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Update on hepatitis E virology: Implications for clinical practice

  • Author Footnotes
    † Current address: Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
    Yannick Debing
    Footnotes
    † Current address: Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
    Affiliations
    Rega Institute for Medical Research, University of Leuven, Belgium
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  • Darius Moradpour
    Affiliations
    Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland
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  • Johan Neyts
    Affiliations
    Rega Institute for Medical Research, University of Leuven, Belgium
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  • Jérôme Gouttenoire
    Correspondence
    Corresponding author. Address: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 48, CH-1011 Lausanne, Switzerland. Tel.: +41 21 314 07 49; fax: +41 21 314 40 95.
    Affiliations
    Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland
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  • Author Footnotes
    † Current address: Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
Published:March 07, 2016DOI:https://doi.org/10.1016/j.jhep.2016.02.045

      Summary

      Hepatitis E virus (HEV) is a positive-strand RNA virus transmitted by the fecal-oral route. The 7.2 kb genome encodes three open reading frames (ORF) which are translated into (i) the ORF1 polyprotein, representing the viral replicase, (ii) the ORF2 protein, corresponding to the viral capsid, and (iii) the ORF3 protein, a small protein involved in particle secretion. Although HEV is a non-enveloped virus in bile and feces, it circulates in the bloodstream wrapped in cellular membranes. HEV genotypes 1 and 2 infect only humans and cause mainly waterborne outbreaks. HEV genotypes 3 and 4 are widely represented in the animal kingdom and are transmitted as a zoonosis mainly via contaminated meat. HEV infection is usually self-limited but may persist and cause chronic hepatitis in immunocompromised patients. Reduction of immunosuppressive treatment or antiviral therapy with ribavirin have proven effective in most patients with chronic hepatitis E but therapy failures have been reported. Alternative treatment options are needed, therefore. Infection with HEV may also cause a number of extrahepatic manifestations, especially neurologic complications. Progress in the understanding of the biology of HEV should contribute to improved control and treatment of HEV infection.

      Abbreviations:

      7mG (7-methylguanylate), CSF (cerebrospinal fluid), gt (genotype), HCV (hepatitis C virus), HEV (hepatitis E virus), ESCRT (endosomal sorting complexes required for transport), IFN (interferon), IRF3 (interferon regulatory factor 3), ISG (interferon-stimulated gene), NF-κB (nuclear factor-κB), ORF (open reading frame), PCP (papain-like cysteine protease), PegIFNα (pegylated interferon-α), PRR (pattern recognition receptor), RdRp (RNA-dependent RNA polymerase), RT-qPCR (reverse transcription quantitative PCR), RIG-I (retinoic acid-inducible gene I), TBK1 (TANK-binding kinase 1), Tsg101 (tumor susceptibility gene 101)

      Keywords

      Introduction

      Hepatitis E virus (HEV) infection is among the most frequent causes of acute hepatitis worldwide, with an estimated 20 million infections and 70,000 deaths attributed to HEV genotypes 1 and 2 every year [
      • 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.
      ]. However, the majority of infections are thought to remain asymptomatic [
      • Kamar N.
      • Bendall R.
      • Legrand-Abravanel F.
      • Xia N.S.
      • Ijaz S.
      • Izopet J.
      • et al.
      Hepatitis E.
      ]. The virus has been recognized as a cause of waterborne hepatitis outbreaks in India not related to hepatitis A and B viruses in the early 1980s [
      • Khuroo M.S.
      Study of an epidemic of non-A, non-B hepatitis. Possibility of another human hepatitis virus distinct from post-transfusion non-A, non-B type.
      ,
      • Wong D.C.
      • Purcell R.H.
      • Sreenivasan M.A.
      • Prasad S.R.
      • Pavri K.M.
      Epidemic and endemic hepatitis in India: evidence for a non-A, non-B hepatitis virus aetiology.
      ]. It was first visualized by immune electron microscopy in a feces sample from a human volunteer infected with stool extracts from presumed cases of epidemic non-A, non-B hepatitis in 1983 [
      • Balayan M.S.
      • Andjaparidze A.G.
      • Savinskaya S.S.
      • Ketiladze E.S.
      • Braginsky D.M.
      • Savinov A.P.
      • et al.
      Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route.
      ]. HEV was molecularly cloned in 1990, allowing the rapid development of serological tests and the investigation of its epidemiology [
      • Reyes G.R.
      • Purdy M.A.
      • Kim J.P.
      • Luk K.C.
      • Young L.M.
      • Fry K.E.
      • et al.
      Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis.
      ,
      • Tam A.W.
      • Smith M.M.
      • Guerra M.E.
      • Huang C.C.
      • Bradley D.W.
      • Fry K.E.
      • et al.
      Hepatitis E virus: molecular cloning and sequencing of the full-length viral genome.
      ].
      HEV has been classified as the sole member of the Orthohepevirus genus within the Hepeviridae family [
      • Emerson S.U.
      • Anderson D.
      • Arankalle A.
      • Meng X.-J.
      • Purdy M.
      • Schlauder G.G.
      • et al.
      Hepevirus.
      ]. The recent development of advanced sequencing technology allowed the identification of novel HEV-related viruses in a variety of animals and led to a revised taxonomic classification of this family (Fig. 1) [
      • Smith D.B.
      • Simmonds P.
      • Jameel S.
      • Emerson S.U.
      • Harrison T.J.
      • Meng X.J.
      • et al.
      Consensus proposals for classification of the family Hepeviridae.
      ].
      Figure thumbnail gr1
      Fig. 1Phylogenetic relationship of hepeviruses identified in various hosts. Nucleotide sequences of 305 full-length hepatitis E virus (HEV) genomes were retrieved from GenBank and aligned with ClustalW, followed by phylogenetic tree building using the neighbor-joining method (Geneious 7.1 software, Biomatters). While genotypes 1 and 2 (gt 1 and 2) are restricted to humans and to endemic regions such as Asia, Africa and Mexico, genotypes 3 and 4 (gt 3 and 4) are also found in a wide range of animal species. Genotype 3 is present worldwide in various hosts such as swine, wild boar, deer, mongoose and Japanese macaques. Genotype 4 is found mainly in China as well as Southeast Asia and infects swine, wild boar and sheep. Viral strains that have not been assigned to one of these 4 genotypes may also infect humans, as documented recently for camel HEV
      [
      • Lee G.H.
      • Tan B.H.
      • Teo E.C.
      • Lim S.G.
      • Dan Y.Y.
      • Wee A.
      • et al.
      Chronic infection with camelid hepatitis E virus in a liver-transplant recipient who regularly consumes camel meat and milk.
      ]
      . Moreover, more distant hepatitis E viruses were identified in birds, bats, rats, ferrets and fish.
      Twenty-five years after the identification of the HEV genome, basic and clinical virology research is gaining momentum due to the increased awareness and perceived importance of hepatitis E as a relevant public health issue [
      • Purcell R.H.
      • Emerson S.U.
      Hepatitis E: an emerging awareness of an old disease.
      ,
      • Pischke S.
      • Heim A.
      • Bremer B.
      • Raupach R.
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      • Ganzenmueller T.
      • et al.
      Hepatitis E: an emerging infectious disease in Germany?.
      ]. Indeed, previously unrecognized HEV infections in industrialized countries by genotypes 3 and 4 are now known to be zoonotically transmitted and to cause persistent infection in immunocompromised patients, especially transplant recipients [
      • Kamar N.
      • Bendall R.
      • Legrand-Abravanel F.
      • Xia N.S.
      • Ijaz S.
      • Izopet J.
      • et al.
      Hepatitis E.
      ,
      • Kamar N.
      • Selves J.
      • Mansuy J.M.
      • Ouezzani L.
      • Peron J.M.
      • Guitard J.
      • et al.
      Hepatitis E virus and chronic hepatitis in organ-transplant recipients.
      ,
      • Behrendt P.
      • Steinmann E.
      • Manns M.P.
      • Wedemeyer H.
      The impact of hepatitis E in the liver transplant setting.
      ]. Patients with chronic HEV may rapidly develop liver cirrhosis. Moreover, the recent availability of cell culture models (e.g., [
      • Okamoto H.
      Hepatitis E virus cell culture models.
      ,
      • 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.
      ]) offers new opportunities for the study of HEV biology and the development of therapeutic and/or prophylactic strategies.
      In this review article, we provide an overview on the molecular virology of HEV and its implications for clinical practice. Current understanding of the HEV life cycle, the viral tissue tropism and host antiviral response as well as potential antivirals shall be discussed.

