• chronic hepatitis b (chb)
• hepatitis b virus (hbv) surface antigen (hbsag)
• dihydropyridophthalazinone (dpp)
• papd5
• papd7

• Disease models
• Hepatitis B Virus
• Hepatocellular carcinoma
• Liver organoids
• Patient-derived organoids

• HBV
• HBx
• HCC
• chronic infection
• host factors
• pathogenesis
• signaling

• cancer
• cat
• hepadnavirus
• hepatitis
• hepatitis B
• liver
• viral

• Cats
• Animals
• Humans
• Hong Kong/epidemiology
• Phylogeny
• Hepadnaviridae/genetics
• Molecular Epidemiology
• Risk Factors

• Marmota monax
• activation of T cells
• chronic hepatitis B
• immune cell subsets
• occult hepadnaviral infection
• programed cell death protein and ligand
• woodchuck model of hepatitis B

• HBV
• IFN
• IFN-α
• ISGs
• Peg-IFN-α

Infection with hepatitis B virus (HBV) is responsible for the increasing global hepatitis burden, with an estimated 296 million people being carriers and living with the risk of developing chronic liver disease and cancer. While the current treatment options for chronic hepatitis B (CHB), including oral nucleos(t)ide analogs and systemic interferon-alpha, are deemed suboptimal, the path to finding an ultimate cure for this viral disease is rather challenging. The lack of suitable laboratory animal models that support HBV infection and associated liver disease progression is one of the major hurdles in antiviral drug development. For more than four decades, experimental infection of the Eastern woodchuck with woodchuck hepatitis virus has been applied for studying the immunopathogenesis of HBV and developing new antiviral therapeutics against CHB. There are several advantages to this animal model that are beneficial for performing both basic and translational HBV research. Previous review articles have focused on the value of this animal model in regard to HBV replication, pathogenesis, and immune response. In this article, we review studies of drug development and preclinical evaluation of direct-acting antivirals, immunomodulators, therapeutic vaccines, and inhibitors of viral entry, gene expression, and antigen release in the woodchuck model of CHB since 2014 until today and discuss their significance for clinical trials in patients.

• Animals
• Antiviral Agents/pharmacology
• Antiviral Agents/therapeutic use
• Disease Models, Animal
• Drug Development
• Hepatitis B/drug therapy
• Hepatitis B virus
• Hepatitis B, Chronic
• Hepatitis C, Chronic/drug therapy
• Humans
• Marmota

• N01AI05399 NIAID NIH HHS
• HHSN272201000011I NIAID NIH HHS

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (⁷⁵NIT⁷⁷ and ⁹⁰NDT⁹²) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.

Hepadnaviruses infect a wide range of hosts. The human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV) are two closely related hepadnaviruses. In contrast to the WHV precore protein, which is N-glycosylated, the HBV precore protein lacks N-glycosylation. As precore and core proteins expressed from the overlapping precore/core genes share most of their sequences but have distinct functions, we investigated the roles of the N-linked glycosylation sequons in HBV and WHV precore/core genes. Our results revealed an important role of the N-linked glycosylation sequons in enhancing precore secretion levels and regulating core protein functions in virion secretion and infection. Furthermore, cross-species complementation assays using HBV and WHV core proteins and HBV or WHV genomes defective in core protein expression indicated that both HBV and WHV core proteins could support intracellular viral replication but not virion secretion of the heterologous virus. These results provide novel insights into the evolution of overlapping genes during host adaptation of hepadnaviruses.

• DNA, Circular
• DNA, Viral
• Genes, Overlapping
• Glycosylation
• Hepadnaviridae/genetics
• Hepatitis B/genetics
• Hepatitis B Virus, Woodchuck
• Hepatitis B virus/genetics
• Host Adaptation
• Humans
• Virus Replication/genetics

• R37 AI043453 NIAID NIH HHS
• National Institute of Arthritis and Musculoskeletal and Skin Diseases

