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Chronic hepatitis E: Advancing research and patient care

Open AccessPublished:May 22, 2022DOI:https://doi.org/10.1016/j.jhep.2022.05.006

      Summary

      The hepatitis E virus (HEV) was initially thought to exclusively cause acute hepatitis. However, the first diagnosis of chronic hepatitis E in transplant recipients in 2008 profoundly changed our understanding of this pathogen. We have now begun to understand that specific HEV genotypes can cause chronic infection in certain immunocompromised populations. Over the past decade, dedicated clinical and experimental research has substantiated knowledge on the epidemiology, transmission routes, pathophysiological mechanisms, diagnosis, clinical features and treatment of chronic HEV infection. Nevertheless, many gaps and major challenges remain, particularly regarding the translation of knowledge into disease prevention and improvement of clinical outcomes. This article aims to highlight the latest developments in the understanding and management of chronic hepatitis E. More importantly, we attempt to identify major knowledge gaps and discuss strategies for further advancing both research and patient care.

      Keywords

      Introduction

      The hepatitis E virus (HEV) was discovered as the causative agent of a hepatitis epidemic in the 1980s,
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      Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route.
      and the genome was subsequently cloned and sequenced (Fig. 1).
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      It is classified into the genus Orthohepevirus of the Hepeviridae family, consisting of 4 species, namely Orthohepevirus A, B, C and D. Orthohepevirus A is commonly associated with human infections. Within this species, there are 8 classified genotypes and 36 subtypes, of which genotypes 1, 2, 3 and 4 are the most frequently reported to infect humans.
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      The species Orthohepevirus C, roughly classified into HEV-C1 and HEV-C2 clades, is widely circulated among rodents, but emerging evidence indicates possible transmission to humans.
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      Chronic hepatitis E in a renal transplant recipient: the first report of genotype 4 hepatitis E virus caused chronic infection in organ recipient.
      Figure thumbnail gr1
      Fig. 1A brief timeline of major developments in the field of chronic hepatitis E.
      The events listed in the upper panel are exclusively related to chronic hepatitis E, whereas the events listed in the lower panel are essentially relevant to chronic hepatitis E but are also important for the HEV field in general. HEV, hepatitis E virus; GT, genotype; EASL, European Association for the Study of the Liver; WHO, World Health Organization; IS, International Standard.
      HEV contains a positive single-stranded RNA genome of about 7.2 kb. The virion is icosahedral in shape and 27-32 nm in diameter. It is a “quasi-enveloped” virus, existing in both non-enveloped and enveloped (“eHEV”) forms. The virus primarily infects hepatocytes in the liver. HEV is shed in faeces as a highly infectious, non-enveloped virus, whereas particles secreted in blood or produced in cell culture contain a lipid envelope with attenuated infectivity.
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      Both naked and quasi-enveloped HEV particles are highly resistant to alcohol-based disinfectants.
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      Hepatitis E virus is highly resistant to alcohol-based disinfectants.
      HEV was initially thought to exclusively cause acute infection, until chronic hepatitis (persistent viraemia beyond 3-6 months) was unexpectedly found in organ transplant recipients (Fig. 1).
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      This has sparked new efforts to study the pathogenesis of chronic HEV infection and identify new therapeutics. Over the past decade, it has become evident that chronic infection predominantly occurs in immunocompromised patients resulting in substantial morbidity and even mortality. However, many challenges remain in clarifying the epidemiology, understanding the pathophysiological mechanisms, developing effective treatments and implementing preventive measures (Box 1). This article seeks to highlight the state-of-the-art in understanding and combating chronic hepatitis E, but more importantly, to identify major knowledge gaps and discuss strategies for advancing both research and patient care in chronic HEV infection.
      Major knowledge gaps and challenges, and outstanding questions. HEV, hepatitis E virus.

      Epidemiology in immunocompromised populations

      Chronic HEV infection was first reported in 2008 in a series of organ transplant recipients infected with genotype 3 HEV.
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      A subsequent case report documented persistent HEV infection in a patient with HIV.
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      Persistent carriage of hepatitis E virus in patients with HIV infection.
      Patients with cancer receiving chemotherapy and/or immunotherapy,
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      The burden of hepatitis E among patients with haematological malignancies: a retrospective European cohort study.
      and patients with autoimmune diseases (e.g. rheumatic diseases) receiving immunosuppressive or immunomodulatory therapies
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      Hepatitis E Virus and rheumatic diseases: what do rheumatologists need to know?.
      are also at risk of developing chronic hepatitis E.
      Overall, chronic hepatitis E is mostly reported in immunocompromised patients with genotype 3 HEV infection in European countries (Fig. 2). In solid organ transplantation settings, active HEV infection can be detected in 1-2% of patients showing viral RNA positivity. Although the exact rate of developing chronicity varies among different studies, overall, HEV infections become chronic in about two-thirds of solid organ transplant recipients based on large cohort studies and meta-analyses.
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      Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants.
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      Hepatitis E seroprevalence and viremia rate in immunocompromised patients: a systematic review and meta-analysis.
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      Epidemiology and management of chronic hepatitis E infection in solid organ transplantation: a comprehensive literature review.
      European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L
      EASL Clinical Practice Guidelines on hepatitis E virus infection.
      Chronic cases reported in patients with cancer and autoimmune disease are scattered, and the exact epidemiological features have yet to be fully understood. Surprisingly, apart from initial cases reported in the United Kingdom
      • 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.
      and the United States,
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      Acute and chronic hepatitis E virus infection in human immunodeficiency virus-infected U.S. Women.
      hardly any additional chronic HEV cases have been documented in HIV populations, despite large series of epidemiological studies conducted in this setting across the globe.
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      • Koppelman M.
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      Acute and chronic hepatitis E virus infection in human immunodeficiency virus-infected U.S. Women.
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      • Ferrara S.
      • Massa S.
      • Bulla F.
      • et al.
      Hepatitis E virus co-infection in HIV-infected patients in Foggia and Naples in southern Italy.
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      • Debes J.D.
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      Prevalence and risk of hepatitis E virus infection in the HIV population of Nepal.
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      Prevalence of hepatitis E infection in HIV/HCV-coinfected patients in Spain (2012-2014).
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      Hepatitis E infection in a longitudinal cohort of hepatitis C virus and HCV/HIV coinfected persons.
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      • Li Y.
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      Hepatitis E virus infection in HIV-infected patients: a large cohort study in Yunnan province, China.
      It is also puzzling to note, that, despite genotype 3 HEV widely circulating in North America where many high-volume transplant centres are located, chronic hepatitis E has rarely been reported in this region.
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      • Feldman D.M.
      • Jacobson I.
      Hepatitis E virus infection in the United States: current understanding of the prevalence and significance in the liver transplant patient population and proposed diagnostic and treatment strategies.
      For example, in a cohort of 145 liver transplant recipients from the United States, patients were found to be at risk of acquiring HEV, but no chronic cases were observed.
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      • et al.
      Prevalence and clinical consequences of Hepatitis E in patients who underwent liver transplantation for chronic Hepatitis C in the United States.
      One particular concern is the fact that a widely accessible commercial assay is not available for HEV diagnosis in North America, and thus the true burden of HEV infection may be underestimated in this region.
      • Whitsett M.
      • Feldman D.M.
      • Jacobson I.
      Hepatitis E virus infection in the United States: current understanding of the prevalence and significance in the liver transplant patient population and proposed diagnostic and treatment strategies.
      Very recently, data on 2 patients with COVID-19 who were infected with genotype 3 HEV were reported in the United Kingdom. One case further progressed to chronic infection, which is likely to have been associated with prolonged lymphopenia and treatment with immunosuppressive medications including tocilizumab and steroids.
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      • Curtis C.
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      Nosocomial transmission of hepatitis E virus and development of chronic infection: the wider impact of COVID-19.
      Genotype 3, 4 and 7 HEV of Orthohepevirus A and rat HEV of Orthohepevirus C are associated with chronic infection.
      Figure thumbnail gr2
      Fig. 2The HEV genotypes attributed to chronic infection.
      Orthohepevirus A (genotype 3, 4, 7) and Orthohepevirus C (HEV-C1; rat HEV) have been associated with the development of chronic hepatitis E in immunocompromised patients based on reports from different countries and regions. Although reverse genetics systems have been constructed for all of them, only the genotype 3 system is widely used to model HEV infection in various in vitro models, as it is capable of sustaining persistent infection, one key aspect of chronic HEV infection. The genotype 4 system is not sufficiently robust in cell culture
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      Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90.
      ,
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      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      and thus requires further optimisation. Genotype 7
      • Li T.C.
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      • Ami Y.
      • Suzaki Y.
      • Nakamura T.
      • et al.
      Production of infectious dromedary camel hepatitis E virus by a reverse genetic system: potential for zoonotic infection.
      and rat HEV
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      • Dallmeier K.
      • Neyts J.
      A rat model for hepatitis E virus.
      have been tested in cell culture but are not commonly used. Human liver chimeric mouse models can support persistent infection of genotype 3 HEV. Monkeys and pigs have been inoculated with genotype 3 HEV and treated with immunosuppressants in attempts to better recapitulate chronic HEV infection. Rabbits can be used to model genotype 3 and 4 HEV when treated with immunosuppressants. Persistent rat HEV infection has been shown in athymic nude rats lacking a normal thymus and functionally mature T cells. Chronic infection with genotype 7 HEV has not yet been demonstrated in animal models. HEV, hepatitis E virus; GT, genotype; INS, immunosuppressant; HLCs, hepatocyte-like cells.
      Chronic infection with genotype 4 HEV was first reported in a Chinese boy with acute lymphoblastic leukaemia during chemotherapy (Fig. 1).
      • Geng Y.
      • Zhang H.
      • Huang W.
      • Harrison T.J.
      • Geng K.
      • Li Z.
      • et al.
      Persistent hepatitis e virus genotype 4 infection in a child with acute lymphoblastic leukemia.
      Chronic infection with this genotype in organ transplant patients was subsequently reported in mainland China
      • Wang Y.
      • Chen G.
      • Pan Q.
      • Zhao J.
      Chronic hepatitis E in a renal transplant recipient: the first report of genotype 4 hepatitis E virus caused chronic infection in organ recipient.
      and Hong Kong.
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      • Chan J.F.W.
      • Yap D.Y.H.
      • Teng J.L.L.
      • Huang C.
      • Yip C.C.Y.
      • et al.
      Genotype 4 hepatitis E virus is a cause of chronic hepatitis in renal transplant recipients in Hong Kong.
      Although genotype 4 is predominantly present in Asian countries such as China and Japan, it has also been identified in Europe.
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      • et al.
      Analyses of clinical and biological data for French and Belgian immunocompetent patients infected with hepatitis E virus genotypes 4 and 3.
      Within species Orthohepevirus A, a single case of chronic infection with genotype 7, a camel HEV strain, was reported in a liver transplant patient from the United Arab Emirates (Fig. 1).
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      • 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.
      This genotype is widely circulating among dromedaries,
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      • Saqib M.
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      • Bornstein S.
      • Zohaib A.
      • Renneker S.
      • et al.
      Hepatitis E virus infection in dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983-2015.
      especially in camel calves during the first year of life,
      • Corman V.M.
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      • Ostermann S.
      • Arloth J.
      • Liljander A.
      • Barua R.
      • et al.
      Hepatitis E virus genotype 7 RNA and antibody kinetics in naturally infected dromedary calves, United Arab Emirates.
      whereas the prevalence in the human population remains largely unknown.
      Although the potential of zoonotic transmission of Orthohepevirus C has yet to be defined, infection with rat HEV (HEV-C1) was first reported in a liver transplant recipient who lived in a rat-infested housing estate in Hong Kong. This was, in fact, defined as a case of chronic infection (Fig. 1).
      • Sridhar S.
      • Yip C.C.Y.
      • Wu S.
      • Cai J.
      • Zhang A.J.
      • Leung K.H.
      • et al.
      Rat hepatitis E virus as cause of persistent hepatitis after liver transplant.
      A second case with severe acute infection was recorded in Canada.
      • Andonov A.
      • Robbins M.
      • Borlang J.
      • Cao J.
      • Hatchette T.
      • Stueck A.
      • et al.
      Rat hepatitis E virus linked to severe acute hepatitis in an immunocompetent patient.
      In total, rat HEV has caused 9 out of 21 hepatitis E cases recorded in immunocompromised patients in Hong Kong. Of those 9 patients, 7 progressed to chronic infection, resulting in a chronicity rate that is similar to that for Orthohepevirus A in this patient population.
      • Sridhar S.
      • Yip C.C.
      • Lo K.H.
      • Wu S.
      • Situ J.
      • Chew N.F.
      • et al.
      Hepatitis E virus species C infection in humans, Hong Kong.
      A very recent study has reported 3 cases of acute hepatitis E caused by Orthohepevirus C infection in Spain,
      • Rivero-Juarez A.
      • Frias M.
      • Perez A.B.
      • Pineda J.A.
      • Reina G.
      • Fuentes-Lopez A.
      • et al.
      Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: first report in Europe.
      urging follow-up studies to investigate whether rat HEV also causes chronic infection in immunocompromised patients in Europe.