      Clinical course of HEV

      Irrespective of the viral genotype, HEV infection leads to a self-limiting illness lasting for few weeks, with a broad range of clinical manifestations ranging from an asymptomatic course to acute liver failure, resulting in fatality rates of 0.2–4%. In general, after a 2–6 week incubation period, liver enzyme elevation occurs and may be accompanied by symptoms such as abdominal pain, nausea and vomiting, anorexia, fever, and jaundice [
      • Kamar N.
      • Bendall R.
      • Legrand-Abravanel F.
      • Xia N.S.
      • Ijaz S.
      • Izopet J.
      • et al.
      Hepatitis E.
      ,
      • Hoofnagle J.H.
      • Nelson K.E.
      • Purcell R.H.
      Hepatitis E.
      ].
      Hepatitis E virus (HEV) is an important cause of acute hepatitis in developing regions, with a high morbidity and mortality in pregnant women.

      Acute infection

      HEV strains infecting humans have been classified into 4 distinct genotypes belonging to a single serotype. Genotypes 1 and 2 are restricted to humans, are spread mainly through contaminated drinking water and represent main causes of waterborne outbreaks of hepatitis in developing regions (Fig. 1). The most severe course of disease is observed in pregnant women infected with HEV genotype 1, with high maternal, fetal and neonatal morbidity and mortality rates as high as 25% [
      • Khuroo M.S.
      • Teli M.R.
      • Skidmore S.
      • Sofi M.A.
      • Khuroo M.I.
      Incidence and severity of viral hepatitis in pregnancy.
      ,
      • Patra S.
      • Kumar A.
      • Trivedi S.S.
      • Puri M.
      • Sarin S.K.
      Maternal and fetal outcomes in pregnant women with acute hepatitis E virus infection.
      ,
      • Krain L.J.
      • Atwell J.E.
      • Nelson K.E.
      • Labrique A.B.
      Fetal and neonatal health consequences of vertically transmitted hepatitis E virus infection.
      ]. Capsid-based recombinant vaccines have proven their efficacy in large studies performed in Nepal and in China [
      • Shrestha M.P.
      • Scott R.M.
      • Joshi D.M.
      • Mammen Jr., M.P.
      • Thapa G.B.
      • Thapa N.
      • et al.
      Safety and efficacy of a recombinant hepatitis E vaccine.
      ,
      • Zhu F.C.
      • Zhang J.
      • Zhang X.F.
      • Zhou C.
      • Wang Z.Z.
      • Huang S.J.
      • et al.
      Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3 trial.
      ,
      • Zhang J.
      • Zhang X.F.
      • Huang S.J.
      • Wu T.
      • Hu Y.M.
      • Wang Z.Z.
      • et al.
      Long-term efficacy of a hepatitis E vaccine.
      ,
      Hepatitis E vaccine: WHO position paper, May 2015.
      ]. However, a vaccine has thus far been licensed only in China.
      HEV genotypes 3 and 4 are now recognized as zoonotic agents with their main reservoir in pigs and game (Fig. 1). Autochthonous infection occurs mostly through the consumption of un/under cooked meat [
      • Kamar N.
      • Bendall R.
      • Legrand-Abravanel F.
      • Xia N.S.
      • Ijaz S.
      • Izopet J.
      • et al.
      Hepatitis E.
      ]. HEV seroprevalence rates in developed regions range between 5 and 20% and peak at 52% in southwestern France [
      • Mansuy J.M.
      • Bendall R.
      • Legrand-Abravanel F.
      • Saune K.
      • Miedouge M.
      • Ellis V.
      • et al.
      Hepatitis E virus antibodies in blood donors, France.
      ]. Of note, we and others have found that middle-aged or elderly men are particularly prone to develop symptomatic autochthonous acute hepatitis E [
      • Kamar N.
      • Bendall R.
      • Legrand-Abravanel F.
      • Xia N.S.
      • Ijaz S.
      • Izopet J.
      • et al.
      Hepatitis E.
      ,
      • Doerig C.
      • Moulin H.
      • Müllhaupt B.
      • Pache I.
      • Bihl F.
      • Telenti A.
      • et al.
      Autochthonous acute hepatitis E in Switzerland: increased rate of severe manifestation in men >50 years.
      ].
      HEV can persist and cause chronic hepatitis in immunocompromised patients.

      Chronic infection

      Chronic HEV infections have been reported in immunocompromised patients such as organ transplant recipients and patients with HIV infection or hematological malignancies undergoing chemotherapy [
      • Kamar N.
      • Selves J.
      • Mansuy J.M.
      • Ouezzani L.
      • Peron J.M.
      • Guitard J.
      • et al.
      Hepatitis E virus and chronic hepatitis in organ-transplant recipients.
      ,
      • Behrendt P.
      • Steinmann E.
      • Manns M.P.
      • Wedemeyer H.
      The impact of hepatitis E in the liver transplant setting.
      ,
      • Colson P.
      • Kaba M.
      • Moreau J.
      • Brouqui P.
      Hepatitis E in an HIV-infected patient.
      ,
      • Dalton H.R.
      • Bendall R.P.
      • Keane F.E.
      • Tedder R.S.
      • Ijaz S.
      Persistent carriage of hepatitis E virus in patients with HIV infection.
      ,
      • Kamar N.
      • Garrouste C.
      • Haagsma E.B.
      • Garrigue V.
      • Pischke S.
      • Chauvet C.
      • et al.
      Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants.
      ,
      • Geng Y.
      • Zhang H.
      • Huang W.
      • Harriso T.J.
      • Geng K.
      • Li Z.
      • et al.
      Persistent hepatitis e virus genotype 4 infection in a child with acute lymphoblastic leukemia.
      ]. These chronic infections are caused by genotype 3 and possibly also genotype 4 and may rapidly evolve to cirrhosis and loss of a liver graft [
      • Kamar N.
      • Selves J.
      • Mansuy J.M.
      • Ouezzani L.
      • Peron J.M.
      • Guitard J.
      • et al.
      Hepatitis E virus and chronic hepatitis in organ-transplant recipients.
      ,
      • Behrendt P.
      • Steinmann E.
      • Manns M.P.
      • Wedemeyer H.
      The impact of hepatitis E in the liver transplant setting.
      ].

      Molecular organization of HEV

      HEV is a non-enveloped, small, icosahedral virus of about 27–34 nm in diameter [
      • Balayan M.S.
      • Andjaparidze A.G.
      • Savinskaya S.S.
      • Ketiladze E.S.
      • Braginsky D.M.
      • Savinov A.P.
      • et al.
      Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route.
      ,
      • Emerson S.U.
      • Purcell R.H.
      Hepatitis E virus.
      ]. Infectious virions contain a 7.2 kb single-stranded, positive-sense RNA genome harboring 3 open reading frames (ORFs). These are translated into (i) the ORF1 protein, comprising the functional domains required for RNA replication, (ii) the ORF2 protein, corresponding to the viral capsid; and (iii) the ORF3 protein, a small, hitherto poorly characterized phosphoprotein involved in viral particle secretion (Fig. 2A). The genomic RNA of HEV has the typical features of a eukaryotic mRNA, including a 7-methylguanylate (7mG) cap at its 5′ end and a poly-A stretch at the 3′ end, followed and preceded, respectively, by short untranslated regions.
      Figure thumbnail gr2
      Fig. 2Genetic organization and life cycle of the hepatitis E virus (HEV). (A) Genetic organization of HEV. The 7.2 kb positive-strand RNA genome has a 5′ 7-methylguanylate (7mG) cap and a 3′ polyadenylated (poly-A) tail. It comprises three open reading frames (ORF). ORF1 encodes a polyprotein of about 190 kDa which harbors methyltransferase (MeT), Y, putative papain-like cysteine protease (PCP), variable (V), macro, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. ORF2 and ORF3 are translated from a 2.2 kb subgenomic RNA generated during viral replication. The capsid protein encoded by ORF2 is N-glycosylated at 3 sites, i.e., Asn 132, Asn 310 and Asn 562. (B) The HEV life cycle includes the following steps: 1) viral attachment to heparin sulfate proteoglycans and entry through as yet unidentified receptor(s); 2) clathrin-mediated endocytosis; 3) release of the viral positive-strand RNA genome into the cytosol; 4) translation to yield the ORF1 protein; 5) replication through a negative-strand RNA intermediate and synthesis of full-length as well as a 2.2 kb subgenomic RNAs; 6) translation of the subgenomic RNA to yield the ORF2 and ORF3 proteins; and 7) packaging, assembly and release of newly formed virus. ORF3 protein is likely associated with intracellular membranes and may trigger virion release via the endosomal sorting complexes required for transport (ESCRT) pathway. Recent studies suggest that virus secreted into the bloodstream is associated with the ORF3 protein and wrapped by cellular membranes while virus secreted into the bile is non-enveloped.
      Two viral RNA species are generated during HEV genome replication, the full-length RNA of 7.2 kb and a subgenomic RNA of 2.2 kb (Fig. 2A) [
      • Graff J.
      • Torian U.
      • Nguyen H.
      • Emerson S.U.
      A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus.
      ,
      • Ichiyama K.
      • Yamada K.
      • Tanaka T.
      • Nagashima S.
      • Jirintai
      • Takahashi M.
      • et al.
      Determination of the 5′-terminal sequence of subgenomic RNA of hepatitis E virus strains in cultured cells.
      ]. The latter species starts a few nucleotides downstream of the ORF1 stop codon [
      • Graff J.
      • Torian U.
      • Nguyen H.
      • Emerson S.U.
      A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus.
      ] and allows the expression of ORF3 and ORF2. The start codons of these two ORFs are separated by only 11 nucleotides and result in frameshifted products (Fig. 2A). The regulation of ORF2 and ORF3 expression remains poorly understood to date. As a consequence of the overlap of the two ORFs, this sequence is highly conserved intra- and intergenotypically, allowing the establishment of a robust and pangenotypic PCR-based diagnostic test. This assay is now widely used to diagnose acute or chronic HEV infection [
      • Aggarwal R.
      Diagnosis of hepatitis E.
      ].

