Keywords

1 Introduction

Hepatitis C virus (HCV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the genus Hepacivirus in the Flaviviridae family. While approximately 150 million people are infected with HCV worldwide, this is likely an underestimate as almost twice as many individuals in the United States may carry the virus, many unknowingly (Edlin 2011). HCV causes persistent infection in 70–80 % of those who become exposed to the virus. While the acute disease is usually asymptomatic, chronic carriers left untreated frequently develop fibrosis, cirrhosis and, in some cases, hepatocellular carcinoma. Treatment for HCV has evolved rapidly in recent years and it is now possible to cure the majority of patients with largely well-tolerated therapies that include a combination of pegylated interferon (IFN)-α, ribavirin and direct acting antiviral (DAA) drugs. However, despite their potency, it remains to be seen whether even the newest DAAs will drastically reduce the global burden of disease due to the high associated costs, logistical challenges of mass deployment and risk of drug resistance. A vaccine, which would prevent infection or delay the onset of pathogenesis during a chronic infection, does not exist. Development of effective therapies has been delayed by the lack of both suitable cell culture systems and animal models. While hepaciviruses similar to HCV have been found in a variety of species, including dogs, horses and outbred mice, HCV appears to have a much more limited host range. Robust infection has only been described in humans and experimentally infected chimpanzees, but some studies have provided evidence for transient and intermittent viremia in a more exotic mammal, tree shrews. The narrow host range of HCV is not completely understood but can in part be explained by differences between species in the sequences of essential host factors at the level of entry as well as in innate antiviral responses. This growing understanding of the barriers to interspecies transmission has aided the development of inbred models with inheritable susceptibility to HCV. As will be discussed here, genetic host adaptation has been and continues to be part of a multipronged approach to develop more tractable animal models for studying HCV infection, immunity and pathogenesis (Fig. 1).

Fig. 1
figure 1

Host adaptation and viral adaptation approaches to create new animal models for the study of hepatitis C. Host adaptation through transplantation of human hepatocytes to create HEAL mice or human liver chimeric mice and/or hematopoietic stem cells (HSCs) to (co-) engraft components of a human immune system (left column). Genetic humanization can be accomplished by identification and expression of human-specific factors or by ablation of restriction factors (middle column). Cell culture passaging strategies are used to adapt HCV to murine or simian hosts (right column). iPS Induced pluripotent stem cells, ES embryonic stem cells, HSC hematopoietic stem cells, HEAL human ectopic artificial liver

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2 Hepatitis C Virus Infection in Non-Human Primates

For many years, studies of hepatitis C were limited to experimentally infected chimpanzees or patient volunteers. While chimpanzees have been instrumental in analyzing HCV infection (reviewed in (Bukh 2004), Fig. 2), studies in this species are challenging due to high costs, genetic heterogeneity, small cohort sizes, limited access to relevant tissue compartments, the inability to genetically manipulate large apes and growing ethical concerns.

Fig. 2
figure 2

Phylogenetic relationship of members of the hepacivirus genus and susceptible host species. Phylogenetic tree of the hepaciviruses is adapted from (Pfaender et al. 2014a) and (Firth et al. 2014) and is based on the nucleotide sequence analysis of the NS3 protease domain and the complete NS5B gene. GHV Guereza hepacivirus, GBV-B George Barker virus B, NPHV non-primate hepacivirus, CHV canine hepacivirus, NrHV Norway rat hepacivirus, RHV rodent hepacivirus, BHV bat hepacivirus

