A mouse model for hepatitis E virus infection

Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluationJournal of HepatologyVol. 64Issue 5PreviewThe hepatitis E virus (HEV) is a small non-enveloped virus (size 27–34 nm) with a positive-sense, single-stranded RNA genome (7.2 kb) belonging to the family of Hepeviridae [1]. The capped and polyadenylated RNA genome comprises a 5′ UTR, three open reading frames (ORF1-3) and a 3′ UTR [2]. ORF 1 encodes non-structural proteins such as the helicase and the RNA-dependent RNA polymerase. ORF2 protein is the viral capsid protein inducing neutralizing antibodies during infection [3], while ORF3 encodes a small phosphorylated protein that is essential for virion release [4,5]. Full-Text PDF Hepatitis E virus (HEV) infection is believed to be the most common cause of acute hepatitis and jaundice in the world [1Hoofnagle J.H. Nelson K.E. Purcell R.H. Hepatitis E.N Engl J Med. 2012; 367: 1237-1244Crossref PubMed Scopus (390) Google Scholar, 2Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Ijaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar]. HEV genotypes 1 and 2 are responsible for waterborne hepatitis outbreaks in regions with low standards of sanitation in Asia, Africa and Central America, infecting about 20 million people and causing 70,000 deaths every year [[3]Rein D.B. Stevens G.A. Theaker J. Wittenborn J.S. Wiersma S.T. The global burden of hepatitis E virus genotypes 1 and 2 in 2005.Hepatology. 2012; 55: 988-997Crossref PubMed Scopus (487) Google Scholar]. HEV genotypes 3 and 4, which are found worldwide and primarily in China as well as Southeast Asia, respectively, have been recognized as zoonotic infections transmitted by the consumption of undercooked or raw pig or game meat, with seroprevalence rates up to 52% in the Southwest of France. This autochthonous form of hepatitis E can persist in immunocompromised individuals, including transplant recipients as well as patients with hematologic malignancies or poorly controlled HIV infection. Hence, HEV infection represents a global health challenge, with two distinct patterns identified in developing and in developed regions. HEV genotypes 1 and 2 infect only humans while genotypes 3 and 4 infect various animal species as well as humans. The course of infection may differ depending on the genotype. For example, high maternal as well as fetal morbidity and mortality have been described in pregnant women infected with HEV genotype 1 while HEV genotype 3 infection is believed to most often occur asymptomatically, can cause a spectrum of extrahepatic manifestations, especially neurologic complications, and can persist in immunocompromised individuals [1Hoofnagle J.H. Nelson K.E. Purcell R.H. Hepatitis E.N Engl J Med. 2012; 367: 1237-1244Crossref PubMed Scopus (390) Google Scholar, 2Kamar N. Bendall R. Legrand-Abravanel F. Xia N.S. Ijaz S. Izopet J. et al.Hepatitis E.Lancet. 2012; 379: 2477-2488Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar]. However, the molecular basis for these differences in the pathogenesis of hepatitis E is unknown and further investigation will depend on the availability of suitable animal models. HEV belongs to the Hepeviridae family [4Emerson S.U. Purcell R.H. Hepatitis E virus.in: Knipe D.M. Howley P.M. Fields virology. 6th ed. Lippincott Williams & Wilkins, Philadelphia2013: 2242-2258Google Scholar, 5Debing Y. Moradpour D. Neyts J. Gouttenoire J. Update on hepatitis E virology: implications for clinical practice.J Hepatol. 2016; ([in press])Google Scholar]. This family has expanded recently, with the isolation of new HEV strains from various animal species, representing potential reservoirs for virus recombination and diversity as well as zoonotic transmission to humans. HEV possesses a 7.2 kb positive-strand RNA genome comprising three open reading frames (ORFs) that are translated into three different products, i.e. (i) the ORF1 replicase, comprising the functional domains required for RNA replication, including a methyltransferase, a helicase and an RNA-dependent RNA polymerase, (ii) the ORF2 capsid protein, and (iii) the ORF3 protein, a small, hitherto poorly characterized protein involved in viral particle secretion and potentially other functions. While important aspects of the viral life cycle remain to be explored, progress has been made in the development of cell culture systems, including infectious clones and replicons [[6]Shukla P. Nguyen H.T. Faulk K. Mather K. Torian U. Engle R.E. et al.Adaptation of a genotype 3 hepatitis E virus to efficient growth in cell culture depends on an inserted human gene segment acquired by recombination.J Virol. 2012; 86: 5697-5707Crossref PubMed Scopus (182) Google Scholar] as well as, among others, induced pluripotent stem cell-derived hepatocyte-like cells as in vitro model for HEV infection [7Helsen N. Debing Y. Paeshuyse J. Dallmeier K. Boon R. Coll M. et al.Stem cell-derived hepatocytes: a novel model for hepatitis E virus replication.J Hepatol. 