• University Grants Commission

Chronic hepatitis and hepatocellular carcinoma (HCC) caused by the hepadnavirus hepatitis B virus (HBV) are significant causes of human mortality. A hepatitis-B-like virus infecting cats, domestic cat hepadnavirus (DCH), was reported in 2018. DCH DNA is hepatotropic and detectable in feline blood or serum (3.2 to 12.3%). Detection of HBV DNA has been reported in sera from 10% of free-roaming dogs in Brazil, whereas 6.3% of sera from dogs in Italy tested positive for DCH DNA by real-time quantitative PCR (qPCR). If DCH, HBV, or another hepadnavirus is hepatotropic in dogs, a role for such a virus in the etiology of canine idiopathic chronic hepatitis (CH) or HCC warrants investigation. This study investigated whether DCH DNA could be detected via qPCR in blood from dogs in Hong Kong and also whether liver biopsies from dogs with confirmed idiopathic CH or HCC contained hepadnaviral DNA using two panhepadnavirus conventional PCRs (cPCR) and a DCH-specific cPCR. DCH DNA was amplified from 2 of 501 (0.4%) canine whole-blood DNA samples. A second sample taken 6 or 7 months later from each dog tested negative in DCH qPCR. DNA extracted from 101 liver biopsies from dogs in Hong Kong or the USA, diagnosed by board-certified pathologists as idiopathic CH (n = 47) or HCC (n = 54), tested negative for DCH DNA and also tested negative using panhepadnavirus cPCRs. This study confirms that DCH DNA can be detected in canine blood by qPCR, although at a much lower prevalence than that reported previously. We identified no evidence to support a pathogenic role for a hepadnavirus in canine idiopathic CH or HCC.

• Hepadnaviridae
• carcinoma
• cats
• chronic
• dogs
• hepatitis
• hepatitis B virus
• hepatocellular

• Animals
• Biopsy
• Carcinoma, Hepatocellular/veterinary
• Cats
• DNA, Viral/genetics
• Dogs
• Hepadnaviridae/genetics
• Hepatitis B virus/genetics
• Hepatitis, Chronic
• Hong Kong
• Humans
• Liver Neoplasms

• Animal Studies
• Chemoembolization
• Interventional-Vascular
• Liver
• Molecular Imaging-Cancer
• Molecular Imaging-Clinical Translation

• Animals
• Carcinoma, Hepatocellular/diagnostic imaging
• Carcinoma, Hepatocellular/therapy
• Chemoembolization, Therapeutic
• Humans
• Liver Neoplasms/diagnostic imaging
• Liver Neoplasms/therapy
• Models, Animal
• Tomography, X-Ray Computed

Conventional treatment of chronic hepatitis B (CHB) is rarely curative due to the immunotolerant status of patients. RG7854 is an oral double prodrug of a toll-like receptor 7 (TLR7) agonist that is developed for the treatment of CHB. The therapeutic efficacy, host immune response, and safety of RG7854 were evaluated in the woodchuck model of CHB. Monotreatment with the two highest RG7854 doses and combination treatment with the highest RG7854 dose and entecavir (ETV) suppressed viral replication, led to loss of viral antigens, and induced seroconversion in responder woodchucks. Since viral suppression and high-titer antibodies persisted after treatment ended, this suggested that a sustained antiviral response (SVR) was induced by RG7854 in a subset of animals. The SVR rate, however, was comparable between both treatment regimens, suggesting that the addition of ETV did not enhance the therapeutic efficacy of RG7854 although it augmented the proliferation of blood cells in response to viral antigens and magnitude of antibody titers. The induction of interferon-stimulated genes in blood by RG7854/ETV combination treatment demonstrated on-target activation of TLR7. Together with the virus-specific blood cell proliferation and the transient elevations in liver enzymes and inflammation, this suggested that cytokine-mediated non-cytolytic and T-cell mediated cytolytic mechanisms contributed to the SVR, in addition to the virus-neutralizing effects by antibody-producing plasma cells. Both RG7854 regimens were not associated with treatment-limiting adverse effects but accompanied by dose-dependent, transient neutropenia and thrombocytopenia. The study concluded that finite, oral RG7854 treatment can induce a SVR in woodchucks that is based on the retrieval of antiviral innate and adaptive immune responses. This supports future investigation of the TLR7 agonist as an immunotherapeutic approach for achieving functional cure in patients with CHB.