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The other non-human primate (NHP) species tested for susceptibility to HCV infection – including cynomolgus monkeys, rhesus monkeys, Japanese monkeys, Green monkeys, Doguera (Abe et al. 1993) and Chacma baboons (Sithebe et al. 2002), cottontop tamarins (Garson et al. 1997) and marmosets – do not seem to support infection. The blocks in HCV transmission in these and potentially other NHP species are not well defined and are likely due to a combination of factors. For example, simian orthologs of essential host factors required for the viral life cycle may be absent or incompatible. Similarly, dominant restriction factors, as observed with HIV, may actively antagonize uptake, replication and/or viral assembly and release. In addition, differences in the kinetics and magnitude of antiviral defenses in many cells may interfere with viral RNA replication. This may be a result of less efficient viral evasion mechanisms that usually enable HCV to establish persistent infection in human cells. For example, in various primate species, differences in the amino acid sequence of the mitochondrial antiviral signal protein (MAVS, also known as IPS1, VISA or Cardif) prevent its proteolytic cleavage by the HCV NS3/4A protease, leaving host antiviral signaling intact (Patel et al. 2012). These incompatibilities may also result in more effective cellular and humoral immune responses in NHPs, contributing to clearance of HCV.

However, these blocks do not appear absolute as stem cell-derived hepatocyte-like cells from pig-tailed macaques (Macaca nemestrina) (Sourisseau et al. 2013) and primary rhesus macaque (Macaca mulatta) hepatocytes can support the entire HCV life cycle (Scull et al. 2015). Additionally, HCV RNA replication in rhesus macaque hepatocytes is enhanced upon blunting antiviral immunity, which is consistent with the observation that the rhesus MAVS ortholog is not cleaved by HCV NS3/4A (Scull et al. 2015). Nonetheless, HCV can establish persistent replication in simianized mice, i.e. immunocompromised xenorecipients engrafted with rhesus hepatocytes (Scull et al. 2015). Yet it still remains to be shown whether rhesus or pig-tailed macaques are actually susceptible to HCV in vivo and if viral persistence can be achieved.

Tree shrews (Tupaia belangeri) – once designated as small, squirrel-like primates but now classified in the separate order Scandentia – have been shown to support intermittent, transient viremia, becoming more permissive to HCV infection when immunosuppressed (Xie et al. 1998). In follow-up studies, acute infection did progress to persistent viremia (Xu et al. 2007; Amako et al. 2010), resulting in clinically symptomatic liver disease, including steatosis, fibrosis and cirrhosis after 3 years (Amako et al. 2010). While these more recent data are promising and may enable studies of HCV immunity and pathogenesis, there are still limitations to their utility as tree shrews are an outbred, genetically diverse organism and few reagents are available for investigating their immune response to viral infection.

3 Potential Surrogate Models: Non-Primate Hepaciviruses

With such a limited host tropism, other closely related viruses have been considered as a proxy for studying HCV. The best characterized of these viruses, GB viruses, named after the surgeon George Barker (Deinhardt et al. 1967), have been used in NHP studies. GB virus B was able to cause hepatitis in marmosets (Callithrix jacchus) (Simons et al. 1995; Lanford et al. 2003) (Fig. 2) as well as other New World monkeys, including tamarins (Saguinus spp.) (Karayiannis et al. 1989; Schaluder et al. 1995) and owl monkeys (Aotus trivirgatus) (Bukh et al. 2001). GBV-B belongs to the genus Hepacivirus in the Flaviviridae family and has the same overall genome organization as HCV. However, the polyproteins of HCV and GBV-B share only 28  % amino acid identity and differ even more in their 5′ and 3′ non-coding regions. The discovery of a GBV-B-like virus, called guereza hepacivirus (GHV) after the colobus species it was identified in, is the first hepacivirus found in a wild NHP and has led to further questions concerning the evolution of hepaciviruses (Lauck et al. 2013).

Other related viruses of either the Pegivirus genus, also a part of the Flaviviridae family, or Hepacivirus genus have been identified in dogs (Kapoor et al. 2011), horses (Burbelo et al. 2012; Kapoor et al. 2013a), wild mice (Kapoor et al. 2013b), bats(Quan et al. 2013) and rats (Firth et al. 2014). Of the non-primate hepaciviruses (NPHV), those observed in horses are the most genetically similar to HCV (Pfaender et al. 2014b) (Fig. 2). However, it remains to be shown whether these viruses indeed cause hepatitis in experimentally inoculated animals before it can be determined whether they might be surrogates for modeling HCV. Importantly, there is currently no experimental evidence of NPHV transmission between horses and humans (Pfaender et al. 2015).