      Modes of HEV transmission relevant to chronic infection and implications for prevention

      Zoonotic transmission

      Chronic HEV infection is predominately attributed to the zoonotic strains, in particular genotypes 3 and 4, which circulate among various mammals including humans, swine, wild boar, deer, rabbits, camels, goats and cattle (Fig. 2). Many other domestic, zoo and wild animals can also be infected through spillover infections, for instance.
      • Spahr C.
      • Knauf-Witzens T.
      • Vahlenkamp T.
      • Ulrich R.G.
      • Johne R.
      Hepatitis E virus and related viruses in wild, domestic and zoo animals: a review.
      Direct and indirect contact with infected animals or consumption of contaminated food products are recognised as the main transmission routes.
      • Zhou J.H.
      • Li X.R.
      • Lan X.
      • Han S.Y.
      • Wang Y.N.
      • Hu Y.
      • et al.
      The genetic divergences of codon usage shed new lights on transmission of hepatitis E virus from swine to human.
      Since the cross-species transmission and zoonotic nature of these genotypes have been extensively characterised,
      • Meng X.J.
      Expanding host range and cross-species infection of hepatitis E virus.
      we will only highlight a few salient points here. Within genotype 3, rabbit HEV was first discovered in Northwest China,
      • Zhao C.
      • Ma Z.
      • Harrison T.J.
      • Feng R.
      • Zhang C.
      • Qiao Z.
      • et al.
      A novel genotype of hepatitis E virus prevalent among farmed rabbits in China.
      and subsequently in the United States
      • Cossaboom C.M.
      • Cordoba L.
      • Cao D.
      • Ni Y.Y.
      • Meng X.J.
      Complete genome sequence of hepatitis E virus from rabbits in the United States.
      and Europe.
      • Lhomme S.
      • Top S.
      • Bertagnoli S.
      • Dubois M.
      • Guerin J.L.
      • Izopet J.
      Wildlife reservoir for hepatitis E virus, Southwestern France.
      In a cohort of 919 HEV-infected patients in France, 5 were infected with the rabbit strain. One immunocompetent individual spontaneously cleared the virus, but 3 out of the 4 immunocompromised patients developed chronic infection.
      • Abravanel F.
      • Lhomme S.
      • El Costa H.
      • Schvartz B.
      • Peron J.M.
      • Kamar N.
      • et al.
      Rabbit hepatitis E virus infections in humans, France.
      In a cohort of 114 patients with confirmed hepatitis E in Switzerland, 3 immunocompromised patients were infected with rabbit HEV and 2 of them developed chronic infection. Strikingly, none of these patients had direct contact with rabbits or had consumed rabbit meat,
      • Abravanel F.
      • Lhomme S.
      • El Costa H.
      • Schvartz B.
      • Peron J.M.
      • Kamar N.
      • et al.
      Rabbit hepatitis E virus infections in humans, France.
      ,
      • Sahli R.
      • Fraga M.
      • Semela D.
      • Moradpour D.
      • Gouttenoire J.
      Rabbit HEV in immunosuppressed patients with hepatitis E acquired in Switzerland.
      which suggests that they likely acquired the infection through other indirect routes yet to be identified.
      Since the first case of chronic infection with genotype 7 HEV was probably acquired through consumption of camel meat or milk (Fig. 1),
      • 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.
      the prevalence of this genotype has been investigated in camels from different geographical regions. The seroprevalence rate ranges from 20-45%,
      • Rasche A.
      • Saqib M.
      • Liljander A.M.
      • Bornstein S.
      • Zohaib A.
      • Renneker S.
      • et al.
      Hepatitis E virus infection in dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983-2015.
      ,
      • El-Kafrawy S.A.
      • Hassan A.M.
      • El-Daly M.M.
      • Qadri I.
      • Tolah A.M.
      • Al-Subhi T.L.
      • et al.
      Seroprevalence of dromedary camel HEV in domestic and imported camels from Saudi Arabia.
      whereas HEV RNA can be detected in about 2% of faecal samples.
      • Rasche A.
      • Saqib M.
      • Liljander A.M.
      • Bornstein S.
      • Zohaib A.
      • Renneker S.
      • et al.
      Hepatitis E virus infection in dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983-2015.
      Longitudinal characterisation of HEV RNA and antibody response in dromedary calves and corresponding dams indicates that the virus primarily infects calves in the first year of life but not adult animals.
      • Corman V.M.
      • Nagy P.
      • Ostermann S.
      • Arloth J.
      • Liljander A.
      • Barua R.
      • et al.
      Hepatitis E virus genotype 7 RNA and antibody kinetics in naturally infected dromedary calves, United Arab Emirates.
      This active enzootic infection pattern is likely maintained by calf-to-calf transmission. Although humans are rarely in contact with camel calves, the potential risk of zoonotic transmission should continue to be assessed.
      A series of cases with HEV-C1 infection reported from Hong Kong,
      • Sridhar S.
      • Yip C.C.Y.
      • Wu S.
      • Cai J.
      • Zhang A.J.
      • Leung K.H.
      • et al.
      Rat hepatitis E virus as cause of persistent hepatitis after liver transplant.
      Canada
      • Andonov A.
      • Robbins M.
      • Borlang J.
      • Cao J.
      • Hatchette T.
      • Stueck A.
      • et al.
      Rat hepatitis E virus linked to severe acute hepatitis in an immunocompetent patient.
      and Spain
      • Rivero-Juarez A.
      • Frias M.
      • Perez A.B.
      • Pineda J.A.
      • Reina G.
      • Fuentes-Lopez A.
      • et al.
      Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: first report in Europe.
      have challenged the initial assumption that rat HEV was non-zoonotic, which was based on the failure to establish infection in rhesus monkeys upon experimental inoculation.
      • Purcell R.H.
      • Engle R.E.
      • Rood M.P.
      • Kabrane-Lazizi Y.
      • Nguyen H.T.
      • Govindarajan S.
      • et al.
      Hepatitis E virus in rats, Los Angeles, California, USA.
      So far, several cases of chronic infection in immunocompromised patients have been recorded in Hong Kong (Fig. 2), and 3 distinct strains are capable of infecting humans, strongly indicating the zoonotic potential of rat HEV. Epidemiological probes in Hong Kong suggest transmission to humans through either direct infection or common exposure such as environmental surfaces contaminated by rat droppings.
      • Sridhar S.
      • Yip C.C.
      • Wu S.
      • Chew N.F.
      • Leung K.H.
      • Chan J.F.
      • et al.
      Transmission of rat hepatitis E virus infection to humans in Hong Kong: a clinical and epidemiological analysis.
      Since food-borne transmission of zoonotic HEV strains appears to be a major route in Europe, immunocompromised patients are advised to avoid undercooked meat and pork-related food products.
      European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L
      EASL Clinical Practice Guidelines on hepatitis E virus infection.
      Intuitively, testing the foods consumed by immunocompromised patients may effectively prevent chronic hepatitis E. However, the current food production and supply chains are complex, and it is neither practical nor feasible to continuously collect food samples for HEV testing in specific patient populations. Furthermore, the exact transmission routes of some chronic cases, for example those infected with rabbit or rat HEV, remain largely elusive. Continuous sampling and testing of these host animals and environmental surfaces contaminated by their droppings could be helpful in clarifying the exact sources of zoonotic transmission, thereby facilitating the development of specific preventive measures.
      • Sridhar S.
      • Yip C.C.
      • Wu S.
      • Chew N.F.
      • Leung K.H.
      • Chan J.F.
      • et al.
      Transmission of rat hepatitis E virus infection to humans in Hong Kong: a clinical and epidemiological analysis.
      Chronic HEV infection universally occurs in immunocompromised patients but predominantly affects organ transplant recipients. This is most likely due to the use of immunosuppressants and the level of immunosuppression in these patients.