• RG7854
• TLR7 agonism
• adaptive immune response
• chronic hepatitis B
• entecavir
• functional cure
• innate immune response
• woodchuck

Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV. Here, we investigated the capabilities of woodchuck hepatic and human non-hepatic cell lines to support HBV infection. DNA transfection assays indicated that all cells tested supported both HBV and WHV replication steps post entry, including the viral covalently closed circular DNA (cccDNA) formation, which is essential for establishing and sustaining infection. Ectopic expression of huNTCP rendered one, but not the other, woodchuck hepatic cell line and the non-hepatic human cell line competent to support productive HBV entry, defined here by cccDNA formation during de novo infection. All huNTCP-expressing cell lines tested became susceptible to infection with hepatitis D virus (HDV) that shares the same entry receptor and initial steps of entry with HBV, suggesting that a late entry/trafficking step(s) of HBV infection was defective in one of the two woodchuck cell lines. In addition, the non-susceptible woodchuck hepatic cell line became susceptible to HBV after fusion with human hepatic cells, suggesting the lack of a host cell-dependent factor(s) in these cells. Comparative transcriptomic analysis of the two woodchuck cell lines revealed widespread differences in gene expression in multiple biological processes that may contribute to HBV infection. In conclusion, other than huNTCP, neither human- nor hepatocyte-specific factors are essential for productive HBV entry. Furthermore, a late trafficking step(s) during HBV infection, following the shared entry steps with HDV and before cccDNA formation, is subject to host cell regulation and thus, a host determinant of HBV infection.

Fundamental studies on, and development of therapies against, chronic hepatitis B virus (HBV) infection, which inflicts hundreds of millions worldwide, are impeded by deficiencies in HBV-susceptible animal models. HBV displays a strict species and cell tropism that are not clearly understood. Here, by studying replication of HBV, and the related woodchuck hepatitis virus, in human and woodchuck hepatic or non-hepatic cells, we found that non-hepatic human cells and some woodchuck hepatic cells could support productive HBV entry after expression of the human cell receptor for HBV. Moreover, by studying the infection of hepatitis D virus, which shares the same entry receptor and initial steps of entry with HBV, we could narrow down a host determinant of HBV infection operating at a late entry/trafficking step(s). Our study thus provides new insights into determinants of HBV host tropism and facilitates the development of HBV-susceptible animal models.

• Animals
• DNA, Circular/metabolism
• DNA, Viral/genetics
• DNA, Viral/metabolism
• Hepatitis B
• Hepatitis B Virus, Woodchuck
• Hepatitis B virus/metabolism
• Hepatitis Delta Virus/genetics
• Hepatitis Delta Virus/metabolism
• Hepatocytes
• Humans
• Marmota
• Virus Replication/genetics

• R37 AI043453 NIAID NIH HHS
• National Institute of Allergy and Infectious Diseases

Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-β and interleukins 1β and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB.

• chronic hepatitis B
• hepatitis B virus
• innate immune response
• pattern recognition receptors
• viral DNA sensing receptors
• woodchuck

• antiviral efficacy
• chronic hepatitis B
• entry inhibitor
• hepatitis B virus
• humanized antibody
• hzVSF
• liver inflammation
• safety
• tenofovir alafenamide fumarate
• vimentin
• woodchuck

• Alanine/pharmacology
• Animals
• Antibodies, Monoclonal, Humanized/pharmacology
• Antiviral Agents/pharmacology
• Disease Models, Animal
• Drug Therapy, Combination
• Endocytosis/drug effects
• Hep G2 Cells
• Hepatitis B/drug therapy
• Hepatitis B/metabolism
• Hepatitis B/virology
• Hepatitis B Virus, Woodchuck/drug effects
• Hepatitis B Virus, Woodchuck/pathogenicity
• Host-Pathogen Interactions
• Humans
• Liver/drug effects
• Liver/metabolism
• Liver/virology
• Marmota
• Tenofovir/analogs & derivatives
• Tenofovir/pharmacology
• Vimentin/antagonists & inhibitors
• Vimentin/metabolism
• Viral Load
• Virus Internalization/drug effects
• Virus Replication/drug effects