4 Rodent Models

Mice are widely used in biomedical research, with many existing analytical tools for dissecting their responses to infection. Furthermore, mice of genetically defined backgrounds are available which are amenable to genetic manipulations. In the following sections, we will summarize the previously established rodent models and discuss some of the recent developments that have been explored to model HCV infection and pathogenesis in rodents.

4.1 HCV Transgenic Mice

HCV does not readily infect mice, and thus early attempts to model aspects of HCV pathogenesis in mice were performed by expressing individual or multiple HCV gene products (Table 1 and reviewed in Kremsdorf and Brezillon (2007)). However, depending on the mouse background, the HCV gene product(s) expressed, and the promoter driving expression of these proteins, the histopathological features observed in these mice differed considerably. When HCV core was expressed under the control of a hepatitis B virus (HBV) promoter, animals developed severe liver disease, culminating in hepatocarcinogenesis (Moriya et al. 1997, 1998). In contrast, driving HCV core and/or E1/E2 expression with a major urinary protein (MUP) or CMV promoter produced a less pronounced and more variable disease phenotype (Pasquinelli et al. 1997; Chiyo et al. 2011; Satoh et al. 2010; Naas et al. 2005; Benali-Furet et al. 2005; Chang et al. 2008, 2009; Lerat et al. 2009; Tanaka et al. 2008; Kamegaya et al. 2005; Jeannot et al. 2012). Likewise, NS5A expression was directly cytopathic in some transgenic lines (Wang et al. 2009), but, when under the control of an apoE or MUP promoter, liver pathologies were not observed (Majumder et al. 2003). Similarly, expression of the HCV serine protease NS3/NS4A or NS4B in mouse models has not been shown to induce liver injury (Desai et al. 2011; Frelin et al. 2006; Wang et al. 2006).

Table 1 HCV transgenic mouse models
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In addition to modeling aspects of HCV-induced liver disease, expression of HCV proteins has been utilized for studying HCV-specific adaptive immune responses in the liver (Disson et al. 2004; Alonzi et al. 2004; Tsukiyama-Kohara et al. 2011; Wegert et al. 2009; Tumurbaatar et al. 2007; Ernst et al. 2007; Furutani et al. 2006; Takaku et al. 2003; Naas et al. 2010; Kriegs et al. 2009; Kanda et al. 2009). Pre-natal expression of HCV proteins in mice causes the murine immune system to become tolerized to viral gene products, but this tolerance can be disrupted via DNA vaccination, which primes CD8 T cells to target hepatocytes expressing HCV NS3/4A (Ahlen et al. 2009). This model lends itself to testing T cell based vaccine candidates.

Undoubtedly, HCV transgenic mice have helped analyze HCV immune responses and viral pathogenesis, but a number of factors still diminish their utility. Transgene copy numbers, and consequently levels and distribution of HCV protein expression, can vary considerably due to random integration in the mouse genome. When driven by strong viral or cellular promoters, expression of HCV gene products can surpass the levels of viral proteins that would be reached by actual infection. Furthermore, interpreting data acquired in transgenic mice is further complicated since HCV proteins are being expressed outside of the inflammatory context of acute and chronic viral infection.

4.2 Xenotransplantation Models

To overcome some of the challenges of HCV transgenic mice, xenotransplantation models have been established in which the murine host is rendered susceptible to HCV infection by xenoengraftment of permissive human cells in the mouse liver (Fig. 1). As described below, a variety of approaches have been taken to accomplish this goal.

4.2.1 Engraftment of Human Hepatoma Cells In Vivo

The simplest mouse models of xenoengraftment are made by intrahepatic injection of human hepatoma cells. To quantify RNA replication and responses to antiviral treatment such as interferon-α (IFN-α) in vivo, Huh7 cells containing an HCV replicon expressing luciferase have been injected into severe combined immunodeficient (SCID)/beige mice and analyzed by whole-body bioluminescence imaging (Zhu et al. 2006). This system is simple with minimal intra- and interexperimental variation but is not a bona fide infection model.