      ORF1

      ORF1, representing about 2/3 of the genome length, encodes the so-called HEV replicase. Domains specifically required for viral RNA replication have been computationally assigned based on sequence homology with other viruses [
      • Koonin E.V.
      • Gorbalenya A.E.
      • Purdy M.A.
      • Rozanov M.N.
      • Reyes G.R.
      • Bradley D.W.
      Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.
      ]. These include methyltransferase (MeT), macro, RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains [
      • Agrawal S.
      • Gupta D.
      • Panda S.K.
      The 3′ end of hepatitis E virus genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp).
      ] (Fig. 2A). The MeT, Hel and RdRp have also been functionally studied. However, the functions of the other (predicted) domains, namely the Y, papain-like cysteine protease (PCP) and variable (V) domains, remain uncertain. Thus, the function of the putative PCP, which shows a weak homology with the Rubella virus protease [
      • Koonin E.V.
      • Gorbalenya A.E.
      • Purdy M.A.
      • Rozanov M.N.
      • Reyes G.R.
      • Bradley D.W.
      Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.
      ], is a subject of debate (Fig. 2A). Indeed, positive-strand RNA viruses commonly encode proteases to process their polyproteins, and several reports suggested that this is the case also for the PCP and the ORF1 protein [
      • Ropp S.L.
      • Tam A.W.
      • Beames B.
      • Purdy M.
      • Frey T.K.
      Expression of the hepatitis E virus ORF1.
      ,
      • Panda S.K.
      • Ansari I.H.
      • Durgapal H.
      • Agrawal S.
      • Jameel S.
      The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious.
      ,
      • Sehgal D.
      • Thomas S.
      • Chakraborty M.
      • Jameel S.
      Expression and processing of the Hepatitis E virus ORF1 nonstructural polyprotein.
      ,
      • Parvez M.K.
      Molecular characterization of hepatitis E virus ORF1 gene supports a papain-like cysteine protease (PCP)-domain activity.
      ,
      • Paliwal D.
      • Panda S.K.
      • Kapur N.
      • Varma S.P.
      • Durgapal H.
      Hepatitis E virus protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.
      ]. However, others have not found any evidence of HEV ORF1 processing [
      • Ansari I.H.
      • Nanda S.K.
      • Durgapal H.
      • Agrawal S.
      • Mohanty S.K.
      • Gupta D.
      • et al.
      Cloning, sequencing, and expression of the hepatitis E virus nonstructural open reading frame 1 (ORF1).
      ,
      • Suppiah S.
      • Zhou Y.
      • Frey T.K.
      Lack of processing of the expressed ORF1 gene product of hepatitis E virus.
      ,
      • Perttila J.
      • Spuul P.
      • Ahola T.
      Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1.
      ]. Using a panel of ORF1 expression systems, including a recently developed HEV replicon [
      • 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.
      ,
      • 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.
      ], we only observed the full-length form of ORF1 protein (VL Dao Thi, T Ahola, DM and JG, unpublished). An emerging hypothesis suggests that the PCP domain, rather than having protease activity, may display broad de-ubiquitination activity, including deISGylation of ISG15-modified proteins [
      • Karpe Y.A.
      • Lole K.S.
      Deubiquitination activity associated with hepatitis E virus putative papain-like cysteine protease.
      ]. In this way, the PCP domain may prevent the proteosomal degradation of selected proteins, possibly those required for viral RNA replication. Clearly, further work is needed in this interesting area.
      Similar to alphavirus replicase proteins, ORF1 exhibits virus-specific methyltransferase and guanylyltransferase activities, allowing the transfer of a methyl group to guanosine triphosphate (GTP), yielding m7GTP (7-methylguanosine), and the covalent coupling of the m7GTP product with the 5′ end of the viral RNA, respectively [
      • Magden J.
      • Takeda N.
      • Li T.
      • Auvinen P.
      • Ahola T.
      • Miyamura T.
      • et al.
      Virus-specific mRNA capping enzyme encoded by hepatitis E virus.
      ]. However, as these observations were made with a construct comprising a large portion of ORF1, including the MeT, Y and PCP domains (ORF1 amino acids [aa] 1-979), one cannot yet formally attribute these enzymatic activities to the methyltransferase domain only.
      The HEV RNA helicase domain is capable of unwinding RNA duplexes with a 5′ single-stranded overhang in the 5′-to-3′ direction in vitro [
      • Karpe Y.A.
      • Lole K.S.
      NTPase and 5′ to 3′ RNA duplex-unwinding activities of the hepatitis E virus helicase domain.
      ]. In addition, the same recombinant HEV helicase protein displays a 5′-nucleoside triphosphatase (NTPase) activity that is required for RNA capping [
      • Karpe Y.A.
      • Lole K.S.
      RNA 5′-triphosphatase activity of the hepatitis E virus helicase domain.
      ]. Therefore, HEV, as other positive-strand RNA viruses including the hepatitis C virus (HCV), may have coupled these two functions in one protein to economize on coding capacity of the viral genome.
      Macro domains, known as ADP-ribose-binding modules [
      • Karras G.I.
      • Kustatscher G.
      • Buhecha H.R.
      • Allen M.D.
      • Pugieux C.
      • Sait F.
      • et al.
      The macro domain is an ADP-ribose binding module.
      ], can interact with proteins modified by ADP-ribosylation. This modification is involved in the regulation of various cellular functions, such as transcription, chromatin organization, organelle assembly, protein degradation and DNA repair [
      • Han W.
      • Li X.
      • Fu X.
      The macro domain protein family: structure, functions, and their potential therapeutic implications.
      ]. While several positive-strand RNA viruses, such as corona- and alphaviruses, encode a macro domain, its exact role in the viral life cycle remains unclear. In vitro characterization of the HEV macro domain revealed its binding to poly(ADP-ribose) and poly(A), possibly simultaneously [
      • Neuvonen M.
      • Ahola T.
      Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.
      ], thereby suggesting the possible recruitment of poly(ADP-ribosyl)ated cellular factors to the replication complex while bound to the viral poly-A tail.

      ORF2

      Given its antigenic properties as well as interest for diagnostic test and vaccine development, the capsid protein encoded by ORF2 is the best studied of the three HEV proteins. ORF2 encodes a 72 kDa protein of 660 aa, which possesses an N-terminal signal peptide driving its secretion into the extracellular compartment. The ORF2 protein is N-glycosylated at three sites when expressed in mammalian cells [
      • Jameel S.
      • Zafrullah M.
      • Ozdener M.H.
      • Panda S.K.
      Expression in animal cells and characterization of the hepatitis E virus structural proteins.
      ,
      • Torresi J.
      • Li F.
      • Locarnini S.A.
      • Anderson D.A.
      Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells.
      ], i.e., Asn 132, Asn 310 and Asn 562 (Fig. 2A) [
      • Zafrullah M.
      • Ozdener M.H.
      • Kumar R.
      • Panda S.K.
      • Jameel S.
      Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein.
      ]. During the viral life cycle, the ORF2 protein may interact with host factors such as heat shock protein 90 [
      • Zheng Z.Z.
      • Miao J.
      • Zhao M.
      • Tang M.
      • Yeo A.E.
      • Yu H.
      • et al.
      Role of heat-shock protein 90 in hepatitis E virus capsid trafficking.
      ], glucose-regulated protein 78 (also known as BiP) [
      • Yu H.
      • Li S.
      • Yang C.
      • Wei M.
      • Song C.
      • Zheng Z.
      • et al.
      Homology model and potential virus-capsid binding site of a putative HEV receptor Grp78.
      ], and heparin sulfate proteoglycans which may serve as initial attachment factors [
      • Kalia M.
      • Chandra V.
      • Rahman S.A.
      • Sehgal D.
      • Jameel S.
      Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection.
      ].