• P30 CA051008 NCI NIH HHS

Since the discovery of hepatitis B virus (HBV) over five decades ago, there have been many independent studies showing presence of HBV genomes in cells of the immune system. However, the nature of HBV lymphotropism and its significance with respect to HBV biology, persistence and the pathogenesis of liver and extrahepatic disorders remains underappreciated. This is in contrast to studies of other viral pathogens in which the capability to infect immune cells is an area of active investigation. Indeed, in some viral infections, lymphotropism may be essential, and even a primary mechanism of viral persistence, and a major contributor to disease pathogenesis. Nevertheless, there are advances in understanding of HBV lymphotropism in recent years due to cumulative evidence showing that: (i) lymphoid cells are a reservoir of replicating HBV, (ii) are a site of HBV-host DNA integration and (iii) virus genomic diversification leading to pathogenic variants, and (iv) they play a role in HBV resistance to antiviral therapy and (v) likely contribute to reactivation of hepatitis B. Further support for HBV lymphotropic nature is provided by studies in a model infection with the closely related woodchuck hepatitis virus (WHV) naturally infecting susceptible marmots. This animal model faithfully reproduces many aspects of HBV biology, including its replication scheme, tissue tropism, and induction of both symptomatic and silent infections, immunological processes accompanying infection, and progressing liver disease culminating in hepatocellular carcinoma. The most robust evidence came from the ability of WHV to establish persistent infection of the immune system that may not engage the liver when small quantities of virus are experimentally administered or naturally transmitted into virus-naïve animals. Although the concept of HBV lymphotropism is not new, it remains controversial and not accepted by conventional HBV researchers. This review summarizes research advances on HBV and hepadnaviral lymphotropism including the role of immune cells infection in viral persistence and the pathogenesis of HBV-induced liver and extrahepatic diseases. Finally, we discuss the role of immune cells in HBV diagnosis and assessment of antiviral therapy efficacy.

• hepatitis B virus
• lymphotyropism
• virus genome integration
• virus variants
• woodchuck model of hepatitis B

The prolonged viral antigen stimulation is the driving force for the development of immune tolerance to chronic hepatitis B virus (HBV) infection. The sustained reduction of viral proteins may allow for the recovery and efficient activation of HBV-specific T and B cells by immune-stimulating agents, checkpoint blockades and/or therapeutic vaccinations. Recently, several therapeutic approaches have been shown to significantly reduce intrahepatic viral proteins and/or circulating HBV surface antigen (HBsAg) with variable impacts on the host antiviral immune responses in animal models or human clinical trials. It remains to be further investigated whether reduction of viral protein expression or induction of intrahepatic viral protein degradation is more efficacious to break the immune tolerance to chronic HBV infection. It is also of great interest to know if the accelerated clearance of circulating HBsAg by antibodies has a long-term immunological impact on HBV infection and disease progression. Although it is clear that removal of antigen stimulation alone is not sufficient to induce the functional recovery of exhausted T and B cells, accumulating evidence suggests that the reduction of viral antigen load appears to facilitate the therapeutic activation of functional antiviral immunity in chronic HBV carriers. Based on a systematic review of the findings in animal models and clinical studies, the research directions toward discovery and development of more efficacious therapeutic approaches to reinvigorate HBV-specific adaptive immune function and achieve the durable control of chronic HBV infection, i.e. a functional cure, in the vast majority of treated patients are discussed.

• HBsAg
• Hepatitis B virus
• immune tolerance
• immunotherapy
• therapeutic vaccination

• hepatitis b virus
• woodchuck hepatitis virus
• precore/core
• cccdna
• hepatitis b core-related antigen

This review describes woodchucks chronically infected with the woodchuck hepatitis virus (WHV) as an animal model for hepatocarcinogenesis and treatment of primary liver cancer or hepatocellular carcinoma (HCC) induced by the hepatitis B virus (HBV). Since laboratory animal models susceptible to HBV infection are limited, woodchucks experimentally infected with WHV, a hepatitis virus closely related to HBV, are increasingly used to enhance our understanding of virus-host interactions, immune response, and liver disease progression. A correlation of severe liver pathogenesis with high-level viral replication and deficient antiviral immunity has been established, which are present during chronic infection after WHV inoculation of neonatal woodchucks for modeling vertical HBV transmission in humans. HCC in chronic carrier woodchucks develops 17 to 36 mo after neonatal WHV infection and involves liver tumors that are comparable in size, morphology, and molecular gene signature to those of HBV-infected patients. Accordingly, woodchucks with WHV-induced liver tumors have been used for the improvement of imaging and ablation techniques of human HCC. In addition, drug efficacy studies in woodchucks with chronic WHV infection have revealed that prolonged treatment with nucleos(t)ide analogs, alone or in combination with other compounds, minimizes the risk of liver disease progression to HCC. More recently, woodchucks have been utilized in the delineation of mechanisms involved in innate and adaptive immune responses against WHV during acute, self-limited and chronic infections. Therapeutic interventions based on modulating the deficient host antiviral immunity have been explored in woodchucks for inducing functional cure in HBV-infected patients and for reducing or even delaying associated liver disease sequelae, including the onset of HCC. Therefore, woodchucks with chronic WHV infection constitute a well-characterized, fully immunocompetent animal model for HBV-induced liver cancer and for preclinical evaluation of the safety and efficacy of new modalities, which are based on chemo, gene, and immune therapy, for the prevention and treatment of HCC in patients for which current treatment options are dismal.

