelope proteins was disrupted upon treatment with the naturally derived components. The glycosylation of the WHV envelope proteins was affected largely by treatment with the silkworm extract than with purified 1-DNJ as well. The mechanism of action for this therapy may lie in the generation of defective particles that are unable to initiate the next cycle of infection as demonstrated by inhibition of GBV-B in vitro. We postulate that the five constituent iminosugars present in the silkworm extract contribute, in a synergistic manner, toward the antiviral effects observed for the inhibition of intact maturation of hepatitis viral particles and may complement conventional therapies. These results indicate that pre-clinical testing of the natural silkworm extract with regards to the efficacy of treatment against viral hepatitis infections can be evaluated in the respective animal models, in preparation for clinical trials in humans.

The role of the woodchuck model in the treatment of hepatitis B virus infection.

Experimental studies of animals with chronic hepadnavirus infection could provide valuable insight into optimal therapeutic strategies for individuals with chronic HBV infection. In this review, we focus on the contributions of the woodchuck model to our understanding of HBV biology and on its role in the development of antiviral drug. Furthermore, we consider the implications of studies focusing on the natural history of WHV infection for the management of HBV and the capacity of treatment to prevent complications of chronic hepatitis B infection.

Chemoimmunotherapy of chronic hepatitis B virus infection in the woodchuck model overcomes immunologic tolerance and restores T-cell responses to pre-S and S regions of the viral envelope protein.

Treatment of chronic hepatitis B virus (HBV) infection could combine potent antiviral drugs and therapeutic vaccines to overcome immunological tolerance and induce the recovery phenotype to protect against disease progression. Conventional vaccination of woodchucks chronically infected with the woodchuck hepatitis virus (WHV) elicited differential T-cell response profiles depending on whether or not carriers were treated with the potent antiviral drug clevudine (CLV), which significantly reduces viral and antigen loads. The differential T-cell responses defined both CLV-dependent and CLV-independent epitopes of the pre-S and S regions of the WHV envelope protein. Only combined treatment involving CLV and conventional vaccine therapeutically restored the T-cell response profile of chronic WHV carrier woodchucks to that seen in prophylactic vaccination and in recovery from acute WHV infection. The results have implications for mechanisms of immunological tolerance operating in chronic HBV infection and suggest that such combined chemoimmunotherapy may be useful for treatment of humans with chronic HBV infection.

Woodchuck dendritic cells generated from peripheral blood mononuclear cells and transduced with recombinant human adenovirus serotype 5 induce antigen-specific cellular immune responses.

Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines.

Quantitative evaluation of 2-deoxy-2[F-18]fluoro-D-glucose-positron emission tomography imaging on the woodchuck model of hepatocellular carcinoma with histological correlation.

PURPOSE: The Eastern woodchuck (Marmota monax) is considered as a naturally occurring animal model of hepatocellular carcinoma (HCC). The performance of 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) for imaging HCC on the woodchuck using Positron emission tomography (PET) was investigated in this study. PROCEDURES: Dynamic FDG-PET scans were performed on five woodchucks with HCC and one healthy woodchuck before removal and processing of the liver tissues for histology. The parameters of a two-tissue compartment model with dual input were estimated using weighted least squares (WLS). RESULTS: Ten HCCs were confirmed histologically. Six HCCs had a tumor-to-liver standardized uptake value (SUV) ratio < or =1.15, a k (4) / k (3) ratio similar to that in hepatic tissues and were well-differentiated. Four HCCs had a tumor-to-liver SUV ratio >1.15, a lower k (4) / k (3) ratio than the hepatic tissues and were moderately differentiated. CONCLUSIONS: Increased FDG uptake was observed in HCCs that were the least differentiated and correlated with a lower k (4) / k (3) ratio.

Gene expression studies of hepatitis virus-induced woodchuck hepatocellular carcinoma in correlation with human results.