      ORF3

      The ORF3 protein is a 13-kDa protein of 113 (genotype 3) or 114 aa (genotypes 1, 2 and 4). It is phosphorylated at the conserved serine residue 71 (Ser 70 in genotype 3) by the cellular mitogen-activated protein kinase (MAPK) [
      • Zafrullah M.
      • Ozdener M.H.
      • Panda S.K.
      • Jameel S.
      The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.
      ]. A yeast two-hybrid study suggested homodimerization of ORF3 protein through a proline-rich C-terminal region [
      • Tyagi S.
      • Jameel S.
      • Lal S.K.
      Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein.
      ]. Primary sequence analyses of the ORF3 protein did not reveal any domains homologous to other proteins or other distinguishing features, except for the presence of two hydrophobic N-terminal domains spanning aa 7 to 23 and aa 28 to 53. Early reports indicated that ORF3 protein associates, via the N-terminal hydrophobic domain [
      • Zafrullah M.
      • Ozdener M.H.
      • Panda S.K.
      • Jameel S.
      The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.
      ], with the cytoskeleton and, more specifically, with microtubules [
      • Kannan H.
      • Fan S.
      • Patel D.
      • Bossis I.
      • Zhang Y.J.
      The hepatitis E virus open reading frame 3 product interacts with microtubules and interferes with their dynamics.
      ]. The protein has also been observed at early and recycling endosomes [
      • Chandra V.
      • Kar-Roy A.
      • Kumari S.
      • Mayor S.
      • Jameel S.
      The hepatitis E virus ORF3 protein modulates epidermal growth factor receptor trafficking, STAT3 translocation, and the acute-phase response.
      ] or multivesicular bodies (MVB) and has been implicated in HEV egress [
      • Nagashima S.
      • Takahashi M.
      • Jirintai S.
      • Tanaka T.
      • Nishizawa T.
      • Yasuda J.
      • et al.
      Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.
      ,
      • Nagashima S.
      • Takahashi M.
      • Jirintai S.
      • Tanggis
      • Kobayashi T.
      • Nishizawa T.
      • et al.
      The membrane on the surface of hepatitis E virus particles is derived from the intracellular membrane and contains trans-Golgi network protein 2.
      ]. Therefore, the subcellular localization, structure and function of the ORF3 protein remain to be fully explored.
      The HEV life cycle is only partly understood, yielding attractive research opportunities.

      HEV life cycle

      As a first contact with the target cell, HEV interacts, similar to many other viruses [
      • Bartlett A.
      • Park P.
      Glycans in diseases and therapeutics. Biology of extracellular matrix.
      ], with heparan sulfate proteoglycans, probably syndecans, allowing initial attachment of the virus [
      • Kalia M.
      • Chandra V.
      • Rahman S.A.
      • Sehgal D.
      • Jameel S.
      Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection.
      ] (Fig. 2B). The receptor(s) governing entry of the virus are still unidentified. The downstream cascade of events includes clathrin-dependent endocytosis involving dynamin-2 and membrane cholesterol pathways [
      • Holla P.
      • Ahmad I.
      • Ahmed Z.
      • Jameel S.
      Hepatitis E virus enters liver cells through a dynamin-2, clathrin and membrane cholesterol-dependent pathway.
      ]. Moreover, cytoskeleton remodeling is crucial for HEV endocytosis [
      • Holla P.
      • Ahmad I.
      • Ahmed Z.
      • Jameel S.
      Hepatitis E virus enters liver cells through a dynamin-2, clathrin and membrane cholesterol-dependent pathway.
      ]. Post-entry steps, including viral genome uncoating and release, have not been addressed. However, recent data suggest a low-pH-independent mechanism [
      • Holla P.
      • Ahmad I.
      • Ahmed Z.
      • Jameel S.
      Hepatitis E virus enters liver cells through a dynamin-2, clathrin and membrane cholesterol-dependent pathway.
      ] (Fig. 2B).
      HEV RNA replication requires, first, translation of the viral replicase by the host translation machinery (Fig. 2B). As for all positive-strand RNA viruses, the mechanism of RNA replication involves the synthesis of a complementary negative-strand RNA by the HEV RdRp and the subsequent synthesis of genomic positive-strand RNA from this negative-strand RNA template. The HEV RNA helicase is expected to fulfill a crucial role in this process by unwinding the two RNA strands [
      • Karpe Y.A.
      • Lole K.S.
      NTPase and 5′ to 3′ RNA duplex-unwinding activities of the hepatitis E virus helicase domain.
      ]. RNA capping likely results from the cooperation of the 5′ triphosphatase activity, harbored by the helicase domain, followed by the transfer of a 7mG to the 5′ end of newly synthesized genomes by the methyltransferase domain [
      • Magden J.
      • Takeda N.
      • Li T.
      • Auvinen P.
      • Ahola T.
      • Miyamura T.
      • et al.
      Virus-specific mRNA capping enzyme encoded by hepatitis E virus.
      ,
      • Karpe Y.A.
      • Lole K.S.
      RNA 5′-triphosphatase activity of the hepatitis E virus helicase domain.
      ].
      The site of RNA replication within the host cell has not been identified yet. However, the ORF1 protein has been shown to be membrane-associated and to localize to an intermediate compartment between the endoplasmic reticulum and the Golgi, suggesting a localization within the early secretory pathway [
      • Perttila J.
      • Spuul P.
      • Ahola T.
      Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1.
      ]. As the formation of a replication complex composed of viral proteins, replicating viral RNA and rearranged cellular membranes is a hallmark of positive-strand RNA viruses [
      • Miller S.
      • Krijnse-Locker J.
      Modification of intracellular membrane structures for virus replication.
      ], one may hypothesize that HEV also induces specific membrane rearrangements which, however, have yet to be identified.
      Further, subgenomic RNA is generated and capped to allow translation of the ORF2 and ORF3 proteins (Fig. 2B). Subgenomic RNA is synthesized by the RdRp. However, the mechanisms regulating genome length vs. subgenomic RNA synthesis are unknown. The capsid protein assembles and packages the capped genomic RNA. Virtually no data are available on the assembly step. However, the spontaneous assembly of RNA and ORF2 protein into virus-like particles in insect cells argues in favor of a self-assembly process involving only a limited number of viral or host factors ([
      • Xing L.
      • Li T.C.
      • Mayazaki N.
      • Simon M.N.
      • Wall J.S.
      • Moore M.
      • et al.
      Structure of hepatitis E virion-sized particle reveals an RNA-dependent viral assembly pathway.
      ]; reviewed in [
      • Mori Y.
      • Matsuura Y.
      Structure of hepatitis E viral particle.
      ]). It implies that newly synthesized genomes must be present in close proximity of the capsid protein to allow virion formation. Investigation of the intraviral interactome revealed a number of protein-protein interactions which support the existence of (a) viral protein complex(es) [
      • Osterman A.
      • Stellberger T.
      • Gebhardt A.
      • Kurz M.
      • Friedel C.C.
      • Uetz P.
      • et al.
      The hepatitis E virus intraviral interactome.
      ].
      Viral egress is believed to require the cellular secretory machinery together with the ORF3 protein. Several studies have demonstrated that a conserved PSAP motif in the ORF3 protein (aa 95-98 in genotype 3) is involved in the interaction with tumor susceptibility gene 101 (Tsg101), a component of the endosomal sorting complexes required for transport (ESCRT) pathway [
      • Nagashima S.
      • Takahashi M.
      • Jirintai S.
      • Tanaka T.
      • Nishizawa T.
      • Yasuda J.
      • et al.
      Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.
      ,
      • Surjit M.
      • Oberoi R.
      • Kumar R.
      • Lal S.K.
      Enhanced alpha1 microglobulin secretion from Hepatitis E virus ORF3-expressing human hepatoma cells is mediated by the tumor susceptibility gene 101.
      ,
      • Nagashima S.
      • Takahashi M.
      • Jirintai
      • Tanaka T.
      • Yamada K.
      • Nishizawa T.
      • et al.
      A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells.
      ]. Moreover, inhibition of HEV release by dominant-negative mutants of the pathway confirmed that HEV hijacks the ESCRT machinery for virion release from infected cells [
      • Nagashima S.
      • Takahashi M.
      • Jirintai S.
      • Tanaka T.
      • Nishizawa T.
      • Yasuda J.
      • et al.
      Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.
      ]. Interestingly, abrogation of ORF3 protein expression in an infectious clone resulted in impaired particle secretion in vitro, with intracellular accumulation of infectious virus with a high density [
      • Yamada K.
      • Takahashi M.
      • Hoshino Y.
      • Takahashi H.
      • Ichiyama K.
      • Nagashima S.
      • et al.
      ORF3 protein of hepatitis E virus is essential for virion release from infected cells.
      ]. Virus of comparable density has been observed in bile or feces while HEV circulating in the blood has a low density [
      • Nagashima S.
      • Takahashi M.
      • Jirintai S.
      • Tanaka T.
      • Nishizawa T.
      • Yasuda J.
      • et al.
      Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.
      ,
      • Yamada K.
      • Takahashi M.
      • Hoshino Y.
      • Takahashi H.
      • Ichiyama K.
      • Nagashima S.
      • et al.
      ORF3 protein of hepatitis E virus is essential for virion release from infected cells.
      ,
      • Nagashima 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.
      ]. Density gradient investigation as well as ultrastructural analyses revealed the presence of host membranes enveloping the HEV particle in cells and culture supernatants, thereby confirming the biochemical evidence for secretion into the bloodstream of quasi-enveloped HEV [
      • Takahashi M.
      • Yamada K.
      • Hoshino Y.
      • Takahashi H.
      • Ichiyama K.
      • Tanaka T.
      • et al.
      Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus can capture HEV particles in culture supernatant and serum but not those in feces.
      ,
      • Takahashi M.
      • Tanaka T.
      • Takahashi H.
      • Hoshino Y.
      • Nagashima S.
      • Jirintai
      • et al.
      Hepatitis E Virus strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation.
      ].
      Hence, while non-enveloped HEV is found in bile and feces, the virion found in blood appears to be wrapped by cellular membranes [
      • Nagashima S.
      • Takahashi M.
      • Jirintai S.
      • Tanaka T.
      • Nishizawa T.
      • Yasuda J.
      • et al.
      Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.
      ,
      • Nagashima 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.
      ]. Similar observations have recently been reported for a rat HEV strain, suggesting that this feature is conserved among hepatitis E viruses [
      • Jirintai S.
      • Tanggis
      • Mulyanto
      • Suparyatmo J.B.
      • Takahashi M.
      • Kobayashi T.
      • et al.
      Rat hepatitis E virus derived from wild rats (Rattus rattus) propagates efficiently in human hepatoma cell lines.
      ]. Interestingly, the secretion of membrane-wrapped virions has also been reported for hepatitis A virus, a member of the Picornaviridae family ([
      • 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.
      ]; reviewed in [
      • Feng Z.
      • Lemon S.M.
      Peek-a-boo: membrane hijacking and the pathogenesis of viral hepatitis.
      ]). While the mechanism underlying HEV membrane envelopment as well as the nature and the composition of the membrane are yet to be further defined, ORF3 protein appears to be at the heart of particle secretion and possibly formation. Importantly, ORF3 protein is present on the secreted membrane-wrapped virion, as demonstrated by capture of HEV particles by ORF3 antibodies in culture supernatant and serum but not in feces [
      • Takahashi M.
      • Tanaka T.
      • Takahashi H.
      • Hoshino Y.
      • Nagashima S.
      • Jirintai
      • et al.
      Hepatitis E Virus strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation.
      ] (Fig. 2B).
      It is tempting to speculate that membrane envelopment protects HEV against neutralizing antibodies when present in the serum, as supported by successful in vitro HEV infection in the presence of anti-ORF2 antibodies [
      • Takahashi M.
      • Tanaka T.
      • Takahashi H.
      • Hoshino Y.
      • Nagashima S.
      • Jirintai
      • et al.
      Hepatitis E Virus strains in serum samples can replicate efficiently in cultured cells despite the coexistence of HEV antibodies: characterization of HEV virions in blood circulation.
      ].