• Woodchuck
• Hepatitis B virus
• Chronic infection
• Liver disease
• Hepatocellular carcinoma
• Cancer treatment

As current interventions for chronic hepatitis B (CHB) rarely induce cure, more effective drugs are needed. Short-term treatment of woodchucks with the novel immunomodulator AIC649, a parapoxvirus-based stimulator of toll-like receptor 9 dependent and independent pathways, has been shown to reduce viral DNA and surface antigen via a unique, biphasic response pattern. The present study evaluated long-term AIC649 treatment in combination with Entecavir for potency and safety in woodchucks. AIC649 monotreatment induced modest reductions in serum viral DNA and surface and e antigens that were associated with the same biphasic response pattern previously observed. Entecavir monotreatment reduced transiently viremia but not antigenemia, while AIC649/Entecavir combination treatment mediated superior viral control. Undetectability of viral antigens and elicitation of antibodies in AIC649/Entecavir-treated woodchucks correlated with the expression of interferons and suppression of viral replication in liver. Combination treatment was well tolerated, and liver enzyme elevations were minor and transient. It was concluded that the AIC649-mediated effects were most likely based on an improvement and/or reconstitution of antiviral immune responses that are typically deficient in CHB. As a combination partner to Entecavir, the antiviral efficacy of AIC649 was markedly enhanced. This preclinical study supports future evaluation of AIC649 for treatment of human CHB.

• TLR9
• antiviral efficacy
• chronic hepatitis B
• entecavir
• immune response
• parapoxvirus
• woodchuck

• Animals
• Antiviral Agents/therapeutic use
• DNA, Viral/blood
• Disease Models, Animal
• Drugs, Investigational/therapeutic use
• Guanine/analogs & derivatives
• Guanine/therapeutic use
• Hepatitis B Virus, Woodchuck/drug effects
• Hepatitis B Virus, Woodchuck/immunology
• Hepatitis B, Chronic/drug therapy
• Hepatitis B, Chronic/immunology
• Immunologic Factors/therapeutic use
• Marmota/virology
• Treatment Outcome
• Viremia/drug therapy
• Virus Replication/drug effects

Woodchucks chronically infected with woodchuck hepatitis virus (WHV), which resembles human hepatitis B virus, develop spontaneous hepatic tumors and may be an important biological and immunological model for human HCC. Nonetheless, this model requires further validation to fully realize its translational potential.

Woodchucks infected at birth with WHV that had developed HCC (n=12) were studied. Computed tomography, ultrasound, and magnetic resonance imaging were performed under anesthesia. LI-RADS scoring and correlative histologic analysis of sectioned tissues were performed. For immune characterization of tumors, CD3 (T cells), CD4 (T helpers), NCAM (Natural killers), FOXP3 (T-regulatory), PDL-1 (inhibitory checkpoint protein), and the human hepatocellular carcinoma (HCC) biomarker alpha-fetoprotein (AFP) immunohistochemical stains were performed.

Forty tumors were identified on imaging of which 29 were confirmed to be HCC with 26 categorized as LR-4 or 5. The remainder of the tumors had benign histology including basophilic foci, adenoma, and lipidosis as well as pre-malignant dysplastic foci. LR-4 and LR-5 lesions showed high sensitivity (90%) and specificity (100%) for malignant and pre-malignant tumors. Natural killers count was found to be 2–5 times lower in tumors relative to normal parenchyma while other immune cells were located in the periphery of tumors. Tumors expressed AFP and did not express PD-L1.

Woodchucks chronically infected with WHV developed diverse hepatic tumor types with diagnostic imaging, pathology, and immune patterns comparable to that in humans. This unique animal model may provide a valuable tool for translation and validation of novel image-guided and immune-therapeutic investigations.