To actually enable the study of anti-HCV immune responses, immunocompetent fetal rats have been tolerized in utero to Huh7 cells and, after birth, transplanted with a larger number of the same cells (Wu et al. 2005). Remarkably, Huh7 cells, which are usually not readily susceptible to HCV infection in vitro, supported viremia of a patient-derived genotype 1a isolate. However, the low levels of observed viremia, complex nature of these experiments, and potential inability of rat T cells to recognize HCV antigens due to the presence of human leukocyte antigen (HLA) on the transplanted Huh7 cells make this model less than ideal.

4.2.2 Ectopic Liver Implantation Models

While it is more desirable to engraft hepatocytes instead of hepatoma cells in the human parenchyma, this is not readily accomplished with primary human hepatocytes. However, pieces of human liver and even artificial human liver organoids have been successfully implanted in ectopic sites. In the so-called “Trimera mouse” (Ilan et al. 2002), small pieces of human liver were maintained under the kidney capsule or the ear of SCID mice. When the liver tissue was taken from HCV positive donors or naïve tissue was infected with HCV prior to transplantation, viremia was maintained for several weeks. This model has been subsequently used to assess the efficacy of neutralizing antibodies (Eren et al. 2006), but the technically and logistically challenging experimental set-up, fairly rapid graft failure and low levels of HCV viremia have hampered the utility of the model.

To overcome the need for primary liver tissue, bioengineering approaches have been undertaken to reconstruct increasingly more complex tissue organoids suitable for transplantation. Human ectopic artificial livers (HEALs) have been created where cryopreserved primary human hepatocytes are supported by polymeric scaffolds, which aid maintenance of the microenvironment and thus stablize these cells. While simpler, polyethylene glycol (PEG)-based polymers were used initially (Chen et al. 2011), newer models allow for even greater control of the scaffold architecture, improving vascularization and, consequently, hepatocyte survival (Miller et al. 2012; Stevens et al. 2013). In mice engrafted intraperitoneally with HEALs (Fig. 1), humanized liver functions could be monitored for several weeks but susceptibility to hepatotropic pathogens, including HCV, has yet to be shown.

4.2.3 Human Liver Chimeric Mice

The most commonly used and best characterized humanized xenotransplantation models for HCV are human liver chimeric mice (Fig. 1). Suitable xenorecipient strains are immunodeficient to avoid graft rejection and also have endogenous liver injury to both promote hepatocyte proliferation and give the donor hepatocytes a growth advantage over the mouse hepatocytes. Donor cells, including hepatoma cell lines, primary hepatocytes and, more recently, stem-cell derived hepatocytes, are injected intrasplenically. Traveling via the portal venous system, the donor cells pass through the liver sinusoidal endothelial cells and form clusters that expand upon induction of liver injury. This can be done via partial hepatectomy or treatment with hepatotoxic chemicals, like retrorsine and carbon tetrachloride. Genetic approaches have also been utilized as they allow for more control over the severity of the liver injury and can limit hepatotoxicity to specifically mouse hepatocytes.

Robust engraftment of human hepatocytes has been shown in a number of immunodeficient liver injury models, including Alb-uPA (Meuleman et al. 2005; Mercer et al. 2001), FAH−/− (Bissig et al. 2010; de Jong et al. 2014), AFC8 (Washburn et al. 2011), MUP-uPA (Tesfaye et al. 2013) and HSV-TK (Kosaka et al. 2013) mice. The resultant human liver chimeric mice are susceptible to several human-tropic pathogens, including HBV, HCV and parasites that cause malaria in humans (reviewed in (Meuleman and Leroux-Roels 2008). Additionally, these mice can be used for monitoring human-like metabolic and toxicological responses in testing antimicrobial compounds.