      Interference of HEV with host antiviral defenses

      While HEV infection is almost always self-limited, persistent infection may be observed in immunocompromised patients. In these, the virus can persist in the liver in the absence of a competent adaptive immune response. Hence, this clinical observation would suggest that innate immunity alone does not suffice to clear viral infection but that a concerted action of innate and adaptive immunity is required. Like many other viruses, HEV may have developed strategies to subvert these host antiviral defenses. However, these strategies are poorly understood to date.
      Viral infection usually elicits an immune response involving the production of type I and III interferon (IFN) after recognition of the viral RNA as a pathogen-associated molecular pattern by pattern recognition receptors (PRR) [
      • Hoffmann H.H.
      • Schneider W.M.
      • Rice C.M.
      Interferons and viruses: an evolutionary arms race of molecular interactions.
      ]. Signaling cascades downstream of PRR lead to the induction and production of type I and III IFN through the activation of key molecules such as TANK-binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB). Secreted IFNs can then induce interferon-stimulated gene (ISG) transcription in other cells via the JAK/STAT signaling cascade (Fig. 3). Few studies tried to clarify the consequences of HEV infection on the host innate immune response. The ORF3 protein has been shown to enhance type I IFN production via a direct interaction with the PRR retinoic acid-inducible gene I (RIG-I) (Fig. 3) [
      • Nan Y.
      • Ma Z.
      • Wang R.
      • Yu Y.
      • Kannan H.
      • Fredericksen B.
      • et al.
      Enhancement of interferon induction by ORF3 product of hepatitis E virus.
      ]. Under similar experimental conditions, the same authors found that the ORF1 protein inhibits RIG-I signaling and prevents IFNβ induction by de-ubiquitination of RIG-I and TBK1 [
      • Nan Y.
      • Yu Y.
      • Ma Z.
      • Khattar S.K.
      • Fredericksen B.
      • Zhang Y.J.
      Hepatitis E virus inhibits type I interferon induction by ORF1 products.
      ]. According to this latter study, the de-ubiquitinating activity of the PCP domain but also the macro domain could be directly involved (Fig. 3). The observation that the virus may exert opposing effects on the host antiviral response via two different viral proteins is intriguing. In addition, transcriptional analyses of A549 human lung epithelial cells infected by HEV revealed an enhanced expression of proinflammatory cytokines, i.e. IL-6, IL-8 and RANTES, as well as activation of the IRF3 and NF-κB pathways (Fig. 3) [
      • Devhare P.B.
      • Chatterjee S.N.
      • Arankalle V.A.
      • Lole K.S.
      Analysis of antiviral response in human epithelial cells infected with hepatitis E virus.
      ], indicating that the control of antiviral responses by HEV is only partly effective.
      Figure thumbnail gr3
      Fig. 3Interference of HEV with host antiviral responses. Upon HEV infection and release of the viral genome into the cytoplasm, host antiviral defenses sense the viral RNA through RIG-I and signal via downstream cascades leading to type I and III interferon (IFN) production. Once translated, the HEV ORF1 protein has been reported to inhibit signaling via retinoic acid-inducible gene I (RIG-I) and to prevent IFN induction by de-ubiquitination of RIG-I and TANK-binding kinase 1 (TBK-1). At the same time, the HEV ORF3 protein may have an opposing effect by enhancing type I IFN production via direct interaction with RIG-I. Consequently, HEV infection leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways, inducing the expression of IFNs and proinflammatory cytokines, i.e., IL-6, IL-8 and RANTES. IFNs activate the JAK/STAT pathway in a paracrine and autocrine manner, resulting in the induction of IFN-stimulated genes (ISGs). In the case of HEV infection, binding of the ORF3 protein to Stat1 has been reported to restrict its phosphorylation and the activation of the downstream cascade, thereby inhibiting ISG expression.
      To overcome the lack of complete blockade of the RNA sensing pathway, interference of HEV with IFN signaling or effector functions would be a plausible hypothesis. In general, after viral RNA sensing and activation of the downstream cascade, type I and III IFN is being produced and functions in a paracrine and autocrine manner. In the case of HEV infection, an inhibition of the IFNα-induced phosphorylation of Stat1 in infected cells as well as a downregulation of ISG expression have been observed [
      • Dong C.
      • Zafrullah M.
      • Mixson-Hayden T.
      • Dai X.
      • Liang J.
      • Meng J.
      • et al.
      Suppression of interferon-alpha signaling by hepatitis E virus.
      ]. In line with this observation, ISG expression induced by either type I, II or III IFNs has been found to be limited in cells replicating an infectious HEV clone [
      • Todt D.
      • Francois C.
      • Anggakusuma
      • Behrendt P.
      • Engelmann M.
      • Knegendorf L.
      • et al.
      Antiviral activity of different interferon (sub-) types against hepatitis E virus replication.
      ]. The proposed mechanism involves the binding of ORF3 protein to Stat1 to restrict its phosphorylation and, thereby, its activation (Fig. 3) [
      • Dong C.
      • Zafrullah M.
      • Mixson-Hayden T.
      • Dai X.
      • Liang J.
      • Meng J.
      • et al.
      Suppression of interferon-alpha signaling by hepatitis E virus.
      ]. Taken together, the ORF3 protein may exert a dual and opposite effect: enhancing IFNα production on the one hand and limiting IFNα signaling and effector functions on the other. Independent confirmation of this function of ORF3 is still awaited, however.
      As IFNα showed strong antiviral activity in vitro and has proven its efficacy to clear the virus in patients with chronic hepatitis E [
      • 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.
      ,
      • Todt D.
      • Francois C.
      • Anggakusuma
      • Behrendt P.
      • Engelmann M.
      • Knegendorf L.
      • et al.
      Antiviral activity of different interferon (sub-) types against hepatitis E virus replication.
      ,
      • Kamar N.
      • Rostaing L.
      • Abravanel F.
      • Garrouste C.
      • Esposito L.
      • Cardeau-Desangles I.
      • et al.
      Pegylated interferon-alpha for treating chronic hepatitis E virus infection after liver transplantation.
      ,
      • Debing Y.
      • Neyts J.
      Antiviral strategies for hepatitis E virus.
      ], we may hypothesize that IFNs play a crucial role in viral clearance mediated by the immune system [
      • Lanford R.E.
      • Feng Z.
      • Chavez D.
      • Guerra B.
      • Brasky K.M.
      • Zhou Y.
      • et al.
      Acute hepatitis A virus infection is associated with a limited type I interferon response and persistence of intrahepatic viral RNA.
      ]. Differences between HCV and HEV infections at the host transcriptional level have been investigated in the chimpanzee model, revealing a lower ISG induction in acute HEV infection as compared to acute HCV infection [
      • Yu C.
      • Boon D.
      • McDonald S.L.
      • Myers T.G.
      • Tomioka K.
      • Nguyen H.
      • et al.
      Pathogenesis of hepatitis E virus and hepatitis C virus in chimpanzees: similarities and differences.
      ]. Such studies performed over the entire course of acute hepatitis E could not be easily conducted in humans. However, ISG levels may be more easily explored in patients with chronic hepatitis E. A preliminary study conducted on a limited number of whole-blood samples from chronically HEV-infected patients showed a modulation of about 30 ISGs. Thus, whereas IFN pathway activation is not sufficient to clear viral infection, basal activation of IFN signaling is detectable in these patients [
      • Moal V.
      • Textoris J.
      • Ben Amara A.
      • Mehraj V.
      • Berland Y.
      • Colson P.
      • et al.
      Chronic hepatitis E virus infection is specifically associated with an interferon-related transcriptional program.
      ]. Investigation of ISG levels in chronic hepatitis E in the blood and in the liver should be explored to further understand the mechanisms of HEV persistence in immunocompromised patients.
      HEV tropism may not be restricted to the liver, possibly explaining some extrahepatic manifestations.