• animal
• carcinoma
• hepatocellular
• immunotherapy
• interventional oncology
• models
• radiology
• tumor microenvironment

Teratomas are germ cell neoplasms composed of a wide variety of tissues. In the woodchuck, only one testicular teratoma has been described in the literature. The objective of this report was to describe the radiologic and pathologic findings in a female woodchuck (Marmota monax) with an ovarian teratoma consisting of mature tissues originating from all three germ layers.

A 2-year-old female woodchuck that had been infected at birth with woodchuck hepatitis virus and subsequently developed hepatocellular carcinoma was incidentally discovered to have a mobile 6.6 × 4.8 × 4.7 cm abdominal mass on computed tomography (CT) imaging. The tumor was predominantly solid and heterogenous on CT with soft tissue, fat, and areas of dense calcification. The teratoma did not enhance with intravenous contrast administration. On ultrasound, the tumor was solid with heterogeneous echogenicity, reflecting the fat content and areas of calcification. Sonolucent areas were present that may have represented cysts. There was heterogeneously increased signal on T1-weighted magnetic resonance imaging (MRI) and heterogeneous hyperintensity in T2-weighted imaging. Fat was evident within the tumor. At necropsy, the tumor was attached to the distal end of the right uterine horn. Histopathology showed mature tissue types representing all three germ layers.

Ovarian teratoma should be considered in the differential diagnosis of ovarian or abdominal masses in woodchucks. The tumor displayed mature tissue derived from all three germ layers. CT, ultrasound, and MRI findings were presented in detail and matched the typical imaging appearance of teratomas.

• Marmota
• Ovarian neoplasm
• Pathology
• Teratoma
• Woodchuck

• Animals
• Antiviral Agents/pharmacokinetics
• Antiviral Agents/therapeutic use
• Disease Models, Animal
• Drug Evaluation, Preclinical/methods
• Female
• Hepatitis B, Chronic/drug therapy
• Hibernation/physiology
• Humans
• Male
• Marmota/physiology
• Metabolic Clearance Rate/physiology
• Seasons

Experimental animals including the ferret, marmoset, woodchuck, mini pig, and tree shrew have been used in biomedical research. However, their gut microbiota have not been fully investigated. In this study, the gut microbiota of these five experimental animals were analyzed with 16S rRNA sequencing. The phyla Firmicutes, Bacteroidetes, and Fusobacteria were present in the gut microbiota of all the species. Specific phyla were present in different animals: Proteobacteria in the ferret, Tenericutes in the marmoset, and Spirochaetes in the mini pig. Fusobacterium and unidentified Clostridiales were the dominant genera in the ferret, whereas Libanicoccus, Lactobacillus, Porphyromonas, and Peptoclostridium were specific to marmoset, mini pig, woodchuck, and tree shrew, respectively. A clustering analysis showed that the overall distribution of microbial species in the guts of these species mirrored their mammalian phylogeny, and the microbiota of the marmoset and tree shrew showed the closest bray_curtis distances to that of humans. PICRUSt functional prediction separated the woodchuck from the other species, which may reflect its herbivorous diet. In conclusion, both the evolutionary phylogeny and daily diet affect the gut microbiota of these experimental animals, which should not be neglected for their usage in biomedical research.

• genome annotation
• genome assembly
• groundhog
• woodchuck

• Animals
• Base Sequence
• Genome
• High-Throughput Nucleotide Sequencing
• Marmota/genetics
• Nanopores
• United States

• R01 HG006677 NHGRI NIH HHS

• Hepatitis B virus
• Hepatitis D virus
• Hepatocyte
• Mouse
• Non-human primate
• Sodium taurocholate co-transporting polypeptide

• Animals
• Disease Models, Animal
• Hepatitis B/virology
• Hepatitis B virus/pathogenicity
• Hepatitis D/virology
• Hepatitis Delta Virus/pathogenicity
• Hepatocytes/virology
• Macaca/virology
• Mice
• Virus Internalization