      Blood transfusion

      The prevalence of HEV in blood donors has been investigated across the globe. The incidence of viraemic donors is surprisingly high, ranging from 0.006% to 0.19% in various European countries.
      • Cordes A.K.
      • Goudeva L.
      • Lutgehetmann M.
      • Wenzel J.J.
      • Behrendt P.
      • Wedemeyer H.
      • et al.
      Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.
      Among 23 HEV-positive plasma samples identified in Spain, the highly infectious non-enveloped HEV was detected in 8 samples, whereas the enveloped form was found in all the samples;
      • Costafreda M.I.
      • Sauleda S.
      • Rico A.
      • Piron M.
      • Bes M.
      Detection of non-enveloped hepatitis E virus in plasma of infected blood donors.
      thus highlighting the risk of transmission via this route. Transfusion-transmitted acute HEV infection was first indicated in Japanese studies in 2004,
      • Matsubayashi K.
      • Nagaoka Y.
      • Sakata H.
      • Sato S.
      • Fukai K.
      • Kato T.
      • et al.
      Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan.
      and chronic HEV infection acquired via blood transfusion was reported later in Japanese liver transplant recipients.
      • Inagaki Y.
      • Oshiro Y.
      • Tanaka T.
      • Yoshizumi T.
      • Okajima H.
      • Ishiyama K.
      • et al.
      A nationwide survey of hepatitis E virus infection and chronic hepatitis E in liver transplant recipients in Japan.
      Subsequently, a series of cases were reported in Europe, particularly in immunocompromised patients, several of whom developed chronic infection.
      • Gallian P.
      • Pouchol E.
      • Djoudi R.
      • Lhomme S.
      • Mouna L.
      • Gross S.
      • et al.
      Transfusion-transmitted hepatitis E virus infection in France.
      ,
      • Westholter D.
      • Hiller J.
      • Denzer U.
      • Polywka S.
      • Ayuk F.
      • Rybczynski M.
      • et al.
      HEV-positive blood donations represent a relevant infection risk for immunosuppressed recipients.
      These cases have been associated with the transfusion of various blood components including fresh frozen plasma, red blood cell samples, platelet preparations and pooled granulocytes.
      • Hewitt P.E.
      • Ijaz S.
      • Brailsford S.R.
      • Brett R.
      • Dicks S.
      • Haywood B.
      • et al.
      Hepatitis E virus in blood components: a prevalence and transmission study in southeast England.
      Because HEV viraemic blood donors are asymptomatic, screening for HEV RNA positivity constitutes the only viable option to control transmission. Several (European) countries including Ireland, the United Kingdom, France, the Netherlands, Germany, Spain, Austria and Luxembourg have implemented mandatory screening of blood donors for HEV RNA.
      • Boland F.
      • Martinez A.
      • Pomeroy L.
      • O'Flaherty N.
      Blood donor screening for hepatitis E virus in the European Union.
      Strategically, HEV screening can be broadly classified into universal screening of all blood donors or selective screening. As demonstrated in Catalonia, Spain, no transfusion-transmitted HEV cases have been reported since the implementation of universal HEV RNA screening.
      • Bes M.
      • Costafreda M.I.
      • Riveiro-Barciela M.
      • Piron M.
      • Rico A.
      • Quer J.
      • et al.
      Effect of hepatitis E virus RNA universal blood donor screening, Catalonia, Spain, 20172020.
      However, universal screening is probably not cost-effective in many countries. In such cases, we recommend a selective screening for blood products used in immunocompromised patients, especially organ transplant recipients. There is ongoing debate regarding the choice of either individual donation- or mini-pool-based protocols.
      • Cordes A.K.
      • Goudeva L.
      • Lutgehetmann M.
      • Wenzel J.J.
      • Behrendt P.
      • Wedemeyer H.
      • et al.
      Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.
      Mini-pool-based techniques are capable of large-scale blood screening, but may not be sufficient to eliminate the risk of HEV transmission.
      • Cordes A.K.
      • Goudeva L.
      • Lutgehetmann M.
      • Wenzel J.J.
      • Behrendt P.
      • Wedemeyer H.
      • et al.
      Risk of transfusion-transmitted hepatitis E virus infection from pool-tested platelets and plasma.
      Individual donation-based protocols have higher sensitivity, but are not suitable for large-scale implementation due to high costs. In our opinion, individual donation-based testing could be the preferred option when applying a selective screening strategy specifically for preventing HEV infection in immunocompromised patients.

      Other possible routes

      In general, the non-zoonotic genotype 1 and 2 HEV is primarily waterborne. However, genotypes 3 and 4 have also been detected in various types of environmental water and in waste and drinking water treatment plants in Europe.
      • Fenaux H.
      • Chassaing M.
      • Berger S.
      • Gantzer C.
      • Bertrand I.
      • Schvoerer E.
      Transmission of hepatitis E virus by water: an issue still pending in industrialized countries.
      ,
      • Cuevas-Ferrando E.
      • Randazzo W.
      • Perez-Cataluna A.
      • Sanchez G.
      HEV occurrence in waste and drinking water treatment plants.
      The presence of HEV has been reported in shellfish,
      • O'Hara Z.
      • Crossan C.
      • Craft J.
      • Scobie L.
      First report of the presence of hepatitis E virus in Scottish-harvested shellfish purchased at retail level.
      fresh vegetables and fruits,
      • Brassard J.
      • Gagne M.J.
      • Genereux M.
      • Cote C.
      Detection of human food-borne and zoonotic viruses on irrigated, field-grown strawberries.
      which could be attributed to contaminated water sources and irrigation. A chronic case from China infected with genotype 4 HEV reported no consumption of pork products, but the individual in question drank water and ate seafood from a river running near a livestock farm.
      • Wang Y.
      • Chen G.
      • Pan Q.
      • Zhao J.
      Chronic hepatitis E in a renal transplant recipient: the first report of genotype 4 hepatitis E virus caused chronic infection in organ recipient.
      In line with these observations, consumption of shellfish (e.g. oysters) has been linked to higher HEV seroprevalence, whereas drinking bottled water was associated with lower seroprevalence in France.
      • Mansuy J.M.
      • Gallian P.
      • Dimeglio C.
      • Saune K.
      • Arnaud C.
      • Pelletier B.
      • et al.
      A nationwide survey of hepatitis E viral infection in French blood donors.
      Nevertheless, the infectivity of HEV from these sources and the potential for transmission to humans have yet to be investigated.