      HEV tissue tropism and associated extrahepatic manifestations

      Although HEV is a primarily hepatotropic virus, it may also replicate to some extent in other tissues, as extrahepatic manifestations such as neurological symptoms, myositis as well as renal and hematologic complications have been observed in the context of hepatitis E [
      • Colson P.
      • Kaba M.
      • Moreau J.
      • Brouqui P.
      Hepatitis E in an HIV-infected patient.
      ,
      • Del Bello A.
      • Arne-Bes M.C.
      • Lavayssiere L.
      • Kamar N.
      Hepatitis E virus-induced severe myositis.
      ,
      • Belbezier A.
      • Deroux A.
      • Sarrot-Reynauld F.
      • Larrat S.
      • Bouillet L.
      Myasthenia gravis associated with acute hepatitis E infection in immunocompetent woman.
      ,
      • Pischke S.
      • Behrendt P.
      • Manns M.P.
      • Wedemeyer H.
      HEV-associated cryoglobulinaemia and extrahepatic manifestations of hepatitis E.
      ] (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Reported sites of HEV replication. HEV infects and replicates primarily in the liver. However, studies performed in animal models reported HEV replication also in the small intestine, colon and lymph nodes as well as kidney, spleen and stomach. Furthermore, replication in the kidney has been recently suggested by the presence of HEV in the urine of patients with acute and chronic HEV as well as experimentally infected monkeys. Among extrahepatic manifestations, neurological complications are the most frequent. HEV RNA has been found in the cerebrospinal fluid of some patients with such complications and evidence for intrathecal antibody production has been provided in one case, suggesting possible infection of the central nervous system. The most severe symptoms are observed in pregnant women, possibly related to the reported infection of placental tissue.
      Experimental HEV infection in animals allowed the detection of negative-strand viral RNA, suggestive of ongoing viral replication, in the liver but also in the small intestine, colon and lymph nodes of pigs [
      • Williams T.P.
      • Kasorndorkbua C.
      • Halbur P.G.
      • Haqshenas G.
      • Guenette D.K.
      • Toth T.E.
      • et al.
      Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model.
      ]. Furthermore, the presence of negative-strand RNA intermediate has been reported in the liver, kidney, small intestine, spleen and stomach in a rabbit HEV model [
      • Liu P.
      • Bu Q.N.
      • Wang L.
      • Han J.
      • Du R.J.
      • Lei Y.X.
      • et al.
      Transmission of hepatitis E virus from rabbits to cynomolgus macaques.
      ]. Given the fecal-oral transmission of HEV, the virus is likely replicating first in the gastro-intestinal tract as a primary site of infection, wherefrom the virus can reach the bloodstream to infect other organs, similarly to what was observed for avian influenza virus H5N1 and rotavirus [
      • Reperant L.A.
      • van de Bildt M.W.
      • van Amerongen G.
      • Leijten L.M.
      • Watson S.
      • Palser A.
      • et al.
      Marked endotheliotropism of highly pathogenic avian influenza virus H5N1 following intestinal inoculation in cats.
      ,
      • Ramig R.F.
      Pathogenesis of intestinal and systemic rotavirus infection.
      ].
      Among the most notorious extrahepatic manifestations of HEV infection are neurological complications such as neuralgic amyotrophy (brachial neuritis, Parsonage-Turner syndrome) and Guillain-Barré syndrome [
      • Dalton H.R.
      • Kamar N.
      • van Eijk J.J.
      • McLean B.N.
      • Cintas P.
      • Bendall R.P.
      • et al.
      Hepatitis E virus and neurological injury.
      ]. Neurological manifestations have been observed in both acute and chronic HEV, but the exact incidence and underlying pathogenic mechanisms are not clear yet [
      • Dalton H.R.
      • Kamar N.
      • van Eijk J.J.
      • McLean B.N.
      • Cintas P.
      • Bendall R.P.
      • et al.
      Hepatitis E virus and neurological injury.
      ]. It has been suggested that immune reactions triggered by HEV infection may play a role, e.g., by the development of antiganglioside antibodies through molecular mimicry [
      • Cheung M.C.
      • Maguire J.
      • Carey I.
      • Wendon J.
      • Agarwal K.
      Review of the neurological manifestations of hepatitis E infection.
      ]. A few studies have reported the presence of HEV RNA in the cerebrospinal fluid (CSF) of some but not all patients with neurological complications, usually at considerably lower titers than in the serum [
      • Kamar N.
      • Izopet J.
      • Cintas P.
      • Garrouste C.
      • Uro-Coste E.
      • Cointault O.
      • et al.
      Hepatitis E virus-induced neurological symptoms in a kidney-transplant patient with chronic hepatitis.
      ,
      • Kamar N.
      • Bendall R.P.
      • Peron J.M.
      • Cintas P.
      • Prudhomme L.
      • Mansuy J.M.
      • et al.
      Hepatitis E virus and neurologic disorders.
      ,
      • Despierres L.A.
      • Kaphan E.
      • Attarian S.
      • Cohen-Bacrie S.
      • Pelletier J.
      • Pouget J.
      • et al.
      Neurologic disorders and hepatitis E, France, 2010.
      ]. In addition, evidence for intrathecal antibody production has been reported (Silva, M. et al. Revised version submitted). Finally, the viral quasispecies was analyzed in the serum and CSF of a single kidney transplant recipient with chronic hepatitis E and neurological symptoms; HEV sequences in the CSF were found to be distinct from those in the serum in this patient [
      • Kamar N.
      • Izopet J.
      • Cintas P.
      • Garrouste C.
      • Uro-Coste E.
      • Cointault O.
      • et al.
      Hepatitis E virus-induced neurological symptoms in a kidney-transplant patient with chronic hepatitis.
      ]. Even though quasispecies compartmentalization requires confirmation at a larger scale, it suggests the existence of neurotropic HEV variants and possibly active viral replication in the central nervous system. Whereas we did not observe robust viral replication in stem cell-derived neural progenitor cells infected with genotype 3 HEV [
      • Helsen 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.
      ], the virus can efficiently replicate in neuroblastoma cells in vitro (VL Dao Thi, DM and JG, unpublished data).
      Potential infection of the human placenta by HEV may explain intrauterine vertical transmission, the often severe course of HEV in pregnant women and the associated obstetric complications (see above). Indeed, active HEV replication in the placenta of HEV-infected pregnant women has been suggested by the detection of both negative-strand viral RNA as well as ORF3 protein and has been associated with fetal loss or maternal acute liver failure [
      • Bose P.D.
      • Das B.C.
      • Hazam R.K.
      • Kumar A.
      • Medhi S.
      • Kar P.
      Evidence of extrahepatic replication of hepatitis E virus in human placenta.
      ]. In line with these findings, we have also observed HEV genotype 3 replication in the human placental cell line JEG3 in vitro (S. Drave, YD, JN and E. Steinmann; unpublished results).
      The kidney tropism of HEV suggested by renal disorders [
      • Kamar N.
      • Mansuy J.M.
      • Esposito L.
      • Legrand-Abravanel F.
      • Peron J.M.
      • Durand D.
      • et al.
      Acute hepatitis and renal function impairment related to infection by hepatitis E virus in a renal allograft recipient.
      ,
      • Kamar N.
      • Weclawiak H.
      • Guilbeau-Frugier C.
      • Legrand-Abravanel F.
      • Cointault O.
      • Ribes D.
      • et al.
      Hepatitis E virus and the kidney in solid-organ transplant patients.
      ], has been recently confirmed by the detection of HEV in urine of acute and chronic HEV-infected patients and monkeys as well as by immunohistochemical evidence of infected cells in the kidney of one of these animals [
      • Geng Y.
      • Zhao C.
      • Huang W.
      • Harrison T.J.
      • Zhang H.
      • Geng K.
      • et al.
      Detection and assessment of infectivity of hepatitis E virus in urine.
      ]. Other reported extrahepatic manifestations include acute pancreatitis and hematological manifestations [
      • Kamar N.
      • Abravanel F.
      • Lhomme S.
      • Rostaing L.
      • Izopet J.
      Hepatitis E virus: chronic infection, extra-hepatic manifestations, and treatment.
      ], but no additional evidence is available regarding HEV tropism in these tissues.
      Future research efforts regarding HEV tissue tropism should focus on (i) the identification of cell entry receptor(s), (ii) confirming viral replication in extrahepatic sites such as neural tissue and kidney, in both animal and human samples; (iii) determining whether specific HEV variants are responsible for neurotropism or other extrahepatic manifestations as well as delineating the responsible genetic determinants; and (iv) the development of in vitro and in vivo models for the most common extrahepatic manifestations to improve our understanding of the underlying mechanisms. Stem cell-derived models may provide attractive alternatives to cancer cell line-based culture models in the near future [
      • Helsen 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.
      ,
      • Rogee S.
      • Talbot N.
      • Caperna T.
      • Bouquet J.
      • Barnaud E.
      • Pavio N.
      New models of hepatitis E virus replication in human and porcine hepatocyte cell lines.
      ].
      A number of approved drugs affect HEV replication in vitro or in patients with hepatitis E.