• R01 AI144008 NIAID NIH HHS

Woodchuck infected with woodchuck hepatitis virus (WHV) represents the pathogenically nearest model of hepatitis B and associated hepatocellular carcinoma (HCC). This naturally occurring animal model also is highly valuable for development and preclinical evaluation of new anti-HBV agents and immunotherapies against chronic hepatitis (CH) B and HCC. Studies in this system uncovered a number of molecular and immunological processes which contribute or likely contribute to the immunopathogenesis of liver disease and modulation of the systemic and intrahepatic innate and adaptive immune responses during hepadnaviral infection. Among them, inhibition of presentation of the class I major histocompatibility complex on chronically infected hepatocytes and a role of WHV envelope proteins in this process, as well as augmented hepatocyte cytotoxicity mediated by constitutively expressed components of CD95 (Fas) ligand- and perforin-dependent pathways, capable of eliminating cells brought to contact with hepatocyte surface, including activated T lymphocytes, were uncovered. Other findings pointed to a role of autoimmune response against hepatocyte asialoglycoprotein receptor in augmenting severity of liver damage in hepadnaviral CH. It was also documented that WHV in the first few hours activates intrahepatic innate immunity that transiently decreases hepatic virus load. However, this activation is not translated in a timely manner to induction of virus-specific T cell response which appears to be hindered by defective activation of antigen presenting cells and presentation of viral epitopes to T cells. The early WHV infection also induces generalized polyclonal activation of T cells that precedes emergence of virus-specific T lymphocyte reactivity. The combination of these mechanisms hinder recognition of virus allowing its dissemination in the initial, asymptomatic stages of infection before adaptive cellular response became apparent. This review will highlight a range of diverse mechanisms uncovered in the woodchuck model which affect effectiveness of the anti-viral systemic and intrahepatic immune responses, and modify liver disease outcomes. Further exploration of these and other mechanisms, either already discovered or yet unknown, and their interactions should bring more comprehensive understanding of HBV pathogenesis and help to identify novel targets for therapeutic and preventive interventions. The woodchuck model is uniquely positioned to further contribute to these advances.

• asialoglycoprotein receptor
• hepatocyte as cytotoxic immune effector
• intrahepatic innate and adaptive immune responses
• major histocompatibility complex presentation
• pre-acute infection
• toll-like receptors
• virus-hepatocyte interaction
• woodchuck model of hepatitis B

• biodistribution
• gold nanoparticles
• hepatocellular carcinoma
• marmota monax
• tumor microenvironment
• woodchuck hepatitis virus

• Animals
• Carcinoma, Hepatocellular
• Disease Models, Animal
• Humans
• Liver
• Liver Neoplasms
• Marmota
• Nanoparticles
• Tissue Distribution

RG7834 is a small‐molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon‐α (wIFN‐α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.57 log₁₀ from baseline and WHV DNA by a mean of 1.71 log₁₀. ETV + wIFN‐α reduced WHsAg and WHV DNA by means of 2.40 log₁₀ and 6.70 log₁₀, respectively. The combination of RG7834, ETV, and wIFN‐α profoundly reduced WHsAg and WHV DNA levels by 5.00 log₁₀ and 7.46 log₁₀, respectively. However, both viral parameters rebounded to baseline after treatment was stopped and no antibody response against WHsAg was observed. Effects on viral RNAs were mainly seen with the triple combination treatment, reducing both pregenomic RNA (pgRNA) and WHsAg RNA, whereas RG7834 mainly reduced WHsAg RNA and ETV mainly affected pgRNA. When WHsAg was reduced by the triple combination, peripheral blood mononuclear cells (PBMCs) proliferated significantly in response to viral antigens, but the cellular response was diminished after WHsAg returned to baseline levels during the off‐treatment period. Consistent with this, Pearson correlation revealed a strong negative correlation between WHsAg levels and PBMC proliferation in response to peptides covering the entire WHsAg and WHV nucleocapsid antigen. Conclusion: A fast and robust reduction of WHsAg by combination therapy reduced WHV‐specific immune dysfunction in the periphery. However, the magnitude and/or duration of the induced cellular response were not sufficient to achieve a sustained antiviral response.

• Himalayan marmot
• hepadnavirus
• immune system
• marmotte de l’Himalaya
• peste
• plague
• système immunitaire

• angiography
• animal model
• hepatocellular carcinoma
• woodchuck

• AIDS
• HBV
• HIV
• animal models
• co-infections
• tuberculosis.

