Title: Animal Models for Hepatitis B: Does the Supply Meet the Demand?
Abstract: Worldwide, more than 257 million people are persistently infected with hepatitis B virus (HBV), which can lead to a broad spectrum of disease outcomes, including cirrhosis and/or hepatocellular carcinoma. HBV can be effectively prevented with a prophylactic vaccine, and currently approved antiviral therapy can suppress viremia but hardly cure the underlying infection. New insights into the viral life cycle and HBV's interactions with the host cell have reignited efforts to devise improved antiviral therapies with the ambitious goal of completely eradicating HBV or permanently inactivating the virus in patients. Systematic testing of approaches to cure HBV has been hampered by the scarcity of animal models faithfully recapitulating infection and the clinical features associated with chronic hepatitis B. HBV is a partially double-stranded DNA virus of the Hepadnaviridae family, genus Orthohepadnavirus. Although viruses genetically similar to HBV have been identified in a variety of species,1Lauber C. Seitz S. Mattei S. et al.Deciphering the origin and evolution of hepatitis B viruses by means of a family of non-enveloped fish viruses.Cell Host Microbe. 2017; 22: 387-399 e6Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar the etiologic agent of HBV in humans has a remarkably narrow tissue and host range, limited to hepatocytes in humans and chimpanzees.2Winer B.Y. Ploss A. Determinants of hepatitis B and delta virus host tropism.Curr Opin Virol. 2015; 13: 109-116Crossref PubMed Scopus (18) Google Scholar,3Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1e00049Crossref PubMed Scopus (1144) Google Scholar The mechanistic basis for this highly restricted tropism has not been fully deciphered, and consequently it has proven difficult to establish the entire viral life cycle in traditionally nonpermissive species. Chronic hepatitis B is a multifarious disease resulting from the intricate interplay between HBV, hepatocytes, nonparenchymal cells and the immune system. Tremendous progress has been made toward modeling HBV infection in increasingly complex cell culture and tissue organoid platforms,4Ramanan V. Scull M.A. Sheahan T.P. et al.New methods in tissue engineering: improved models for viral infection.Annu Rev Virol. 2014; 1: 475-499Crossref PubMed Scopus (17) Google Scholar but none of them adequately capture the complexity of the disease. The highly restricted species tropism of HBV, which is limited to chimpanzees and humans,2Winer B.Y. Ploss A. Determinants of hepatitis B and delta virus host tropism.Curr Opin Virol. 2015; 13: 109-116Crossref PubMed Scopus (18) Google Scholar,3Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1e00049Crossref PubMed Scopus (1144) Google Scholar has made the in vivo study of this virus notoriously difficult. This work has become even more challenging with the National Institutes of Health's moratorium on research in chimpanzees, underscoring the need for alternative small animal models suitable for studying HBV pathogenesis. Considerable progress has been made in understanding the basic mechanisms of HBV replication, but the precise host factors needed for completion of the virus's replication cycle remain to be fully elucidated. We have insufficient insights into how HBV is sensed by the host's innate and adaptive immune responses and how host defenses may contribute to the markedly severe pathogenesis caused by HBV, which is widely considered a noncytopathic virus. Although the HBV replication cycle can be mimicked by other genetically related viruses, such as woodchuck hepatitis virus (WHBV) in WHBV–infected woodchucks,5Summers J. Smolec J.M. Snyder R. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks.Proc Natl Acad Sci U S A. 1978; 75: 4533-4537Crossref PubMed Scopus (454) Google Scholar the genetic diversity of woodchucks, and lack of immunologic tools precludes their use as a practical model. Nonetheless, studies using these surrogate models have been very informative because they have provided important pathogenic insights and have been invaluable testing antiviral treatment approaches against HBV infection.6Allweiss L. Strick-Marchand H. In-vitro and in-vivo models for hepatitis B cure research.Curr Opin HIV AIDS. 2020; 15: 173-179Crossref PubMed Scopus (5) Google Scholar Without an established small animal model that supports the entire life cycle of HBV and recapitulates human disease phenotypes, our ability to fully understand HBV interactions with infected cell and the immune system and to develop effective antiviral treatment against these dreadful infections, remains limited. Immunocompetent animal models may also lend themselves to evaluating improved HBV prophylactic (ie, single dose) and therapeutic vaccines. Distinct (new) biomarkers to track HBV infection in peripheral circulation can be further evaluated in animal models. These include but are not limited to circulating HBV RNA (pgRNA), quantitative hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) as well as anti-HBs and -HBc antibodies. Establishing