At least 130 million individuals are chronically infected with hepatitis C virus (HCV). Chronic HCV carriers are at risk of developing severe liver disease including fibrosis, cirrhosis and hepatocellular carcinoma. HCV is a positive-sense, single stranded RNA virus of the Flaviviridae
family. Current standard of care, consisting of pegylated interferon alpha (peg-IFN), ribavirin (RBV) and one of two inhibitors of the HCV RNA NS3/4A protease results in sustained virological response rates in 60–70% of patients in clinical trial cohorts[1
]. However, this therapy is poorly tolerated and not equally effective in all patient cohorts and against all HCV genotypes. New treatment modalities, which are currently being tested in clinical trials have raised the hope that HCV can be cured in the majority of patients.
Since the discovery of HCV in 1989[3
] much has been learned about the functions of HCV proteins and RNA elements and host factor requirement using cell culture systems (reviewed in[4
]). However, analysis of systemic host responses to viral infection has been hampered by the lack of suitable small animal models. HCV has narrow host range, infecting only humans and chimpanzees. Chimpanzees are susceptible to patient-derived virus and infectious clones, and recapitulate the natural course of infection[5
]. The ability to experimentally infect these animals has shed light on the role of the host immune response and aided the preclinical assessment of drug and vaccine candidates. However, studies in chimpanzees are hampered by limited availability, high costs, and are not considered ethically justifiable in many jurisdictions. Several distinct and potentially complementary strategies have been proposed to bridge this gap and to create more accessible small animal models[6
]. One approach focuses on adapting HCV to use essential host factors from non-permissive species, such as rodents or smaller non-human primates. These efforts are based on the remarkable genetic plasticity of HCV, which fuelled by its high replicative capacity and error prone replication machinery allowing the virus to evade selection pressure exerted by the host immune system or drug regimens. This, however, can also be exploited to adapt viral genomes to use essential host molecules of other species. For example, it was previously demonstrated that certain mutations in the viral envelope proteins E1 and E2 result in a gain of function allowing HCV to efficiently engage murine CD81 and occludin (OCLN)[7
] which otherwise do not facilitate efficient viral uptake[8
]. While a fully mouse adapted HCV genome has not been constructed these studies provided important proof-of-concept for the feasibility of this approach.
Alternatively, the murine host can be manipulated to provide an environment that is more conducive to HCV infection. Transplantation of human hepatocytes into highly immunocompromized liver injury mouse strains can result in a high degree of human hepatocyte engraftment. Such liver chimeric mice are susceptible to human hepatotropic pathogens, including hepatitits B virus[9
], HCV [9
] and human malaria parasites[12
]. However, humanized xenotransplantation models are low in throughput, costly, vary from donor-to-donor and pathogenesis studies are hampered due to the severe immunodeficiency of the recipient. The latter can be potentially overcome humanizing both the liver and the immune system in a single recipient. Recently, it was demonstrated that mice dually engrafted with human fetal hepatoblasts and components of a human immune system support HCV replication at low levels and are capable of mounting antigen-specific human immune responses which eventually result in onset of fibrosis[14
], a hallmark of advancing hepatitis C. This study established important proof-of-concept for the approach but the system requires additional refinements to improve human cell chimerism, levels of HCV viremia and the functionality of the human immune system.
A genetically-engineered, inbred mouse model with inheritable susceptibility to HCV would overcome the technical difficulties of xenotransplantation. However, the determinants limiting HCV species tropism remain poorly defined. In mouse cells, the HCV life-cycle is blocked at multiple steps. HCV utilizes numerous cellular factors to enter its primary target cell, the human hepatocyte. Those include the scavenger receptor class B type I (SCARB1)[15
], the tetraspanin CD81 [16
], tight junction proteins, claudin-1 (CLDN1) [17
] and OCLN [8
] and the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) [19
] and the cholesterol uptake receptor Niemann Pick C1 like 1 (NPC1L1)[20
]. We have previously shown that CD81, SCARB1, CLDN1 and OCLN are all required for uptake into rodent cells but only CD81 and OCLN need to be of human origin[8
]. This discovery opened the door for constructing a genetically humanized mouse model. Indeed, we recently demonstrated that adenoviral delivery of human CD81 and OCLN renders mice susceptible to HCV infection with diverse HCV genotypes[21
]. In order to visualize HCV uptake into murine hepatocytes, which generally do not readily support HCV RNA replication, we constructed a highly sensitive detection system where an in vivo reporter is activated by CRE recombinase expressed in the context of the incoming recombinant HCV genome. We have shown that viral uptake can be blocked by passive immunization strategies and that inoculation of these animals with a vectored vaccine induces humoral immunity and confers partial protection to heterologous challenge. We have also demonstrated proof-of-principle for combining this system with gene knockout analysis to begin to dissect viral entry in vivo.
Unfortunately, this current model supports only HCV uptake and genome translation. The ability to recapitulate the entire viral life-cycle in mice would facilitate studies on immune responses and pathogenesis. There is evidence, that can HCV replicate in murine cells, albeit to low levels, using recombinant genomes expressing a dominant selectable marker[22
]. This suggests that all essential host factors for RNA replication are expressed in mouse cells but the rodent orthologues may interact less efficiently with the viral proteins. HCV replication is substantially increased in mouse embryonic fibroblasts derived from PKR and IRF3-deficient mice[24
]. Thus, the inability of HCV to overcome murine defenses may also impair replication.
Adenoviral delivery to express exogenous human entry factors in mice is high-throughput and allows rapid evaluation of mutant genes but induces strongly antiviral interferon stimulated genes (ISGs), creating an environment which is not conducive for viral replication. It is conceivable that stable expression of entry factors in the form of transgenic or knock-in mice would overcome this caveat and may allow identification of a mouse background, which supports RNA replication. HCV assembly and egress in the HCV life cycle are likely to be supported in mice, as it was recently reported that infectious particles can assemble in murine hepatoma cell lines [26
Here, we provide a detailed protocol for the construction of genetically humanized mice and highlight some of their applications in studying HCV entry and preclinical assessment of drug and vaccine candidates.