• Animals
• Coinfection/microbiology
• Disease Models, Animal
• Guinea Pigs
• HIV Infections/immunology
• HIV Infections/pathology
• HIV-1/immunology
• Hepatitis B/immunology
• Hepatitis B/pathology
• Hepatitis B virus/immunology
• Humans
• Macaca mulatta
• Marmota
• Mice
• Mycobacterium tuberculosis/immunology
• National Institute of Allergy and Infectious Diseases (U.S.)
• Rabbits
• Tuberculosis/immunology
• Tuberculosis/pathology
• United States

• R01 HL129881 NHLBI NIH HHS
• R33 AI138328 NIAID NIH HHS
• HHSN272201100001G NIAID NIH HHS

The Eastern woodchuck (Marmota monax) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

• Chronic Hepatitis B
• Eastern Woodchuck
• Genome Assembly
• Hepatocellular Carcinoma
• Immune Response
• Marmota monax
• Whole Genome Sequencing

• Adaptive Immunity
• Aging
• Animals
• Hepatitis B/virology
• Hepatitis B Virus, Woodchuck/immunology
• Hepatitis B Virus, Woodchuck/pathogenicity
• Immunity, Innate
• Interferon-gamma/metabolism
• Killer Cells, Natural/metabolism
• Killer Cells, Natural/virology
• Liver/pathology
• Liver/virology
• Marmota/virology
• T-Lymphocytes/immunology
• T-Lymphocytes/virology
• Virus Replication/immunology

• R01 CA166213 NCI NIH HHS
• National Cancer Institute

• HHSN27200007 AHRQ HHS
• HHSN272201000011I NIAID NIH HHS
• UL1 TR001409 NCATS NIH HHS

• HBV
• WHV
• self-assembly

• HDV-host interactions
• Hepatitis delta virus
• Regulation of HDV infection outcome

• Bat
• Habitat loss
• Hepatitis B virus
• Orthohepadnavirus
• TBHBV
• Uroderma bilobatum

• Animals
• Chiroptera/virology
• Ecosystem
• Female
• Hepadnaviridae/isolation & purification
• Hepadnaviridae Infections/veterinary
• Hepadnaviridae Infections/virology
• Male
• Panama
• Species Specificity

• Immune Response
• Mouse
• Treatment
• Viral Infection

Woodchucks infected with woodchuck hepatitis virus (WHV) represent a highly valuable model of human hepatitis B virus (HBV) infection, chronic hepatitis (CH), and virus induced-primary liver cancer. Toll-like receptors (TLRs) are important mediators of immune responses playing pivotal roles in the pathogenesis of viral diseases; however, their expression profiles in different forms of infection and stages of hepatitis, and in healthy animals remain essentially unknown. In this study, woodchuck TLRs 1–10 exon fragments were identified and TLR genes transcription quantified in livers, primary hepatocytes, peripheral blood mononuclear cells (PBMC), and in selected organs during experimental WHV infection. Among others, liver biopsies from acute hepatitis (AH) and CH showed significantly augmented expression of the majority of TLRs when compared to healthy and woodchucks prior to AH, with resolved AH or primary occult infection. In contrast to the liver tissue, significant upregulation of TLR3, TLR4, and TLR10, but downregulation of TLR7, characterized hepatocytes derived from livers of animals with resolved AH accompanied by secondary occult infection. Hepatocytes from CH showed significantly lower expression or a trend toward suppression of several TLRs when compared to hepatocytes from healthy animals and woodchucks with other forms of infection or hepatitis, suggesting that hepatocyte innate immune response is downregulated during CH. Contrastingly, upregulated transcription of some TLRs characterized PBMC throughout CH. Our study uncovered that TLR expression significantly varies between different forms of hepadnaviral infection and whether infection is accompanied or not by hepatitis. The results showed that the profiles of TLRs’ expression in circulating lymphomononuclear cells do not mirror accurately those of livers and hepatocytes from infected animals. These findings are of importance to the understanding of immune process operating at different sites targeted by virus in the course of hepadnaviral infection and evaluation of future therapies modifying antiviral innate responses in the woodchuck model.

• PBMC
• hepadnaviral infection
• hepatocytes
• liver
• lymphatic organs
• toll-like receptors
• woodchuck model of hepatitis B

• Antiviral
• HBV
• Hepatitis B
• Nucleoside analogues
• Woodchuck hepatitis virus (WHV)

• Animals
• Antiviral Agents/pharmacology
• Cell Line
• Dideoxynucleosides/pharmacology
• Hepatitis B Virus, Duck/drug effects
• Hepatitis B Virus, Woodchuck/drug effects
• Hepatitis B virus/drug effects
• Hepatocytes/drug effects
• Hepatocytes/virology
• Humans
• Nucleic Acid Synthesis Inhibitors/pharmacology
• Nucleosides/pharmacology
• RNA, Viral/drug effects
• Virus Replication/drug effects