      Vaccination for preventing chronic HEV infection

      The hope for better prevention of HEV infection lies in the availability of safe and effective vaccines. The only approved vaccine (Hecolin) was licensed in China in 2011 (Fig. 1). It is well-tolerated and effective in the prevention of genotype 1 and 4 HEV in the general population with long-lasting protective immunity.
      • 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.
      In order for Hecolin to prevent chronic HEV infection at a population level, it will also be important to assess the protection it offers against genotypes 3 and 7, as well as HEV-C1. Immunisation with Hecolin in a rabbit model has been shown to confer full protection against genotype 3,
      • Li M.
      • Li S.
      • He Q.
      • Liang Z.
      • Wang L.
      • Wang Q.
      • et al.
      Hepatitis E-related adverse pregnancy outcomes and their prevention by hepatitis E vaccine in a rabbit model.
      whereas it is only partially protective against HEV-C1 infection in rats.
      • Sridhar S.
      • Situ J.
      • Cai J.P.
      • Yip C.C.
      • Wu S.
      • Zhang A.J.
      • et al.
      Multimodal investigation of rat hepatitis E virus antigenicity: implications for infection, diagnostics, and vaccine efficacy.
      Universal implementation of the HEV vaccine is clearly unnecessary, but the cost-effectiveness of selectively vaccinating vulnerable populations such as immunocompromised patients and pregnant women should be carefully assessed. The World Health Organization (WHO) calls for further studies to evaluate the safety and immunogenicity of Hecolin in special populations, and an ongoing phase IV trial is testing protection in pregnant women in Bangladesh.
      • Zaman K.
      • Dudman S.
      • Stene-Johansen K.
      • Qadri F.
      • Yunus M.
      • Sandbu S.
      • et al.
      HEV study protocol : design of a cluster-randomised, blinded trial to assess the safety, immunogenicity and effectiveness of the hepatitis E vaccine HEV 239 (Hecolin) in women of childbearing age in rural Bangladesh.
      Interestingly, mathematical modelling has provided proof-of-concept that targeted vaccination of a subpopulation with a high frequency of pork-related food consumption in Germany would dramatically reduce cases of HEV infection.
      • Ji Y.
      • Li P.
      • Jia Y.
      • Wang X.
      • Zheng Q.
      • Peppelenbosch M.P.
      • et al.
      Estimating the burden and modeling mitigation strategies of pork-related hepatitis E virus foodborne transmission in representative European countries.
      Emerging evidence from COVID-19 vaccination has shown reduced effectiveness in transplant patients attributed to the use of immunosuppressive medications.
      • Kamar N.
      • Abravanel F.
      • Marion O.
      • Couat C.
      • Izopet J.
      • Del Bello A.
      Three doses of an mRNA covid-19 vaccine in solid-organ transplant recipients.
      ,
      • Embi P.J.
      • Levy M.E.
      • Naleway A.L.
      • Patel P.
      • Gaglani M.
      • Natarajan K.
      • et al.
      Effectiveness of 2-dose vaccination with mRNA COVID-19 vaccines against COVID-19-associated hospitalizations among immunocompromised adults - nine states, January-September 2021.
      Similarly, vaccination with Hecolin in rabbits prior to administration of immunosuppressive medication fully protected them against genotype 3 and 4 HEV infection, whereas only partial protection was achieved when the animals were already receiving immunosuppressants.
      • He Q.
      • Zhang F.
      • Shu J.
      • Li S.
      • Liang Z.
      • Du M.
      • et al.
      Immunocompromised rabbit model of chronic hepatitis E reveals liver fibrosis and distinct efficacy of different vaccination strategies.
      From a clinical practice perspective, one could argue in favour of preferentially vaccinating patients on the waiting list for organ transplantation.
      Nosocomial transmission of HEV and development of chronic infection have been reported in patients affected by COVID-19.
      Although a HEV vaccine for animals is not available, vaccinating animal reservoirs, particularly farm pigs, represents an alternative approach. However, HEV infection does not cause diseases in pigs or affect the economic performance of swine herds, thereby challenging the rationale for developing such a vaccine. Therefore, future studies should evaluate the cost-benefit of vaccinating animals by also considering the public health consequences on human populations.
      • Ji Y.
      • Li P.
      • Jia Y.
      • Wang X.
      • Zheng Q.
      • Peppelenbosch M.P.
      • et al.
      Estimating the burden and modeling mitigation strategies of pork-related hepatitis E virus foodborne transmission in representative European countries.
      First and foremost, healthcare workers, researchers and regulatory authorities must expeditiously review the evidence to determine if the currently available HEV vaccine (Hecolin) can effectively prevent chronic hepatitis E in immunocompromised populations.

      Diagnosis and clinical features of chronic HEV infection

      Diagnosis

      Anti-HEV antibodies are often undetectable in immunosuppressed patients chronically infected with the virus. For these patients, nucleic acid amplification technique-based detection of viral RNA in blood and/or stool samples is the only reliable method for diagnosis. Chronic HEV infection is defined by detection of viral RNA for more than 3 months. The viral load calculated from quantification of HEV RNA is also used to evaluate the treatment response.
      European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L
      EASL Clinical Practice Guidelines on hepatitis E virus infection.
      However, the widely available molecular tests are designed primarily to detect Orthohepevirus A, meaning that Orthohepevirus C infections could be missed; thus, these tests will require further optimisation.
      • Rivero-Juarez A.
      • Frias M.
      • Perez A.B.
      • Pineda J.A.
      • Reina G.
      • Fuentes-Lopez A.
      • et al.
      Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: first report in Europe.
      Diagnosis is usually only considered when infection is clinically suspected, but this is clearly insufficient to fully recognise the burden of HEV infection in immunocompromised patients. Questions have been raised as to whether structured systematic screening of HEV infection for transplant recipients living in endemic areas should be considered,
      • Ankcorn M.J.
      • Ijaz S.
      • Poh J.
      • Elsharkawy A.M.
      • Smit E.
      • Cramb R.
      • et al.
      Toward systematic screening for persistent hepatitis E virus infections in transplant patients.
      but future research – especially cost-effectiveness analysis – is required to address this issue.

      Intrahepatic manifestations

      Persistent HEV infection causes chronic liver inflammation, and rapid progression to fibrosis and cirrhosis. Although the exact rate of disease progression remains unclear, in a large retrospective multicentre study, 8 out of 56 organ recipients with chronic hepatitis E developed cirrhosis. Among them, 2 liver transplant recipients required a second liver transplantation and 2 died of decompensated cirrhosis.
      • 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.
      Systematic analysis of the liver histology of patients with hepatitis E classified 3 clusters with distinct histopathologies. Liver biopsies from patients with chronic hepatitis E mainly belonged to the cluster displaying smouldering hepatitis; despite mild to moderate inflammatory activity, a subset of these patients had already developed fibrosis and cirrhosis.
      • Lenggenhager D.
      • Pawel S.
      • Honcharova-Biletska H.
      • Evert K.
      • Wenzel J.J.
      • Montani M.
      • et al.
      The histologic presentation of hepatitis E reflects patients' immune status and pre-existing liver condition.
      Within the liver, destructive cholangitis has been observed in a subset of patients with chronic HEV. Immunohistochemical staining detected the virus in both hepatocytes and bile duct epithelia within the same liver biopsy, suggesting that direct infection in cholangiocytes could be a potential cause of cholangitis.
      • Beer A.
      • Holzmann H.
      • Pischke S.
      • Behrendt P.
      • Wrba F.
      • Schlue J.
      • et al.
      Chronic Hepatitis E is associated with cholangitis.
      Since chronic hepatitis B or C virus infections are major causes of hepatocellular carcinoma (HCC), it is prudent to question whether chronic hepatitis E can also cause HCC.
      • Klohn M.
      • Schrader J.A.
      • Bruggemann Y.
      • Todt D.
      • Steinmann E.
      Beyond the usual suspects: hepatitis E virus and its implications in hepatocellular carcinoma.
      There is one documented case of an immunocompromised patient who developed HCC after 8 years of chronic HEV infection without other major aetiologies for cirrhosis or HCC.
      • Borentain P.
      • Colson P.
      • Bolon E.
      • Gauchez P.
      • Coso D.
      • Gerolami R.
      Hepatocellular carcinoma complicating hepatitis E virus-related cirrhosis.
      Given the limited number of reported cases, whether HEV joins hepatitis B and C viruses as a potential causative agent of HCC in chronically infected patients must be closely monitored.