With the exception of AFC8 mice, all the above-mentioned xenorecipient strains have demonstrated robust human hepatic chimerism when using adult hepatocytes. Due to genetic differences between hepatocyte donors, host responses can differ. To minimize inter-experimental variations and create a renewable resource for human donor cells, stem cell-derived hepatocytes have been explored as a possible solution. Hepatocyte-like cells (HLCs) can now be routinely generated from embryonic stem (ES) or induced pluripotent stem (iPS) cells (Touboul et al. 2010; Si-Tayeb et al. 2010). These HLCs express hepatocyte-specific markers, support hepatocyte-specific metabolic functions and can be infected with HCV (Schwartz et al. 2012; Wu et al. 2012; Roelandt et al. 2012). Recent studies suggest that HLCs can also be engrafted reasonably well in vivo and support persistent HCV infection (Carpentier et al. 2014). However, engraftment efficiency seems to depend strongly on the xenorecipient strain, as immunodeficient MUP-uPA mice, but not other liver injury models, support robust in vivo expansion.

4.2.4 HCV Immunity and Pathogenesis in Humanized Xenotransplantation Models

Human liver chimeric mice are currently the only experimental models besides chimpanzees that are readily susceptible to HCV. These mice have been used to study innate host responses to HCV and for testing the efficacy of novel therapeutic regimens. To expand the use of these mice in analyzing human immune responses to HCV, protocols are being refined so that mice are co-engrafted with human hepatocytes and components of a human immune system (Fig. 1). Initial attempts co-injected a mixture of human fetal hepatoblasts, non-parenchymal cells and hematopoietic stem cells (HSCs) into AFC8 mice, yielding reasonable immune cell engraftment but low human hepatic chimerism (Washburn et al. 2011). Nonetheless, dually engrafted mice did become chronically infected following inoculation with HCV patient isolates and exhibited an HCV-specific T cell response, which appeared to be responsible for observed signs of early liver fibrosis. While these data are encouraging, protocols need to be refined further to improve dual chimerism and minimize inter- and intravariability of experiments. More recent reports have demonstrated that extensive double humanization of both the liver and immune system can be achieved with mature hepatocytes and HSCs (Gutti et al. 2014; Wilson et al. 2014). Long-term dual reconstitution, without any evidence of hepatocyte rejection by the human immune system, was sustained even when the human cells were mismatched in their major histocompatibility complex (MHC, (Gutti et al. 2014). The latter observation is consistent with the limited HLA matching in human liver transplantations, presumably due to the tolerogenic microenvironment of the liver. However, the limited function of the transplanted human immune system at least partially contributes to the lack of allogeneic graft rejection in dually engrafted humanized mice. To improve both the cellular complexity and functionality of the engrafted human immune system, several modifications are being tested. These include, but are not limited to: the expression of human orthologs of non-redundant cytokines with limited biological cross-reactivity to foster development of human immune cell lineages which currently do not develop efficiently in conventional humanized mice; expression of human MHC in the absence of mouse MHC to ensure faithful presentation of self- and virally derived peptides to human T cells and to reduce graft-versus-host-disease; co-transplantation of HSC donor-matched human thymic cortical epithelium to facilitate proper T cell selection; the improvement of lymphoid architecture organization, especially in the spleen and lymph-nodes, to allow for adequate T and B cell priming; genetic replacement of non-compatible immune cell receptors and chemokines expressed on non-hematopoietically derived cells to improve e.g. immune cell trafficking; the introduction of a human microbiome to account for effects of species-specific commensals on the immune system (reviewed in Shultz et al. 2012).