      Treatment and antivirals

      Soon after the discovery of chronic HEV in immunocompromised patients, the first case reports were published describing the efficacy of ribavirin or pegylated IFNα (PegIFNα) monotherapy or a combination of both to treat chronic HEV (Table 1) [
      • Kamar N.
      • Rostaing L.
      • Abravanel F.
      • Garrouste C.
      • Esposito L.
      • Cardeau-Desangles I.
      • et al.
      Pegylated interferon-alpha for treating chronic hepatitis E virus infection after liver transplantation.
      ,
      • Mallet V.
      • Nicand E.
      • Sultanik P.
      • Chakvetadze C.
      • Tesse S.
      • Thervet E.
      • et al.
      Brief communication: case reports of ribavirin treatment for chronic hepatitis E.
      ,
      • Kamar N.
      • Rostaing L.
      • Abravanel F.
      • Garrouste C.
      • Lhomme S.
      • Esposito L.
      • et al.
      Ribavirin therapy inhibits viral replication on patients with chronic hepatitis E virus infection.
      ,
      • Dalton H.R.
      • Keane F.E.
      • Bendall R.
      • Mathew J.
      • Ijaz S.
      Treatment of chronic hepatitis E in a patient with HIV infection.
      ]. In the following years, ribavirin has become the drug of choice and its efficacy has been validated in larger studies [
      • Kamar N.
      • Izopet J.
      • Tripon S.
      • Bismuth M.
      • Hillaire S.
      • Dumortier J.
      • et al.
      Ribavirin for chronic hepatitis E virus infection in transplant recipients.
      ], although a controlled trial is still lacking to date. Ribavirin was also found to be an effective treatment for severe acute HEV [
      • Pischke S.
      • Hardtke S.
      • Bode U.
      • Birkner S.
      • Chatzikyrkou C.
      • Kauffmann W.
      • et al.
      Ribavirin treatment of acute and chronic hepatitis E: a single-centre experience.
      ,
      • Robbins A.
      • Lambert D.
      • Ehrhard F.
      • Brodard V.
      • Hentzien M.
      • Lebrun D.
      • et al.
      Severe acute hepatitis E in an HIV infected patient: Successful treatment with ribavirin.
      ]. Both drugs were shown to inhibit HEV replication in vitro, in subgenomic replicon as well as in full-length infectious systems [
      • Debing Y.
      • Emerson S.U.
      • Wang Y.
      • Pan Q.
      • Balzarini J.
      • Dallmeier K.
      • et al.
      Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon.
      ]. Interestingly, a moderate but significant synergistic effect was noted for the combination of ribavirin and IFNα in vitro.
      Table 1Overview of approved drugs affecting hepatitis E virus (HEV) replication.
      eIF4E, eukaryotic initiation factor 4E; GTP, guanosine triphosphate; mTOR, mammalian target of rapamycin; PegIFNα, pegylated interferon-α.
      Depletion of intracellular GTP pools was shown to be a mechanism of action of ribavirin in vitro [
      • Debing Y.
      • Emerson S.U.
      • Wang Y.
      • Pan Q.
      • Balzarini J.
      • Dallmeier K.
      • et al.
      Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon.
      ], although it remains to be established to what extent this and other potential mechanisms apply in the liver of infected patients [
      • Paeshuyse J.
      • Dallmeier K.
      • Neyts J.
      Ribavirin for the treatment of chronic hepatitis C virus infection: a review of the proposed mechanisms of action.
      ]. Although ribavirin treatment results in viral clearance in most patients, cases of treatment failure have been reported, often linked to dose reductions because of anemia [
      • Kamar N.
      • Izopet J.
      • Tripon S.
      • Bismuth M.
      • Hillaire S.
      • Dumortier J.
      • et al.
      Ribavirin for chronic hepatitis E virus infection in transplant recipients.
      ,
      • Pischke S.
      • Hardtke S.
      • Bode U.
      • Birkner S.
      • Chatzikyrkou C.
      • Kauffmann W.
      • et al.
      Ribavirin treatment of acute and chronic hepatitis E: a single-centre experience.
      ,
      • Debing Y.
      • Gisa A.
      • Dallmeier K.
      • Pischke S.
      • Bremer B.
      • Manns M.
      • et al.
      A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients.
      ]. These cases either present viral recurrence after therapy cessation or development of an apparent “resistance” to ribavirin. A G1634R mutation in the RdRp domain of HEV ORF1 protein was detected at the time of treatment failure in two cases [
      • Debing Y.
      • Gisa A.
      • Dallmeier K.
      • Pischke S.
      • Bremer B.
      • Manns M.
      • et al.
      A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients.
      ]. This mutation has also been observed in a third patient experiencing ribavirin treatment failure [
      • Ramière C.
      • Debing Y.
      • Trabaud M.-A.
      • Scholtes C.
      • Ritter J.
      • Lebossé F.
      • et al.
      Near full-length hepatitis E virus genome sequencing analysis in a chronically infected patient following ribavirin treatment failure.
      ]. In vitro assessment of this mutation indicated a similar sensitivity to ribavirin but an increased fitness as compared to the wild-type, which may explain the failure of ribavirin treatment [
      • Debing Y.
      • Gisa A.
      • Dallmeier K.
      • Pischke S.
      • Bremer B.
      • Manns M.
      • et al.
      A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients.
      ]. A recent study found an increased presence of the G1634R mutation in patients with ribavirin treatment failure (48%) compared to patients showing with a sustained virological response (31%), although this difference was not statistically significant [
      • Lhomme S.
      • Kamar N.
      • Nicot F.
      • Ducos J.
      • Bismuth M.
      • Garrigue V.
      • et al.
      Mutation in the hepatitis E virus polymerase and outcome of ribavirin therapy.
      ]. The clinical implications of this finding and the potential use of the G1634R mutation as a prognostic marker for ribavirin treatment outcome remain to be explored.
      Cases of ribavirin treatment failure in transplant patients, most of which cannot be treated with PegIFNα, highlight the need for alternative treatment options for chronic hepatitis E. We have recently shown that the HCV polymerase inhibitor sofosbuvir inhibits RNA replication of HEV genotype 3 in vitro and that it has an additive antiviral effect when combined with ribavirin (Table 1) [
      • 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.
      ]. Sofosbuvir, a nucleotide analog with a pangenotypic inhibitory effect on the HCV RdRp [
      • Sofia M.J.
      • Bao D.
      • Chang W.
      • Du J.
      • Nagarathnam D.
      • Rachakonda S.
      • et al.
      Discovery of a beta-d-2′-deoxy-2′-alpha-fluoro-2′-beta-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus.
      ], leads to sustained virological response in the majority of patients with chronic hepatitis C when combined with other antivirals [
      • Lawitz E.
      • Mangia A.
      • Wyles D.
      • Rodriguez-Torres M.
      • Hassanein T.
      • Gordon S.C.
      • et al.
      Sofosbuvir for previously untreated chronic hepatitis C infection.
      ]. The excellent tolerability of sofosbuvir, including in solid organ transplant recipients or cirrhotic patients [
      • Charlton M.
      • Gane E.
      • Manns M.P.
      • Brown Jr., R.S.
      • Curry M.P.
      • Kwo P.Y.
      • et al.
      Sofosbuvir and ribavirin for treatment of compensated recurrent hepatitis C virus infection after liver transplantation.