      Extrahepatic manifestations

      A number of extrahepatic manifestations related to HEV infection have been reported, as extensively reviewed elsewhere.
      • Wu J.
      • Xiang Z.
      • Zhu C.
      • Yao Y.
      • Bortolanza M.
      • Cao H.
      • et al.
      Extrahepatic manifestations related to hepatitis E virus infection and their triggering mechanisms.
      ,
      • Pischke S.
      • Hartl J.
      • Pas S.D.
      • Lohse A.W.
      • Jacobs B.C.
      • Van der Eijk A.A.
      Hepatitis E virus: infection beyond the liver?.
      Although most of these cases occur in acute infection, neurological and renal manifestations have also been observed in chronically infected patients.
      • Abravanel F.
      • Nicot F.
      • Lhomme S.
      • Cazabat M.
      • Drumel T.
      • Velay A.
      • et al.
      Hepatitis E virus quasispecies in cerebrospinal fluid with neurological manifestations.
      ,
      • 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.
      Guillain-Barré syndrome, neuralgic amyotrophy, myelitis and encephalitis are the most common neurological disorders associated with HEV. Viral RNA has been detected in the cerebrospinal fluid of patients with chronic HEV presenting neurological symptoms with evidence of HEV quasi-species compartmentalisation.
      • Abravanel F.
      • Nicot F.
      • Lhomme S.
      • Cazabat M.
      • Drumel T.
      • Velay A.
      • et al.
      Hepatitis E virus quasispecies in cerebrospinal fluid with neurological manifestations.
      The susceptibility of neuronal cells to HEV infection has been widely demonstrated in cell culture models.
      • Zhou X.
      • Huang F.
      • Xu L.
      • Lin Z.
      • de Vrij F.M.S.
      • Ayo-Martin A.C.
      • et al.
      Hepatitis E virus infects neurons and brains.
      ,
      • Drave S.A.
      • Debing Y.
      • Walter S.
      • Todt D.
      • Engelmann M.
      • Friesland M.
      • et al.
      Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells.
      Furthermore, HEV infection in nervous tissues has been shown in different animal models.
      • Zhou X.
      • Huang F.
      • Xu L.
      • Lin Z.
      • de Vrij F.M.S.
      • Ayo-Martin A.C.
      • et al.
      Hepatitis E virus infects neurons and brains.
      ,
      • Tian J.
      • Shi R.
      • Liu T.
      • She R.
      • Wu Q.
      • An J.
      • et al.
      Brain infection by hepatitis E virus probably via damage of the blood-brain barrier due to alterations of tight junction proteins.
      However, the neurological pathology may be attributed to both direct infection and other indirect mediators, and this could also apply in HEV-induced kidney injury. HEV-infected monkeys and patients have been shown to excrete quasi-enveloped virions in the urine, but whether this is associated with renal injury remains inconclusive.
      • 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.
      ,
      • Marion O.
      • Capelli N.
      • Lhomme S.
      • Dubois M.
      • Pucelle M.
      • Abravanel F.
      • et al.
      Hepatitis E virus genotype 3 and capsid protein in the blood and urine of immunocompromised patients.
      In a chronic case, HEV infection in the dermal endothelium induced a cutaneous T-cell lymphoproliferative disorder.
      • Mallet V.
      • Bruneau J.
      • Zuber J.
      • Alanio C.
      • Leclerc-Mercier S.
      • Roque-Afonso A.M.
      • et al.
      Hepatitis E virus-induced primary cutaneous CD30(+) T cell lymphoproliferative disorder.
      HEV virions have been detected in the ejaculate originating from the male reproductive system of chronically infected patients. It has been suggested that the testis and prostate are the sites for persistent HEV replication, but the pathophysiological impact on the male reproductive system requires further clarification. Furthermore, the infectivity of ejaculate-derived HEV, in the form of enveloped virions similar to those in serum but different from the non-enveloped particles in faeces, has not been determined. Thus, it is not yet known whether HEV can be considered a sexually transmitted pathogen.
      • Horvatits T.
      • Wissmann J.E.
      • Johne R.
      • Groschup M.H.
      • Gadicherla A.K.
      • Schulze Zur Wiesch J.
      • et al.
      Hepatitis E virus persists in the ejaculate of chronically infected men.
      The key transmission routes contributing to chronic HEV infection have been recognised, thereby facilitating the development of preventive strategies.

      Pathophysiological mechanisms

      Immune response to HEV infection

      Host innate immunity acts as the first line of defence against viral infections. The innate antiviral immune response involves the conventional types of innate immune cells, including natural killer cells, dendritic cells, granulocytes, monocytes and macrophages, but HEV can also elicit an innate immune response in host cells. HEV viral RNA has been shown to trigger a robust interferon response in the hepatocytes of HEV-infected patients, in cell line and organoid models.
      • Wang W.
      • Wang Y.
      • Qu C.
      • Wang S.
      • Zhou J.
      • Cao W.
      • et al.
      The RNA genome of hepatitis E virus robustly triggers an antiviral interferon response.
      The innate immune system detects viral invasion and triggers the production of antiviral cytokines such as interferon.
      It has become clear that innate immunity alone does not provide adequate defence against HEV as chronic infection has been frequently reported in immunocompromised patients in whom the adaptive immune system is usually impaired. Activation of HEV-specific CD4 and CD8 T-cell responses has been widely detected in patients with acute HEV, and the functionalities of these T cells have been proven ex vivo.
      • Brown A.
      • Halliday J.S.
      • Swadling L.
      • Madden R.G.
      • Bendall R.
      • Hunter J.G.
      • et al.
      Characterization of the specificity, functionality, and durability of host T-cell responses against the full-length hepatitis E virus.
      ,
      • Tripathy A.S.
      • Das R.
      • Rathod S.B.
      • Arankalle V.A.
      Cytokine profiles, CTL response and T cell frequencies in the peripheral blood of acute patients and individuals recovered from hepatitis E infection.
      However, dysfunction of HEV-specific T-cell responses occurs in patients with chronic HEV. HEV-specific CD4 and CD8 T-cell responses were undetectable in the majority of chronically infected patients, but appeared shortly after viral clearance.
      • Suneetha P.V.
      • Pischke S.
      • Schlaphoff V.
      • Grabowski J.
      • Fytili P.
      • Gronert A.
      • et al.
      Hepatitis E virus (HEV)-specific T-cell responses are associated with control of HEV infection.
      This corroborates reports of an inverse correlation between HEV-specific T-cell responses and HEV-antigen levels.
      • Al-Ayoubi J.
      • Behrendt P.
      • Bremer B.
      • Suneetha P.V.
      • Gisa A.
      • Rinker F.
      • et al.
      Hepatitis E virus ORF 1 induces proliferative and functional T-cell responses in patients with ongoing and resolved hepatitis E.
      In rhesus macaques, depletion of CD8 T cells delays viral clearance but is not sufficient to establish chronic infection.
      • Bremer W.
      • Blasczyk H.
      • Yin X.
      • Salinas E.
      • Grakoui A.
      • Feng Z.
      • et al.
      Resolution of hepatitis E virus infection in CD8+ T cell-depleted rhesus macaques.
      HEV clearance in these macaques is accompanied by a neutralising antibody response and liver infiltration of functional HEV-specific CD4 and CD8 T cells.
      • Bremer W.
      • Blasczyk H.
      • Yin X.
      • Salinas E.
      • Grakoui A.
      • Feng Z.
      • et al.
      Resolution of hepatitis E virus infection in CD8+ T cell-depleted rhesus macaques.
      Clinical and experimental evidence collectively suggests that the development of chronic HEV infection is probably attributed to the dysfunction of multiple immune compartments including innate immunity, T-cell functionality and antibody response. In addition, HEV persistence further facilitates immune evasion especially through interactions with viral proteins.
      • Li Y.
      • Qu C.
      • Yu P.
      • Ou X.
      • Pan Q.
      • Wang W.
      The interplay between host innate immunity and hepatitis E Virus.