profiles of such markers that can unambiguously discern between, for example, viral suppression and a (functional) cure would be invaluable for interpreting clinical trial data. Given the paucity of patient liver biopsies, which are rarely taken during clinical trials owing to safety concerns, animal models also play a key role in translational research because they permit to assess the impact of novel antiviral therapies on virologic (eg, intrahepatic replication intermediated) and host parameters (eg, immune cell infiltration, transcriptional, proteomic of metabolomic changes) within the infected liver. Previous work in humans suggested that intrahepatic covalently closed circular DNA (cccDNA) levels may be studied through evaluating circulating blood cell free DNA or in circulating cells that have been released from the liver.7Bhan I. Mosesso K. Goyal L. et al.Detection and analysis of circulating epithelial cells in liquid biopsies from patients with liver disease.Gastroenterology. 2018; 155: 2016-2018 e11Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Studies in animal models would undoubtedly help to determine how well such peripheral surrogates correlate with intrahepatic replication levels. Optimally, any animal model for hepatitis B should closely mimic as many of the relevant clinical features observed in patients as possible. HBV animal models should be readily susceptible to all viral genotypes, support formation of cccDNA and result in persistent viremia, in the majority of exposed animals. The model should be fully immune-competent to mechanistically dissect correlates of protective immunity, persistence and immune-mediated pathogenesis. Because, in humans, decades can pass between the acute phase of infection and the development of severe liver pathologies, it is impractical, and perhaps even unrealistic, to aim for experimental models to follow this same extended progression timeline. To overcome this challenge, it would be desirable if the model(s) was/were amenable to genetic manipulation that possibly allowed for accelerated and/or exacerbated development of clinically relevant symptoms. From a practical perspective, an animal model for hepatitis B should be highly reproducible, easy to propagate, high throughput, and affordable to produce. Currently, no single animal model exists for HBV that combines all of the desired features outlined above. However, there are several models that at least recapitulate aspects of the viral life cycle (recently reviewed in detail6Allweiss L. Strick-Marchand H. In-vitro and in-vivo models for hepatitis B cure research.Curr Opin HIV AIDS. 2020; 15: 173-179Crossref PubMed Scopus (5) Google Scholar; see also Table 1) and have served as valuable models for evaluating various antivirals in preclinical studies.Table 1Comparison Of Different Animal Models for HBVHBV life-cycle supported?SpeciesModelentryReplicationassembly/releaseImmune statusThroughputcostsutilityCaveatsMouse1.3x HBV transgenicnoyes∗does not occur exactly as de novo HBV replication.Yesimmunocompetent but tolerized to HBVhighlowimmune-responses, some pathogenesisnot an authentic infection modelAAV-/AdV-HBVnoyes∗does not occur exactly as de novo HBV replication.Yesimmunocompetenthighlowimmune responses, immunomodulatorsnot an authentic infection modelhNTCP transgenic/knock-inyesNoNoimmunocompetenthighlowentry inhibitors?only entryhuman liver chimericyesyesdenotes clear evidence for cccDNA formationYesimmunodeficientmediummediumtesting of DAAs and some HTAsimmunodeficientdually engrafted human liver/immune system miceyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentmediummediumimmune-responses, some pathogenesis, immunomodulatorsheterogeneity, limited immune functionalityRhesus macaqueAAV-hNTCPyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentlowhighimmune-responsesonly low level, transient viremiaTree shrewsHBVyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentlowmediumTBDweak, short-lived viremia with limited viral replicationWoolly monkeyWMHBVyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentlown/an/asurrogate virus, endangered species, not accessibleSpider monkeysWMHBVyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentlown/an/asurrogate virus, endangered species, not routinely available for researchSquirrel monkeyHBV/WMHBVyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentlowhighn/asurrogate virus, only low level, transient WMHBV viremia, limited availability of toolswoodchuckWHBVyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentlowhighvaccine development, assessing immunologic aspects of the disease, and evaluating antiviral therapiessurrogate virus, difficult to access, limited availability of toolsducksDHBVyesyesdenotes clear evidence for cccDNA formationYesimmunocompetentHighhighvaccine development, assessing immunologic aspects of the disease, and evaluating antiviral therapiessurrogate virus, limited availability of toolsAdV, adenovirus; AAV, adeno-associated virus; cccDNA, covalently closed circular DNA; DAA, directly acting antiviral; DHBV, duck HBV; HBV, hepatitis B virus; HTA, host targeting antiviral; WMHBV, woolly monkey HBV; WHBV, woodchuck HBV.