2016; 64: 565-573Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Dao Thi V.L. Debing Y. Wu X. Rice C.M. Neyts J. Moradpour D. et al.Sofosbuvir inhibits hepatitis E virus replication in vitro and results in an additive effect when combined with ribavirin.Gastroenterology. 2016; 150: 82-85Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar]. Several animal models for HEV infection are currently available. These include natural hosts such as swine, rabbits, rats, ferrets and chickens. However, rats, ferrets and chickens are only infected by genetically distant HEV strains, and swine as well as rabbits experience only acute, self-limited HEV infection by some but not all human-pathogenic strains [[9]Yugo D.M. Cossaboom C.M. Meng X.J. Naturally occurring animal models of human hepatitis E virus infection.ILAR J. 2014; 55: 187-199Crossref PubMed Scopus (39) Google Scholar]. As an alternative, nonhuman primates such as cynomolgus and rhesus macaques as well as chimpanzees support infection by all HEV strains infecting humans [[9]Yugo D.M. Cossaboom C.M. Meng X.J. Naturally occurring animal models of human hepatitis E virus infection.ILAR J. 2014; 55: 187-199Crossref PubMed Scopus (39) Google Scholar]. They also develop only acute, self-limited infection, although a recent Letter to the Editor published in the Journal of Hepatology reported the persistence of HEV genotype 4 in two experimentally infected rhesus macaques [[10]Huang F. Yang C. Zhou X. Yu W. Pan Q. Rhesus macaques persistently infected with hepatitis E shed virus into urine.J Hepatol. 2016; (in press)Google Scholar]. However, primate models remain very restricted for ethical and financial reasons as well as in terms of experimental tools. In this issue of the Journal, Allweiss et al. describe the first mouse model of HEV infection [[11]Allweiss L. Gass S. Giersch K. Groth A. Kah J. Volz T. et al.Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluation.J Hepatol. 2016; 64: 1033-1040Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. The authors infected uPA/SCID/beige (USB) mice repopulated with primary human hepatocytes with serum or fecal extracts from patients with acute or chronic hepatitis E (Fig. 1). These mice carry a transgene governing the expression of the urokinase-type plasminogen activator under the control of the albumin enhancer/promotor (Alb-uPA). Hepatocyte-specific expression of the uPA induces extensive liver injury, providing a terrain for the repopulation with human hepatocytes. In order to avoid rejection of the human hepatocytes, Alb-uPA mice were crossed with severe combined immunodeficiency (SCID) mice, yielding USB mice (Fig. 1). These mice have chimeric livers when repopulated with human hepatocytes soon after birth. This and related models have been used successfully to study hepatitis B, C as well as D viruses and to explore novel therapeutic or preventive strategies [12Petersen J. Dandri M. Gupta S. Rogler C.E. Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma.Proc Natl Acad Sci U S A. 1998; 95: 310-315Crossref PubMed Scopus (109) Google Scholar, 13Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (748) Google Scholar, 14Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (278) Google Scholar, 15Washburn M.L. Bility M.T. Zhang L. Kovalev G.I. Buntzman A. Frelinger J.A. et al.A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease.Gastroenterology. 2011; 140: 1334-1344Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Mailly L. Xiao F. Lupberger J. Wilson G.K. Aubert P. Duong F.H. et al.Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.Nat Biotechnol. 2015; 33: 549-554Crossref PubMed Scopus (105) Google Scholar, 17Vercauteren K. Brown R.J. Mesalam A.A. Doerrbecker J. Bhuju S. Geffers R. et al.Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice.Gut. 2016; (in press)PubMed Google Scholar]. Infection of human liver chimeric USB mice was attempted by the oral, intraperitoneal and intravenous routes, using serum or fecal extracts from patients with HEV genotype 1 or 3 infection. In addition, infected mice were co-housed with naïve mice. Interestingly, intravenous injection of fecal extracts and co-housing were the only successful routes of transmission. However, the overall number of animals and inocula studied remains relatively limited to date. Hence, it is not possible yet to draw firm conclusions as to the efficacy of the different transmission routes. In the infected animals, stable viral RNA titers were measured in serum, liver, bile and feces for more than 25 weeks, demonstrating chronic infection in these immunocompromised mice lacking functional cellular immune responses. In line with observations made in patients with hepatitis E, viral titers were higher in bile and feces as compared to serum. Infection with HEV genotype 1 resulted in approximately 1-log