      The multifaceted role of immunosuppressive medications

      The majority of reported chronic HEV cases are organ transplant recipients and the link between chronicity and universal use of immunosuppressants has been well-documented.
      • Wang Y.
      • Metselaar H.J.
      • Peppelenbosch M.P.
      • Pan Q.
      Chronic hepatitis E in solid-organ transplantation: the key implications of immunosuppressants.
      In order to prevent organ rejection, the primary aim of using immunosuppressants is to inhibit T-cell proliferation. However, the role of immunosuppressive medications in the development of chronic HEV infection probably extends beyond T-cell inhibition to the suppression of multiple compartments of the immune system. This may explain why chronic HEV infection is rare in patients with HIV, who typically experience a progressive loss of CD4 T cells in isolation. Patients with inflammatory bowel disease are often immunocompromised due to the disease or treatment with immunomodulatory agents, but chronic HEV infection has hardly been reported.
      • Senosiain C.
      • Gonzalez-Tallon A.A.
      • Lopez-Sanroman A.
      • Mateos M.L.
      • Perez-Gracia M.T.
      • Garcia-Sanchez M.C.
      • et al.
      Hepatitis E seroprevalence in inflammatory bowel disease.
      This may be attributed to the lower level of immunosuppression in these patients.
      Different immunosuppressive regimens can differentially affect the immune system and regulate HEV infection based on their distinct mechanism of action.
      • Pan Q.
      • Tilanus H.W.
      • Metselaar H.J.
      • Janssen H.L.
      • van der Laan L.J.
      Virus-drug interactions--molecular insight into immunosuppression and HCV.
      For example, the use of tacrolimus but not cyclosporine A (both calcineurin inhibitors) in organ transplant recipients is significantly associated with the onset of chronic hepatitis E.
      • 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.
      In a cohort of heart transplant recipients, HEV clearance was found to be significantly associated with the use of mycophenolate mofetil (MMF).
      • Pischke S.
      • Stiefel P.
      • Franz B.
      • Bremer B.
      • Suneetha P.V.
      • Heim A.
      • et al.
      Chronic hepatitis e in heart transplant recipients.
      In cell culture models, the mTOR inhibitors, rapamycin and everolimus, have been shown to facilitate HEV replication,
      • 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.
      whereas MMF
      • 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.
      inhibits it. One peculiar study documented persistent HEV infection in a patient with tuberous sclerosis complex treated with everolimus, but the virus was cleared within 3 months following the withdrawal of everolimus.
      • van Dijk W.E.M.
      • Vergeer M.
      • Arends J.E.
      Persistent hepatitis E infection in a patient with tuberous sclerosis complex treated with everolimus: a case report.
      In a clinical trial testing sofosbuvir in patients with chronic HEV (to be discussed further in a following section), the potential effects on HEV viral load appear to be affected by the specific immunosuppressive medication. The use of MMF compared to mTOR inhibitors is related to a stronger reduction in HEV RNA.
      • Cornberg M.
      • Pischke S.
      • Muller T.
      • Behrendt P.
      • Piecha F.
      • Benckert J.
      • et al.
      Sofosbuvir monotherapy fails to achieve HEV RNA elimination in patients with chronic hepatitis E - The HepNet SofE pilot study.
      Although the patient number for this subgroup analysis is too small to draw firm conclusions, these results are in line with aforementioned experimental observations.
      • 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.
      ,
      • 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.
      In general, the number of patients with chronic HEV is often too small to be stratified by the use of different immunosuppressants. Therefore, it is essential to integrate both clinical and experimental approaches to better clarify the differential effects of different immunosuppressants on chronic hepatitis E. A greater understanding of the impact of immunosuppressive medications on different immune compartments including innate and adaptive immune systems as well as on the virus per se will help us to elucidate the pathophysiological mechanisms behind chronicity and to devise new strategies for the improved management of patients with chronic HEV.
      Various experimental models have been established to (partially) recapitulate chronic HEV infection, providing essential tools for studying pathophysiological mechanisms and testing antiviral treatments.

      Inflammatory response in disease progression

      A substantial proportion of chronic HEV cases experience rapid disease progression and can develop cirrhosis within 2 years.
      • Zhou X.
      • de Man R.A.
      • de Knegt R.J.
      • Metselaar H.J.
      • Peppelenbosch M.P.
      • Pan Q.
      Epidemiology and management of chronic hepatitis E infection in solid organ transplantation: a comprehensive literature review.
      The underlying fibrogenic mechanisms specific to HEV infection remain poorly investigated, but this pathogenic process is known to involve different cell types in viral hepatitis more generally. Macrophages are the key cell types in sensing pathogen and tissue damage. They exert robust pro-inflammatory and pro-fibrogenic functions and orchestrate other cell types in the pathogenesis of liver fibrosis.
      • van der Heide D.
      • Weiskirchen R.
      • Bansal R.
      Therapeutic targeting of hepatic macrophages for the treatment of liver diseases.
      Inflammasome activation is a prominent mechanism that drives pathogenic inflammation. Currently, 5 receptor proteins have been identified to assemble inflammasomes. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is the best characterised form, which is most relevant to viral infections. A recent study showed that macrophages effectively initiate NLRP3 inflammasome activation in response to active HEV infection but also inactivated viral particles.
      • Li Y.
      • Yu P.
      • Kessler A.L.
      • Shu J.
      • Liu X.
      • Liang Z.
      • et al.
      Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.
      This study provided further in vivo evidence of inflammasome activation in rabbits and patients acutely infected with HEV.
      • Li Y.
      • Yu P.
      • Kessler A.L.
      • Shu J.
      • Liu X.
      • Liang Z.
      • et al.
      Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.
      Future investigation in chronically infected animals and patients is highly relevant. Interestingly, pharmacological targeting of NLRP3 by steroids can inhibit HEV-triggered inflammasome activation.
      • Li Y.
      • Yu P.
      • Kessler A.L.
      • Shu J.
      • Liu X.
      • Liang Z.
      • et al.
      Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.
      Considering the emerging evidence that steroid treatment can improve COVID-19-related outcomes,
      • Group R.C.
      • Horby P.
      • Lim W.S.
      • Emberson J.R.
      • Mafham M.
      • Bell J.L.
      • et al.
      Dexamethasone in hospitalized patients with Covid-19.
      whether anti-inflammatory treatment can alleviate disease severity and the progression of chronic hepatitis E warrants investigation.

      Experimental models

      In vitro

      Hepatotropic viruses intrinsically have narrow tropism and poor replication efficiency in vitro, thereby challenging the development of robust cell culture models. Identifying HEV strains that can efficiently replicate in vitro is a prerequisite. The discovery of the genotype 3 Kernow-C1 strain in a patient with chronic HEV is a landmark development. After 6 passages in cell culture, insertion of the human genome-derived S17 sequence was identified in the hypervariable region of the HEV genome, termed Kernow-C1 p6, which further enhanced replication in vitro.
      • 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.
      By constructing reverse genetics systems, p6-based genotype 3 HEV models have been widely used in the field, and are capable of sustaining persistent replication or infection in various human cell lines.
      • Zhou X.
      • Huang F.
      • Xu L.
      • Lin Z.
      • de Vrij F.M.S.
      • Ayo-Martin A.C.
      • et al.
      Hepatitis E virus infects neurons and brains.
      ,
      • Li Y.
      • Miao Z.
      • Li P.
      • Zhang R.
      • Kainov D.E.
      • Ma Z.
      • et al.
      Ivermectin effectively inhibits hepatitis E virus replication, requiring the host nuclear transport protein importin alpha1.
      Although reverse genetics systems are also available for genotype 4,
      • Nimgaonkar I.
      • Archer N.F.
      • Becher I.
      • Shahrad M.
      • LeDesma R.A.
      • Mateus A.
      • et al.
      Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90.
      7105 and rat HEV,
      • Debing Y.
      • Mishra N.
      • Verbeken E.
      • Ramaekers K.
      • Dallmeier K.
      • Neyts J.
      A rat model for hepatitis E virus.
      these models are either not sufficiently robust or have not been widely validated.
      Interestingly, a subclone derived from the human hepatocarcinoma cell line HepG2/C3A is able to grow as a polarised monolayer in culture, and most of the infectious HEV particles are released from the apical side in this model.
      • Capelli N.
      • Marion O.
      • Dubois M.
      • Allart S.
      • Bertrand-Michel J.
      • Lhomme S.
      • et al.
      Vectorial release of hepatitis E virus in polarized human hepatocytes.
      However, these immortalised (cancer) cell lines harbour numerous genetic, epigenetic and functional alterations that compromise the authenticity with which they recapitulate HEV-host interactions. Primary human hepatocytes
      • Todt D.
      • Friesland M.
      • Moeller N.
      • Praditya D.
      • Kinast V.
      • Bruggemann Y.
      • et al.
      Robust hepatitis E virus infection and transcriptional response in human hepatocytes.
      and pluripotent stem cell-derived hepatocyte-like cells
      • Dao Thi V.L.
      • Wu X.
      • Belote R.L.
      • Andreo U.
      • Takacs C.N.
      • Fernandez J.P.
      • et al.
      Stem cell-derived polarized hepatocytes.
      have been explored as alternatives. In these polarised hepatocyte-like cells, quasi-enveloped HEV particles are secreted basolaterally, whereas naked virions are released apically, resembling the natural life cycle of HEV release.
      • Dao Thi V.L.
      • Wu X.
      • Belote R.L.
      • Andreo U.
      • Takacs C.N.
      • Fernandez J.P.
      • et al.
      Stem cell-derived polarized hepatocytes.
      Primary human enterocytes polarised in culture have been shown to support HEV replication and release quasi-enveloped HEV particles mainly into the apical compartment.
      • Marion O.
      • Lhomme S.
      • Nayrac M.
      • Dubois M.
      • Pucelle M.
      • Requena M.
      • et al.
      Hepatitis E virus replication in human intestinal cells.
      However, primary human hepatocytes or enterocytes are not easy to obtain, and are technically difficult to be maintained in culture. Hepatocyte-like cells can be routinely generated from pluripotent stem cells but are also not capable of sustaining long-term culture, thus limiting their utility for the modelling of persistent HEV infection.
      Innate immunity is essential but not sufficient in defending against HEV, and chronic infection is largely associated with impairment of the adaptive immune system.
      The advent of organoid technology represents a unique opportunity to move the field forward. These 3D cultured “mini-organs” are better at recapitulating the architecture, functionality and genetic signature of the original tissues. Human livers harbour bipotent progenitors that can be readily cultured into organoids, which resemble a cholangiocyte phenotype, but can be differentiated towards a hepatocyte-like phenotype by specific culture conditions.
      • Marsee A.
      • Roos F.J.M.
      • Verstegen M.M.A.
      • Consortium H.P.B.O.
      • Gehart H.
      • de Koning E.
      • et al.
      Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.
      Recently, both cholangiocyte-lineage organoids and hepatocyte-differentiated organoids have been shown to be highly susceptible to HEV infection, which is consistent with clinical observations that both cholangiocytes and hepatocytes can be infected by HEV in the livers of patients.
      • Beer A.
      • Holzmann H.
      • Pischke S.
      • Behrendt P.
      • Wrba F.
      • Schlue J.
      • et al.
      Chronic Hepatitis E is associated with cholangitis.
      ,
      • Wang W.
      • Wang Y.
      • Qu C.
      • Wang S.
      • Zhou J.
      • Cao W.
      • et al.
      The RNA genome of hepatitis E virus robustly triggers an antiviral interferon response.
      The establishment and advantages of liver organoid-based HEV models have been demonstrated in mapping virus-host interactions and discovering anti-HEV agents.
      • Li P.
      • Li Y.
      • Wang Y.
      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      Nevertheless, HEV replication in liver organoids gradually diminishes upon passaging in culture, which may be attributed to the robust innate immune defence. Overall, in vitro models are capable of mimicking persistent infection, but this is only one feature of chronic hepatitis E. Disease progression in patients with chronic HEV inevitably involve many cell types, in particular immune cells. Co-culture systems, for example augmenting liver organoids with macrophages and hepatic stellate cells, would further advance these in vitro models and better recapitulate chronic HEV infection.