∗ does not occur exactly as de novo HBV replication.∗∗ denotes clear evidence for cccDNA formation Open table in a new tab AdV, adenovirus; AAV, adeno-associated virus; cccDNA, covalently closed circular DNA; DAA, directly acting antiviral; DHBV, duck HBV; HBV, hepatitis B virus; HTA, host targeting antiviral; WMHBV, woolly monkey HBV; WHBV, woodchuck HBV. Rodent models would undoubtedly be most convenient owing to their low cost, rapid propagation, and the prevalence of experimental tools for their genetic manipulation. However, mice and rats are resistant to HBV infection. The discovery of the human sodium taurocholate cotransporting peptide (hNTCP or SLC10A) as the receptor for HBV and hepatitis delta virus3Yan H. Zhong G. Xu G. et al.Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Elife. 2012; 1e00049Crossref PubMed Scopus (1144) Google Scholar raised hopes that murine models with inbred susceptibility to HBV infection could be generated. However, although HBV can enter hNTCP-expressing hepatocytes in mice, blocks in subsequent steps of the viral life cycle hinder further progression of infection.8Winer B.Y. Shirvani-Dastgerdi E. Bram Y. et al.Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection.Sci Transl Med. 2018; 10Crossref PubMed Scopus (20) Google Scholar In vitro evidence suggests that cccDNA formation is supported in certain murine hepatoma cell clones,9Cui X. Guo J.T. Hu J. Hepatitis B virus covalently closed circular dna formation in immortalized mouse hepatocytes associated with nucleocapsid destabilization.J Virol. 2015; 89: 9021-9028Crossref PubMed Scopus (38) Google Scholar which is consistent with the fact that the minimal factors required for this step10Wei L. Ploss A. Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation.Nat Microbiol. 2020; 5: 715-726Crossref PubMed Scopus (22) Google Scholar are highly conserved across species. These data indicate that other steps in the HBV infection cycle, likely before cccDNA formation, may not be supported. Systematic dissection of the blocks in interspecies transmission holds the key toward establishing an HBV-susceptible mouse model. Transgenic or viral (eg, adeno-associated virus or adenovirus) vector-mediated expression of the HBV genome has proven effective in bypassing some of these restrictions. Although such models have shown some usefulness in testing therapeutics and studying aspects of infection,11Michler T. Kosinska A.D. Festag J. et al.Knockdown of virus antigen expression increases therapeutic vaccine efficacy in high-titer hepatitis B virus carrier mice.Gastroenterology. 2020; 158: 1762-1775 e9Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,12Wooddell C.I. Rozema D.B. Hossbach M. et al.Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.Mol Ther. 2013; 21: 973-985Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar such as HBV-specific immune responses in rodents, one should keep in mind that an actual infection is not established because the virus cannot spread: the infectious virions produced and released from cells harboring the viral genome are unable to enter naive cells. Humanized xenotransplantation models represent a valuable complementary system. Robust engraftment of human hepatocytes into various immunodeficient liver injury models has been reported (reviewed in13Douglas D.N. Kneteman N.M. Mice with chimeric human livers and their applications.Methods Mol Biol. 2019; 1911: 459-479Crossref PubMed Scopus (2) Google Scholar). Progress has been made in engrafting in vivo expanded primary human hepatocytes14Michailidis E. Vercauteren K. Mancio-Silva L. et al.Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes.Proc Natl Acad Sci U S A. 2020; 117: 1678-1688Crossref PubMed Scopus (16) Google Scholar,15Azuma H. Paulk N. Ranade A. et al.Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice.Nat Biotechnol. 2007; 25: 903-910Crossref PubMed Scopus (540) Google Scholar and stem cell–derived hepatocytes,16Yusa K. Rashid S.T. Strick-Marchand H. et al.Targeted gene correction of alpha1-antitrypsin deficiency in induced pluripotent stem cells.Nature. 2011; 478: 391-394Crossref PubMed Scopus (499) Google Scholar which will aid in increasing throughput, opens the possibility of manipulating genetically human hepatocytes and decreases donor-to-donor variability, although a shortcoming of the current models is that other nonparenchymal, hepatic cell populations critical for cellular crosstalk are not humanized. Regardless, the resultant human liver chimeric mice are susceptible to HBV17Dandri M. Burda M.R. Torok E. et al.Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (303) Google Scholar and several other human hepatotropic pathogens13Douglas D.N. Kneteman N.M. Mice with chimeric human livers and their applications.Methods Mol Biol. 2019; 1911: 459-479Crossref PubMed Scopus (2) Google Scholar and are a valuable tool for testing antiviral therapeutics.18Petersen J. Dandri M. Mier W. et al.Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.Nat Biotechnol. 