higher viremia as compared to genotype 3. By consequence, both the ORF2 and ORF3 proteins could be reliably detected by immunofluorescence microscopy only in the livers of mice infected with genotype 1. Importantly, HEV infection per se did not appear to cause significant liver injury, as alanine transaminase (ALT) levels remained normal, underpinning an immune-mediated pathogenesis of hepatitis E in humans, as for the other forms of viral hepatitis. This observation also highlights an important limitation of the current version of this in vivo model system, i.e. the lack of a functional adaptive immune system. The authors made use of their novel animal model to investigate some aspects of the HEV life cycle. In situ hybridization revealed the presence of HEV RNA in almost all human hepatocytes within the chimeric livers of genotype 1-infected animals, indicating highly efficient infection. Furthermore, the authors were capable, by immunofluorescence microscopy, to localize ORF2 in the cytoplasm of infected cells as broad cytoplasmic or punctate signal, as well as ORF3 at the apical hepatocyte membrane and within bile caniculi of adjacent human hepatocytes. In addition, they studied the buoyant density of viral particles in serum and bile. In this context, HEV has previously been found with a low density in blood and a high density in feces. It is believed, therefore, that HEV, similar to hepatitis A virus [[18]Feng Z. Hensley L. McKnight K.L. Hu F. Madden V. Ping L. et al.A pathogenic picornavirus acquires an envelope by hijacking cellular membranes.Nature. 2013; 496: 367-371Crossref PubMed Scopus (477) Google Scholar], is wrapped by cellular membranes ('quasi-enveloped') when secreted into the bile but not into bloodstream [5Debing Y. Moradpour D. Neyts J. Gouttenoire J. Update on hepatitis E virology: implications for clinical practice.J Hepatol. 2016; ([in press])Google Scholar, 19Nagashima S. Jirintai S. Takahashi M. Kobayashi T. Tanggis Nishizawa T. et al.Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies.J Gen Virol. 2014; 95: 2166-2175Crossref PubMed Scopus (125) Google Scholar]. Similar to that described in patients with hepatitis E, mouse-derived HEV presented with higher density in feces than in serum. Altogether, these observations validate human liver chimeric USB mice as an attractive model to further investigate the HEV life cycle in vivo. As a further application of their model and as a proof of concept for preclinical antiviral development, the authors treated four mice infected with HEV genotype 1 with ribavirin, which is used to treat chronic hepatitis E in patients, with an efficacy of about 75% after 12 weeks of administration [[20]Kamar N. Izopet J. Tripon S. Bismuth M. Hillaire S. Dumortier J. et al.Ribavirin for chronic hepatitis E virus infection in transplant recipients.N Engl J Med. 2014; 370: 1111-1120Crossref PubMed Scopus (342) Google Scholar]. They observed a relatively modest antiviral effect after 2 weeks in two animals and a more robust, mean 2.5-log drop of viremia after 6 weeks in two others. However, longer treatment durations and follow-up have not been reported, and it is unknown whether ribavirin can eliminate HEV from the serum and feces of human liver chimeric USB mice. This model appears very attractive to study mechanisms, especially the viral determinants, of ribavirin failure as well as to explore novel antiviral and preventive strategies. However, investigators using this model will have to take into account potential differences between humans and rodents in the extrahepatic metabolism of drugs. For example, we have shown recently that the hepatitis C virus polymerase inhibitor sofosbuvir can inhibit HEV replication in vitro and has an additive effect with ribavirin [[8]Dao Thi V.L. Debing Y. Wu X. Rice C.M. Neyts J. Moradpour D. et al.Sofosbuvir inhibits hepatitis E virus replication in vitro and results in an additive effect when combined with ribavirin.Gastroenterology. 2016; 150: 82-85Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar]. However, as discussed in this report, the high esterase activity present in the serum and gastrointestinal tract of rodents will likely preclude the evaluation of sofosbuvir in a murine model. In conclusion, human liver chimeric USB mice represent an attractive small animal model for the study of HEV infection and replication as well as the evaluation of novel antiviral and preventive strategies in vivo. Ongoing efforts with the aim to reconstitute such mice with a functional human immune system may allow investigation of the pathogenesis of hepatitis E in the future. The authors acknowledge support by the Swiss National Science Foundation (grant 31003A-156030 to DM) and the Gilead Sciences International Research Scholars Program in Liver Disease (Award 2015 to JG). The authors declare no conflict of interest related to this article. JG and DM wrote the manuscript.