      ], suggests this drug may be used as an add-on to ribavirin in the treatment of chronic HEV. A clinical proof-of-concept study will be required to explore the antiviral potential of sofosbuvir in combination with ribavirin in chronic HEV, especially in patients who fail to achieve HEV elimination with ribavirin alone. In the same study [
      • 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.
      ], we have reported that nucleoside analogs such as 2′-C-methyladenosine or 2′-C-methylcytidine can also inhibit HEV infection, albeit with lower potency, opening the door to the possible future development of more specific compounds derived from the same classes of inhibitors.
      As solid organ transplant recipients represent the majority of patients with chronic HEV, research focusing on the effect of immunosuppressive drugs and regimens on clearance of HEV infection is of prime importance [
      • Wang Y.
      • Metselaar H.J.
      • Peppelenbosch M.P.
      • Pan Q.
      Chronic hepatitis E in solid-organ transplantation: the key implications of immunosuppressants.
      ]. For instance, analysis of a limited number of cases suggests that the use of the immunosuppressant mycophenolate mofetil is associated with HEV clearance [
      • Pischke S.
      • Stiefel P.
      • Franz B.
      • Bremer B.
      • Suneetha P.V.
      • Heim A.
      • et al.
      Chronic hepatitis E in heart transplant recipients.
      ] (Table 1). Patients receiving the calcineurin inhibitor tacrolimus have a higher risk of developing chronic HEV than those receiving cyclosporine A [
      • Kamar N.
      • Garrouste C.
      • Haagsma E.B.
      • Garrigue V.
      • Pischke S.
      • Chauvet C.
      • et al.
      Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants.
      ]. The importance of the particular choice of immunosuppressive therapy is to some extent supported by in vitro data. Mycophenolic acid, the active component of mycophenolate mofetil, was found to exert potent in vitro anti-HEV activity, mediated by depletion of intracellular GTP pools [
      • Debing Y.
      • Emerson S.U.
      • Wang Y.
      • Pan Q.
      • Balzarini J.
      • Dallmeier K.
      • et al.
      Ribavirin inhibits in vitro hepatitis E virus replication through depletion of cellular GTP pools and is moderately synergistic with alpha interferon.
      ,
      • Wang Y.
      • Zhou X.
      • Debing Y.
      • Chen K.
      • Van Der Laan L.J.
      • Neyts J.
      • et al.
      Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis E virus.
      ]. It remains questionable, however, whether such GTP depletion occurs in vivo and to what extent the direct antiviral activity is offset by mycophenolic acid’s immunosuppressive effects. In addition, steroids were found not to influence HEV replication in vitro, while calcineurin and mTOR inhibitors both stimulate HEV replication [
      • Wang Y.
      • Zhou X.
      • Debing Y.
      • Chen K.
      • Van Der Laan L.J.
      • Neyts J.
      • et al.
      Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis E virus.
      ,
      • Zhou X.
      • Wang Y.
      • Metselaar H.J.
      • Janssen H.L.
      • Peppelenbosch M.P.
      • Pan Q.
      Rapamycin and everolimus facilitate hepatitis E virus replication: revealing a basal defense mechanism of PI3K-PKB-mTOR pathway.
      ]. The proviral effect of mTOR inhibitors such as rapamycin and everolimus was found to be mediated by blocking an antiviral signaling pathway downstream of mTOR dependent on eIF4E-binding protein 1 [
      • Zhou X.
      • Wang Y.
      • Metselaar H.J.
      • Janssen H.L.
      • Peppelenbosch M.P.
      • Pan Q.
      Rapamycin and everolimus facilitate hepatitis E virus replication: revealing a basal defense mechanism of PI3K-PKB-mTOR pathway.
      ], while the calcineurin inhibitors, cyclosporine A and tacrolimus, promote HEV replication through inhibition of cyclophilins A and B [
      • Wang Y.
      • Zhou X.
      • Debing Y.
      • Chen K.
      • Van Der Laan L.J.
      • Neyts J.
      • et al.
      Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis E virus.
      ]. Although these findings provide interesting indications on what therapies may be beneficial or detrimental in preventing and treating chronic hepatitis E in transplant patients, they require verification in vivo, either in a suitable animal model or preferentially in sufficiently large well-designed clinical studies [
      • Debing Y.
      • Neyts J.
      MTOR-inhibitors may aggravate chronic hepatitis E.
      ]. However, a recent study provided in vivo evidence of significantly higher HEV RNA level in patients with chronic HEV receiving an mTOR inhibitor as compared to those receiving calcineurin inhibitors as immunosuppressive regimen [
      • Kamar N.
      • Lhomme S.
      • Abravanel F.
      • Cointault O.
      • Esposito L.
      • Cardeau-Desangles I.
      • et al.
      An early viral response predicts the virological response to ribavirin in hepatitis E virus organ transplant patients.
      ]. In addition, the use of mycophenolic acid did not affect the response to ribavirin [
      • Kamar N.
      • Lhomme S.
      • Abravanel F.
      • Cointault O.
      • Esposito L.
      • Cardeau-Desangles I.
      • et al.
      An early viral response predicts the virological response to ribavirin in hepatitis E virus organ transplant patients.
      ].
      Interest to develop specific new HEV antivirals is, because of the limited potential return on investment, limited. Hence, research efforts may focus on (i) optimizing the current antiviral treatments (ribavirin and/or PegIFNα) as well as immunosuppressive regimens; (ii) the clinical evaluation of other drugs with proven in vitro inhibition of HEV replication, such as sofosbuvir; and finally (iii) determining the most adapted immunosuppressive regimens to prevent chronic HEV in transplant recipients.

      Conclusions

      HEV is a remarkable virus, with relatively simple genetic organization, a positive-strand RNA genome resembling cellular mRNAs, and only three ORFs and (poly)protein products. Given its efficient clearance by a competent immune system, HEV has to propagate rapidly. Therefore, HEV possesses characteristics of a virus employing the “hit-and-run” strategy, which favors its genetic evolution. This may explain the broad representation of HEV in the animal kingdom and the crossing of species barriers. To follow this strategy, the virus had to acquire a high stability in the environment for its survival and efficient propagation. A deeper understanding of the structure of the viral particle as well as the identification of the receptor(s) for HEV should shed new light on its zoonotic transmission and tissue tropism. Advances on current challenges, such as the epidemiology of autochthonous hepatitis E, the optimization and standardization of diagnostic assays, the pathogenesis and clinical management of extrahepatic manifestations, the prevention of HEV infection in populations at risk, and the treatment of chronic hepatitis E may be facilitated by parallel progress in the understanding of fundamental aspects of HEV biology and the clinical investigation of hepatitis E.

      Financial support

      The authors acknowledge support by the Research Foundation Flanders (PhD Fellowship to YD), the Swiss National Science Foundation (grant 31003A-156030 to DM), the Belgian Science Policy Interuniversity Attraction Poles (grant VII–P7/45 to JN), the Flemish Institute for Science & Technology SBO project (grant HLIM3D to JN) and the Gilead Sciences International Research Scholars Program in Liver Disease (Award 2015 to JG).

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Authors’ contributions

      YD, DM, JN and JG jointly wrote the manuscript.

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