      In vivo

      Human liver chimeric mouse models based on immunodeficient mice repopulated with human hepatocytes have been shown to support persistent infection of genotype 1 and 3 HEV and are suitable for antiviral drug testing. Because of the severely immunodeficient background, this model does not replicate immunopathogenesis, a critical feature seen in patients with chronic HEV.
      • 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.
      ,
      • van de Garde M.D.
      • Pas S.D.
      • van der Net G.
      • de Man R.A.
      • Osterhaus A.D.
      • Haagmans B.L.
      • et al.
      Hepatitis E virus (HEV) genotype 3 infection of human liver chimeric mice as a model for chronic HEV infection.
      This limitation may be partially overcome by adoptive transfer of human immune cells, which is technically difficult but feasible.
      • Douam F.
      • Ziegler C.G.K.
      • Hrebikova G.
      • Fant B.
      • Leach R.
      • Parsons L.
      • et al.
      Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses.
      In athymic nude rats lacking a normal thymus and functionally mature T cells, inoculation with rat HEV can result in persistent infection, but with limited evidence of an inflammatory response in the liver.
      • Debing Y.
      • Mishra N.
      • Verbeken E.
      • Ramaekers K.
      • Dallmeier K.
      • Neyts J.
      A rat model for hepatitis E virus.
      Cynomolgus monkeys were treated with tacrolimus to better mimic immunosuppression in transplant patients, and were then inoculated with genotype 3 HEV. Chronic infection was confirmed by persistent viraemia and faecal viral shedding, a slight increase in serum liver enzymes and typical liver histopathology with focal inflammation.
      • Gardinali N.R.
      • Guimaraes J.R.
      • Melgaco J.G.
      • Kevorkian Y.B.
      • Bottino F.O.
      • Vieira Y.R.
      • et al.
      Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy.
      A similar approach has been applied in pigs to establish chronic infection of genotype 3 HEV, in which active suppression of cell-mediated immune responses has been mechanistically attributed to chronicity.
      • Cao D.
      • Cao Q.M.
      • Subramaniam S.
      • Yugo D.M.
      • Heffron C.L.
      • Rogers A.J.
      • et al.
      Pig model mimicking chronic hepatitis E virus infection in immunocompromised patients to assess immune correlates during chronicity.
      Nevertheless, the ethical constraints of using monkeys and the large size of farm pigs have hampered their application in the research field. Very recently, chronic infection in rabbits was successfully established by immunosuppressant treatment and inoculation with genotype 3 or 4 HEV. This model recapitulates key features of viral kinetics, chronic disease progression and extrahepatic manifestations.
      • He Q.
      • Zhang F.
      • Shu J.
      • Li S.
      • Liang Z.
      • Du M.
      • et al.
      Immunocompromised rabbit model of chronic hepatitis E reveals liver fibrosis and distinct efficacy of different vaccination strategies.
      It is suitable for testing both antiviral drugs and HEV vaccines,
      • He Q.
      • Zhang F.
      • Shu J.
      • Li S.
      • Liang Z.
      • Du M.
      • et al.
      Immunocompromised rabbit model of chronic hepatitis E reveals liver fibrosis and distinct efficacy of different vaccination strategies.
      thus expanding the potential for wide-ranging applications in the HEV field in the near future.