The lack of good molecular markers for diagnosis as well as treatment assessment has rendered the hepatocellular carcinoma (HCC) a major challenge in health care. In this study, woodchucks were used as an animal model for hepatitis virus-induced HCC, and gene expression studies were performed using a human oligonucleotide microarray. An analysis approach combing supervised significant analysis of microarray (SAM), prediction analysis of microarray (PAM), and unsupervised hierarchical cluster methodologies statistically determined 211 upregulated and 78 downregulated genes between liver cancer and non-cancer liver tissues, and demonstrated > or = 93% accuracy in classifying the tissue samples. RT-PCR results confirmed the differential expression of selected sequenced woodchuck genes (SAT, IDH3B, SCD) in the microarray. Our study showed that differentially expressed genes were involved in transcription, RNA splicing, translation, cell cycle, metabolism, protein folding and degradation, apoptosis, immune response, metal binding, etc. Interestingly, some genes were involved with signaling pathways such as Ras/MAPK (MAPKAP1), Src-dependent pathways (CSK), hedgehog signaling pathway (HHIP), while Wnt signaling pathway may not be dominant in woodchuck HCC as shown by the downregulation of beta-catenin (TNNB1) and the upregulation of CXXC4 and CSNK2B. Numerous genes found in this study were also differentially expressed in human HCC and many other human cancers including breast, prostate and lung cancers, etc., serving as tumor suppressors, promoters, prognostic markers or chemotherapy targets. In conclusion, this study has demonstrated the robustness of the data analysis and the potential of using human microarrays on woodchuck samples. In particular, some of the differentially expressed genes in the woodchuck HCC can be further explored for possible molecular imaging targets or biological markers in human HCC.

Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays.

AIM: To demonstrate the feasibility of using woodchuck samples on human microarrays, to provide insight into pathways involving positron emission tomography (PET) imaging tracers and to identify genes that could be potential molecular imaging targets for woodchuck hepatocellular carcinoma. METHODS: Labeled cRNA from woodchuck tissue samples were hybridized to Affymetrix U133 plus 2.0 GeneChips. Ten genes were selected for validation using quantitative RT-PCR and literature review was made. RESULTS: Testis enhanced gene transcript (BAX Inhibitor 1), alpha-fetoprotein, isocitrate dehydrogenase 3 (NAD+) beta, acetyl-CoA synthetase 2, carnitine palmitoyltransferase 2, and N-myc2 were up-regulated and spermidine/spermine N1-acetyltransferase was down-regulated in the woodchuck HCC. We also found previously published results supporting 8 of the 10 most up-regulated genes and all 10 of the 10 most down-regulated genes. CONCLUSION: Many of our microarray results were validated using RT-PCR or literature search. Hence, we believe that woodchuck HCC and non-cancerous liver samples can be used on human microarrays to yield meaningful results.

Genetic changes in hepatitis delta virus from acutely and chronically infected woodchucks.

A woodchuck-derived hepatitis delta virus (HDV) inoculum was created by transfection of a genotype I HDV cDNA clone directly into the liver of a woodchuck that was chronically infected with woodchuck hepatitis virus. All woodchucks receiving this inoculum became positive for HDV RNA in serum, and 67% became chronically infected, similar to the rate of chronic HDV infection in humans. Analysis of HDV sequences obtained at 73 weeks postinfection indicated that changes had occurred at a rate of 0.5% per year; many of these modifications were consistent with editing by host RNA adenosine deaminase. The appearance of sequence changes, which were not evenly distributed on the genome, was correlated with the course of HDV infection. A limited number of modifications occurred in the consensus sequence of the viral genome that altered the sequence of the hepatitis delta antigen (HDAg). All chronically infected animals examined exhibited these changes 73 weeks following infection, but at earlier times, only one of the HDV carriers exhibited consensus sequence substitutions. On the other hand, sequence modifications in animals that eventually recovered from HDV infection were apparent after 27 weeks. The data are consistent with a model in which HDV sequence changes are selected by host immune responses. Chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.

Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core alpha-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.

Hepatocellular carcinoma in the woodchuck model of hepatitis B virus infection.

The Eastern woodchuck ( Marmota monax ) harbors a DNA virus (Woodchuck hepatitis virus [WHV]) that is similar in structure and replicative life cycle to the human hepatitis B virus (HBV). Like HBV, WHV infects the liver and can cause acute and chronic hepatitis. Furthermore, chronic WHV infection in woodchucks usually leads to development of hepatocellular carcinoma (HCC) within the first 2-4 years of life. The woodchuck model has been important in the preclinical evaluation of safety and efficacy of the antiviral drugs now in use for treatment of HBV infection and continues to serve as an important, predictive model for innovative forms of therapy of hepatitis B using antiviral nucleosides and immune response modifiers alone or in combination. Almost all woodchucks that become chronic WHV carriers after experimental neonatal inoculation develop HCC with a median HCC-free survival of 24 months and a median life expectancy of 30-32 months. The woodchuck model of viral-induced HCC has been used effectively for the development of new imaging agents for enhancement of detection of hepatic neoplasms by ultrasound and magnetic resonance imaging. The chemoprevention of HCC using long-term antiviral nucleoside therapy has been shown in the woodchuck, and "proof of principal" has been established for some of the innovative, molecular methods for treatment of HCC. The model is available for fundamental investigations of the viral and molecular mechanisms responsible for hepatocarcinogenesis and should have substantial value for future development of innovative methods for chemoprevention and gene therapy of human HCC.