2008; 26: 335-341Crossref PubMed Scopus (309) Google Scholar, 19Klumpp K. Shimada T. Allweiss L. et al.Efficacy of NVR 3-778, alone and in combination with pegylated interferon, vs entecavir in uPA/SCID mice with humanized livers and HBV infection.Gastroenterology. 2018; 154: 652-662 e8Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Amblard F. Boucle S. Bassit L. et al.Novel hepatitis B virus capsid assembly modulator induces potent antiviral responses in vitro and in humanized mice.Antimicrob Agents Chemother. 2020; 64Crossref Scopus (0) Google Scholar However, their highly immunocompromised nature limits their usefulness for evaluating any antiviral approaches relying on immune stimulation and/or modulation. Protocols are being continuously refined to achieve robust co-engraftment of hepatocytes and components of a human immune system (HIS). Such dually engrafted mice can support HBV infection, and studies have shown that viral infection triggers activation of the engrafted HIS,21Dusseaux M. Masse-Ranson G. Darche S. et al.Viral Load Affects the Immune Response to HBV in mice with humanized immune system and liver.Gastroenterology. 2017; 153: 1647-1661 e9Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar particularly natural killer cells22Billerbeck E. Mommersteeg M.C. Shlomai A. et al.Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells.J Hepatol. 2016; 65 (334–243)Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar and M2 macrophages,23Bility M.T. Cheng L. Zhang Z. et al.Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.PLoS Pathog. 2014; 10e1004032Crossref PubMed Scopus (136) Google Scholar and leads to some virally induced histopathology.23Bility M.T. Cheng L. Zhang Z. et al.Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.PLoS Pathog. 2014; 10e1004032Crossref PubMed Scopus (136) Google Scholar It should be noted, however, that sourcing donor-matched hepatocytes, nonparenchymal cells, and hematopoietic stem cells remains challenging at present. Although stem cell–derived cells may provide a possible solution, currently available protocols for differentiating embryonic stem cells or induced pluripotent cells into the hepatic lineage and hematopoietic stem cells do not yield cells that can be engrafted reproducibly and robustly into commonly used xenorecipient strains. Future refinements will focus on improving the limited the functionality of the engrafted HIS. Numerous strategies to do this have been proposed (reviewed in24Walsh N.C. Kenney L.L. Jangalwe S. et al.Humanized mouse models of clinical disease.Annu Rev Pathol. 2017; 12: 187-215Crossref PubMed Scopus (252) Google Scholar,25Douam F. Ploss A. The use of humanized mice for studies of viral pathogenesis and immunity.Curr Opin Virol. 2018; 29: 62-71Crossref PubMed Scopus (15) Google Scholar); several human cell lineages remain under-represented, in part owing to the orthologs of nonredundant cytokines exhibiting limited biological species cross-reactivity. It was previously shown that selective expansion of under-represented cell types, such as dendritic cells, natural killer cells, and granulocytes, leads to immune responses to the live attenuated yellow fever virus (YFV) vaccine more akin to those observed in YFV vaccinees.26Douam F. Ziegler C.G.K. Hrebikova G. et al.Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses.Nat Commun. 2018; 9: 5031Crossref PubMed Scopus (18) Google Scholar Promisingly, the expression of murine thymic stromal lymphopoietin can restore lymph node development in immunocompromised mouse strains used for xenotransplantation and drastically augments human immunity to model antigens.27Li Y. Masse-Ranson G. Garcia Z. et al.A human immune system mouse model with robust lymph node development.Nat Methods. 2018; 15: 623-630Crossref PubMed Scopus (41) Google Scholar Additionally, the development of functional adaptive immune responses has been limited by the lack of HLA gene expression. Expressing a human major histocompatibility complex class I allele has multiple benefits allowing for more faithful development of CD4+ and CD8+ T cells in the thymus, enabling recognition of (viral) antigens in peripheral tissues by human T cells and facilitating the tracking of antigen-specific CD8+ T cells with major histocompatibility complex multimers, as previously shown for EBV, dengue virus and YFV.28Shultz L.D. Saito Y. Najima Y. et al.Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice.Proc Natl Acad Sci U S A. 2010; 107: 13022-13027Crossref PubMed Scopus (260) Google Scholar, 29Strowig T. Gurer C. Ploss A. et al.Priming of protective T cell responses against virus-induced tumors in mice with human immune system components.J Exp Med. 2009; 206: 1423-1434Crossref PubMed Scopus (214) Google Scholar, 30Jaiswal S. Pearson T. Friberg H. et al.Dengue virus infection and virus-specific HLA-A2 restricted immune responses in humanized NOD-scid IL2rgammanull mice.PLoS One. 2009; 4e7251Crossref PubMed Scopus (103) Google Scholar Co-engraftment of improved xenorecipient strains with additional hematopoietic stem cell donor-matched human tissues, such as liver, thymus and/or lymph nodes, could also significantly augment the immune response. Such co-engraftments could enhance T- and B-cell selection, intrahepatic T-cell priming,31Huang L.R. Wohlleber D. Reisinger F. et al.Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection.Nat Immunol. 2013; 14: 574-583Crossref PubMed Scopus (149) Google Scholar and liver-mediated secretion of key human immune components.32Sander L.E. Sackett S.D. Dierssen U. et al.Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function.J Exp Med. 2010; 207: 1453-1464Crossref PubMed Scopus (233) Google Scholar Finally, engraftment of second-generation humanized mice with a human-like microbiome represents another valuable approach to enhance immunity, as previously suggested.33Gulden E. Vudattu N.K. Deng S. et al.Microbiota control immune regulation in humanized mice.JCI Insight. 2017; 2Crossref PubMed Scopus (14) Google Scholar Nonhuman primates (NHPs) would likely provide the strongest immune data for translation into clinical trials, given their similar immune systems and the wealth of reagents available. Work in macaques has shown that the expression of hNTCP in the liver results in HBV viremia, albeit transiently and at low levels.34Burwitz B.J. Wettengel J.M. Muck-Hausl M.A. et al.Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques.Nat Commun. 2017; 8: 2146Crossref PubMed Scopus (30) Google Scholar Ideally, additional steps toward making an improved NHP model for HBV include the creation of transgenic rhesus macaques susceptible to HBV and alteration of HBV preS1 to accommodate binding and entry using the macaque NTCP receptor. The bottom line is that the scarcity of accessible immunocompetent models of chronic HBV infection is a critical roadblock in the journey to find a cure for HBV. Currently available models are either immunodeficient, do not truly model infection, or use surrogate viruses in rare species. Each of these weaknesses create distinct challenges in translating preclinical results to humans. Ideally, immunocompetent chronic infection models would be established in both mice and NHPs. Developing such models would allow for more relevant studies in species for which immunologic reagents and protocols are already available for a broad range of targets and pharmacodynamic markers. Furthermore, studies could initially be run at a lower cost and higher throughput in mice before being subsequently confirmed in the costlier, but likely more relevant, primate system. To decrease the disease burden caused by HBV, it will undoubtedly be key to continue educating the public to overcome the stigma associated with this disease, to systematically identify HBV carriers, and to promote the widespread use of the available prophylactic HBV vaccine. However, these measures fall short in helping the large number of patients already persistently infected. There is irrefutable, albeit sporadic, evidence that chronic hepatitis B can be cured, providing hope for patients and motivation for scientists and clinicians alike that novel therapeutics can result in a (functional) cure. This arguably ambitious goal will take an all hands on deck approach to develop critical reagents such as HBV animal models in line with the following key principles. (1) To be truly transformative, any animal model development efforts need to focus on the actual needs of potential end users and meet the highest standards of rigor and reproducibility. (2) Whenever possible, necessary tools and reagents ought to be made accessible through public repositories such as BEI, a repository developed by the National Institute of Allergy and Infectious Diseases (https://www.beiresources.org). (3) To support multipronged approaches to developing a HBV animal model, robust, long-term investments—from both the private and public sectors—remain crucial. (4) The evaluation process for publicly funded research must be insulated from politics and allowed to impartially identify promising research proposals that are ethically sound as well as scientifically and technically rigorous. Installing, for example, a National Institutes of Health Human Fetal Tissue Ethics Advisory Board that has the predetermined objective of blocking the use of critically needed human fetal tissue is impeding promising biomedical research that has the potential to save lives and decrease human suffering caused by hepatitis B and many other diseases. (5) Finally, efficient information exchange not only within academia as well as with industry will be important to keep all parties abreast of key developments. To this end, the International Coalition to Eliminate HBV (ICE-HBV) workshop and other outlets serve as a viable and increasingly important platform. We thank the many colleagues in the field for providing critical input for the content of this manuscript during and after the ICE-HBV sponsored HBV animal model workshop at the 2019 HBV meeting in Melbourne, in particular Scott Balsitis, Jan Martin Berke, Benjamin Burwitz, Pei Jer Chen, Maura Dandri, Lu Gao, Dieter Glebe, Luca Guidotti, Jianming Hu, Jake Liang, Ulrike Protzer, and Peter Revill.