5 Genetically Humanized Mouse Models for HCV Infection

An inbred mouse model with inheritable susceptibility to HCV would overcome the technical difficulties of the xenotransplantation model (Fig. 1). The challenge is to systematically identify and overcome any restrictions to HCV infection in murine cells. HCV’s narrow host range is not completely understood, and the viral life cycle is blocked or insufficiently supported in murine cells at multiple steps. Productive HCV uptake into human hepatocytes relies on a large number of human host molecules (reviewed in Ding et al. 2014). These include glycosaminoglycans (GAGs) present on heparan sulfate proteoglycans (HSPGs), low-density-lipoprotein receptor (LDLR) (Agnello et al. 1999), CD81 (Pileri et al. 1998), scavenger receptor class B member 1 (SCARB1) (Scarselli et al. 2002), the tight junction proteins claudin-1 (CLDN1) (Evans et al. 2007) and occludin (OCLN) (Liu et al. 2009; Ploss et al. 2009), the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) (Lupberger et al. 2011), the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) (Sainz et al. 2012), transferrin receptor 1 (TfR1) (Martin and Uprichard 2013) and the cell death-inducing DFFA-like effector b (CIDEB) (Wu et al. 2014). The block of HCV entry in rodent cells can be explained by differences in critical residues in the second extracellular loops of CD81 (Flint et al. 2006; Higginbottom et al. 2000) and OCLN (Michta et al. 2010). Consequently, expression of human CD81 and OCLN, along with human or mouse SCARB1 and CLDN1, facilitates HCV uptake in mouse cells in vitro (Ploss et al. 2009). Other human entry factors appear to contribute minimally to HCV species tropism at the level of entry, but their individual roles still need to be experimentally tested.

Establishing HCV glycoprotein-mediated uptake into mouse livers adenovirally transduced with CD81 and OCLN opened the door for genetically overcoming the barrier to HCV entry in mice. Indeed, expression of human CD81 and OCLN appears sufficient for HCV entry into hepatocytes of fully immunocompetent inbred mice. This genetically humanized mouse model allows dissection of the HCV entry process in the 3D context of the liver in vivo and has been applied to test pre-clinically the efficacy of neutralizing antibodies and vaccine candidates (Giang et al. 2012; Dorner et al. 2011; de Jong et al. 2014). Transgenic mice have also been developed in which human CD81, SCARB1, CLDN1 and OCLN expression are driven by liver-specific promoters (Hikosaka et al. 2011). However, initial reports have suggested that these lines are resistant to HCV infection in vivo (Hikosaka et al. 2011). This observation is likely due to the lower level of entry factor expression in the transgenic mice and the need for a very sensitive reporter system to quantify viral entry (Dorner et al. 2011).

As an alternative to the genetic host adaptations described above, previous studies have shown that the block of HCV at the level of entry can also be overcome through viral adaptation (Fig. 1). Using an in vitro selection approach, mutations within HCV E1 and E2 that increased the affinity of the viral envelope for mouse CD81 were identified. These mutations appeared to more broadly affect the conformation of the viral envelope, as the resulting mouse CD81-adapted strain is also less dependent on human OCLN and can enter cell lines expressing only mouse CD81, SCARB1, CLDN1 and OCLN (Bitzegeio et al. 2010). It has yet to be demonstrated if this mouse-adapted HCV strain can enter mouse primary hepatocytes in vitro or in vivo.

Establishing HCV entry in vivo has some utility, but what is ultimately needed is a model that supports all steps of the viral life cycle. More than a decade ago, it was shown that HCV RNA is translated, but not readily replicated, following entry into murine cells (Dorner et al. 2011; McCaffrey et al. 2002). Subsequent studies in cell culture demonstrated that dominant negative inhibitors are not present and that the murine orthologs of host factors critical for HCV replication cooperate sufficiently with the viral replication machinery, as HCV replicons, i.e. selectable HCV RNA genomes, can replicate in murine cell lines (Zhu et al. 2003; Uprichard et al. 2006; Frentzen et al. 2011). Nevertheless, the efficiency of post-entry steps of the viral life cycle could conceivably be improved with human host factors important for HCV replication, assembly and/or egress. However, previous gain- and loss-of-function studies converged on only a few critical host factors, namely miR-122, cyclophilin A, phosphatidylinositol 4 kinase IIIα (PI4KIIIα), and apolipoprotein E (reviewed in (Bartenschlager et al. 2010)) – all of whose sequences are largely conserved between mice and humans. Thus, additional proviral factors that enhance HCV replication and/or assembly in mouse cells have yet to be identified.

Numerous studies have shown that innate antiviral responses play a critical role in limiting HCV infection in human cells, including hepatoma cell lines and human primary hepatocytes (Andrus et al. 2011; Marukian et al. 2011). Likewise, HCV replication is drastically enhanced in cell lines with strong impairments in type I and III interferon signaling, such as mouse cells lacking MAVS (Frentzen et al. 2014), protein kinase R (PKR; (Chang et al. 2006)), interferon regulatory factor 3 (IRF3; (Lin et al. 2010)) or STAT1 (Vogt et al. 2013). Known mechanisms by which HCV evades antiviral defenses, such as the cleavage of MAVS (Meylan et al. 2005) or Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF or TICAM; (Li et al. 2005)), appear to function in mouse cells (Vogt et al. 2013). However, differences in the kinetics and/or magnitude of virally induced innate defenses may restrict HCV RNA replication more efficiently in mouse compared to human cells. Consistent with these in vitro data, mice expressing human HCV entry factors crossed to genetic backgrounds impaired in antiviral innate defenses support low level HCV RNA replication (Dorner et al. 2013). In these genetically humanized mice, infectious HCV is detectable in circulation, confirming previous studies that demonstrated late stages of the HCV life cycle are supported in mouse cells if sufficient ApoE is present (Long et al. 2011).

Recapitulating the entire HCV lifecycle in inbred immunocompetent mice is an important next step in developing a mouse model suitable for mechanistic studies of HCV immunity and pathogenesis. The previously published model required immune-suppression to establish low-level viremia. However, more recent work suggests that this may be strain-dependent. In fact, mice expressing human CD81, SCARB1, CLDN1 and OCLN on the fully immunocompetent ICR mouse background not only supported persistent infection with various HCV isolates very efficiently but also developed clinically apparent liver disease (Chen et al. 2014). While these data are somewhat difficult to reconcile with most previously published literature, it is conceivable that a fortuitous allele combination in the genetically variable outbred ICR stock favors susceptibility to HCV.

6 Summary and Outlook

The advent of highly potent DAAs holds promise to effectively treat the great majority of patients. However, current treatments are very expensive and mandate strict adherence to dosing to prevent the outgrowth of resistant viral variants. To provide simpler and more cost-effective interventions and to optimally prevent infections, a HCV vaccine may ultimately be needed. Testing and prioritization of immunotherapies and vaccines is delayed by the lack of (a) readily accessible animal model(s). More tractable in vivo platforms could also be deployed to answer questions of basic virology, HCV pathogenesis, and correlates of protective immunity (Fig. 3). A variety of partially complementary approaches are currently being pursued to develop better small animal models for HCV infection. While most other NHP species besides chimpanzees were thought to be resistant to HCV infection, recent studies show that the HCV life cycle can be established in HLCs derived from pig-tailed macaques and primary hepatocytes from rhesus macaques. This suggests that certain NHP species may indeed be permissive to HCV infection. In addition, the discovery of hepaciviruses genetically closely related to HCV in outbred mice, rats, dogs and horses may provide further avenues for studying HCV. The barriers of HCV’s narrow host tropism are now better understood and have spurred a combination of viral adaptations and/or genetic host humanizations to establish inbred rodent models with inheritable susceptibility to HCV infection. Xenotransplantation approaches are being continuously refined, and it has become possible to reproducibly generate human liver chimeric mice at fairly high throughput. These mice can then be used to analyze all aspects of the viral life cycle with genetically diverse HCV isolates. Improvements in protocols yielding HLCs from directed differentiation of ES and iPS cells hold promise to develop renewable hepatocyte sources of genetically defined backgrounds. Furthermore, these advances may enable the generation of humanized mouse avatars engrafted with patient-specific hepatocytes to model clinically relevant disease phenotypes. In proof-of-concept studies, human liver and components of a human immune system were robustly engrafted in a single xenorecipient, paving the way for modeling HCV-associated hepatitis, including relevant co-infections with HBV and/or HIV. Undoubtedly, as new techniques and protocols are perfected, it will remain important to continue evaluating the ability of any new HCV model to faithfully recapitulate aspects of HCV pathogenesis and its consequences in humans.

Fig. 3
figure 3

Comparison of different animal models for hepatitis C

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