      Therapeutic development

      First-line treatment

      The current management of patients with chronic HEV has been extensively described in the EASL guidelines.
      European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L
      EASL Clinical Practice Guidelines on hepatitis E virus infection.
      Briefly, dose reduction of immunosuppressive medications is the first-line option, which can achieve sustained viral clearance in about one-third of chronically infected transplant recipients. However, reducing immunosuppression is not feasible in all transplant patients because it can lead to an increased risk of acute rejection, especially in heart, lung or pancreas transplant patients. Reducing immunosuppressive therapy has been reported to be associated with mortality in HEV-infected haematopoietic stem cell transplant recipients.
      • von Felden J.
      • Alric L.
      • Pischke S.
      • Aitken C.
      • Schlabe S.
      • Spengler U.
      • et al.
      The burden of hepatitis E among patients with haematological malignancies: a retrospective European cohort study.
      Although no approved treatment is available, ribavirin monotherapy can be administered when immunosuppressant dose reduction is not possible and/or if it does not result in HEV clearance (Table 1).
      • von Felden J.
      • Alric L.
      • Pischke S.
      • Aitken C.
      • Schlabe S.
      • Spengler U.
      • et al.
      The burden of hepatitis E among patients with haematological malignancies: a retrospective European cohort study.
      ,
      • Sridhar S.
      • Yip C.C.
      • Lo K.H.
      • Wu S.
      • Situ J.
      • Chew N.F.
      • et al.
      Hepatitis E virus species C infection in humans, Hong Kong.
      ,
      • Affeldt P.
      • Di Cristanziano V.
      • Grundmann F.
      • Wirtz M.
      • Kaiser R.
      • Benzing T.
      • et al.
      Monitoring of hepatitis E virus RNA during treatment for chronic hepatitis E virus infection after renal transplantation.
      • Kamar N.
      • Abravanel F.
      • Behrendt P.
      • Hofmann J.
      • Pageaux G.P.
      • Barbet C.
      • et al.
      Ribavirin for hepatitis E virus infection after organ transplantation: a large European retrospective multicenter study.
      • Mulder M.B.
      • de Man R.A.
      • Kamar N.
      • Durmaz G.
      • de Bruijne J.
      • Vanwolleghem T.
      • et al.
      Determining the therapeutic range for ribavirin in transplant recipients with chronic hepatitis E virus infection.
      • Ankcorn M.
      • Said B.
      • Morgan D.
      • Elsharkawy A.M.
      • Maggs J.
      • Ryder S.
      • et al.
      Persistent hepatitis E virus infection across England and Wales 2009-2017: demography, virology and outcomes.
      • Owada Y.
      • Oshiro Y.
      • Inagaki Y.
      • Harada H.
      • Fujiyama N.
      • Kawagishi N.
      • et al.
      A nationwide survey of hepatitis E virus infection and chronic hepatitis in heart and kidney transplant recipients in Japan.
      • Friebus-Kardash J.
      • Eisenberger U.
      • Ackermann J.
      • Kribben A.
      • Witzke O.
      • Wenzel J.
      • et al.
      Prevalence of active hepatitis E virus infection and efficacy of ribavirin treatment in renal allograft recipients.
      • Low E.X.S.
      • Tripon E.
      • Lim K.
      • Tan P.S.
      • Low H.C.
      • Dan Y.Y.
      • et al.
      Risk factors for ribavirin treatment failure in Asian organ transplant recipients with chronic hepatitis E infection.
      • Choi M.
      • Hofmann J.
      • Kohler A.
      • Wang B.
      • Bock C.T.
      • Schott E.
      • et al.
      Prevalence and clinical correlates of chronic hepatitis E infection in German renal transplant recipients with elevated liver enzymes.
      • Cordts S.E.
      • Schneble L.
      • Schnitzler P.
      • Wenzel J.J.
      • Vinke T.
      • Rieger S.
      • et al.
      Prevalence, morbidity, and therapy of hepatitis E virus infection in pediatric renal allograft recipients.
      • 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.
      Large series in solid organ transplant patients have shown that treatment with ribavirin monotherapy leads to sustained virological response (SVR) in up to 90% of patients.
      • Kamar N.
      • Abravanel F.
      • Behrendt P.
      • Hofmann J.
      • Pageaux G.P.
      • Barbet C.
      • et al.
      Ribavirin for hepatitis E virus infection after organ transplantation: a large European retrospective multicenter study.
      ,
      • Kamar N.
      • Izopet J.
      • Tripon S.
      • Bismuth M.
      • Hillaire S.
      • Dumortier J.
      • et al.
      Ribavirin for chronic hepatitis E virus infection in transplant recipients.
      ,
      • 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.
      The majority (∼80%) of patients receive 600 mg per day for 3 months, but this treatment duration was empirically determined. For patients who relapse after 3 months of therapy, prolonged treatment (6 months or even longer) can lead to SVR.
      • Kamar N.
      • Abravanel F.
      • Behrendt P.
      • Hofmann J.
      • Pageaux G.P.
      • Barbet C.
      • et al.
      Ribavirin for hepatitis E virus infection after organ transplantation: a large European retrospective multicenter study.
      ,
      • Kamar N.
      • Izopet J.
      • Tripon S.
      • Bismuth M.
      • Hillaire S.
      • Dumortier J.
      • et al.
      Ribavirin for chronic hepatitis E virus infection in transplant recipients.
      Patients with negative serum HEV RNA and persisting HEV RNA in stools after 3 months of therapy are known to be at high risk of relapse after ceasing therapy.
      • Abravanel F.
      • Lhomme S.
      • Rostaing L.
      • Kamar N.
      • Izopet J.
      Protracted fecal shedding of HEV during ribavirin therapy predicts treatment relapse.
      Prolonging treatment duration has been shown to improve the SVR rate in this context.
      • Marion O.
      • Lhomme S.
      • Del Bello A.
      • Abravanel F.
      • Esposito L.
      • Hebral A.L.
      • et al.
      Monitoring hepatitis E virus fecal shedding to optimize ribavirin treatment duration in chronically infected transplant patients.
      Ribavirin monotherapy is effective in treating chronic hepatitis E in organ transplant recipients. However, some patients cannot tolerate or do not adequately respond to ribavirin, thus highlighting the need to develop novel therapies.
      Table 1Summary of the key findings from major/representative clinical studies assessing ribavirin monotherapy for treating chronic hepatitis E.
      StudyPublication yearOriginHEV genotypePatient characteristicsNumber of treated patientsOutcome (patient number)Remark
      Sridhar et al.
      • Sridhar S.
      • Yip C.C.
      • Lo K.H.
      • Wu S.
      • Situ J.
      • Chew N.F.
      • et al.
      Hepatitis E virus species C infection in humans, Hong Kong.
      2021Hong Kong, ChinaHEV-C1Immunocompromised54 (SVR)One patient only responded after >6 months of ribavirin and was pending SVR at the time of this study
      Affeldt et al.
      • Affeldt P.
      • Di Cristanziano V.
      • Grundmann F.
      • Wirtz M.
      • Kaiser R.
      • Benzing T.
      • et al.
      Monitoring of hepatitis E virus RNA during treatment for chronic hepatitis E virus infection after renal transplantation.
      2021GermanyN/ASOT64 (HEV clearance)Renal transplant recipients
      Kamar et al.
      • Kamar N.
      • Abravanel F.
      • Behrendt P.
      • Hofmann J.
      • Pageaux G.P.
      • Barbet C.
      • et al.
      Ribavirin for hepatitis E virus infection after organ transplantation: a large European retrospective multicenter study.
      2020Europe (multi-centre)GT3SOT255207 (SVR; a first course)18 out of 36 re-treated patients also achieved SVR
      Milder et al.
      • Mulder M.B.
      • de Man R.A.
      • Kamar N.
      • Durmaz G.
      • de Bruijne J.
      • Vanwolleghem T.
      • et al.
      Determining the therapeutic range for ribavirin in transplant recipients with chronic hepatitis E virus infection.
      2020Europe (multi-centre)SOT; HSCT9671 (SVR)Determined the therapeutic range for ribavirin between 1.8 and 2.3 mg/L
      Ankcorn et al.
      • Ankcorn M.
      • Said B.
      • Morgan D.
      • Elsharkawy A.M.
      • Maggs J.
      • Ryder S.
      • et al.
      Persistent hepatitis E virus infection across England and Wales 2009-2017: demography, virology and outcomes.
      2020United KingdomN/AImmunocompromised6548 (HEV clearance)A study across England and Wales 2009-2017
      Owada et al.
      • Owada Y.
      • Oshiro Y.
      • Inagaki Y.
      • Harada H.
      • Fujiyama N.
      • Kawagishi N.
      • et al.
      A nationwide survey of hepatitis E virus infection and chronic hepatitis in heart and kidney transplant recipients in Japan.
      2020JapanGT3SOT33 (SVR)A nationwide survey of HEV infection in heart and kidney transplant recipients
      Friebus-Kardash et al.
      • Friebus-Kardash J.
      • Eisenberger U.
      • Ackermann J.
      • Kribben A.
      • Witzke O.
      • Wenzel J.
      • et al.
      Prevalence of active hepatitis E virus infection and efficacy of ribavirin treatment in renal allograft recipients.
      2019GermanyGT3SOT1615 (SVR)One developed ribavirin resistance and revealed multiple mutations of HEV polymerase, K1383N, G1364R/K, and D1384N
      Low et al.
      • Low E.X.S.
      • Tripon E.
      • Lim K.
      • Tan P.S.
      • Low H.C.
      • Dan Y.Y.
      • et al.
      Risk factors for ribavirin treatment failure in Asian organ transplant recipients with chronic hepatitis E infection.
      2019SingaporeGT3; GT7SOT94 (SVR)The GT7 infected patient came from United Arab Emirates
      • 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.
      von Felden et al.
      • von Felden J.
      • Alric L.
      • Pischke S.
      • Aitken C.
      • Schlabe S.
      • Spengler U.
      • et al.
      The burden of hepatitis E among patients with haematological malignancies: a retrospective European cohort study.
      2019Europe (multi-centre)GT3Haematological malignancy107 (HEV clearance)Treated patients include 4 alloHSCT and 3 of them cleared HEV
      Choi et al.
      • Choi M.
      • Hofmann J.
      • Kohler A.
      • Wang B.
      • Bock C.T.
      • Schott E.
      • et al.
      Prevalence and clinical correlates of chronic hepatitis E infection in German renal transplant recipients with elevated liver enzymes.
      2018GermanyGT3SOT1613 (SVR)Renal transplant recipients; one developed ribavirin resistance
      Cordts et al.
      • Cordts S.E.
      • Schneble L.
      • Schnitzler P.
      • Wenzel J.J.
      • Vinke T.
      • Rieger S.
      • et al.
      Prevalence, morbidity, and therapy of hepatitis E virus infection in pediatric renal allograft recipients.
      2018GermanyGT3SOT44 (SVR)Paediatric renal allograft recipients
      Kamar et al.
      • Kamar N.
      • Izopet J.
      • Tripon S.
      • Bismuth M.
      • Hillaire S.
      • Dumortier J.
      • et al.
      Ribavirin for chronic hepatitis E virus infection in transplant recipients.
      2014FranceGT3SOT5946 (SVR)6 of the 10 patients who had a recurrence were re-treated and cleared the virus
      Pischke et al.
      • 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.
      2013GermanyN/ASOT119 (HEV clearance)Kidney, heart and lung transplant recipients
      The treatment measures/outcomes are described as defined/reported by these original studies. Only information related to treatment of chronic HEV infection is presented in the table.
      alloHSCT, allogeneic haematopoietic stem cell transplantation; GT, genotype; HEV, hepatitis E virus; HSCT, haematopoietic stem cell transplantation; N/A, not available; SOT, solid organ transplant; SVR, sustained virological response.
      Lymphopenia reflecting deep-seated immunosuppression, as evidenced in some transplant patients, such as heart and lung transplant recipients, has been linked to a reduction in the SVR rate. Ribavirin dose reduction and the need for blood transfusions were also associated with a decreased virological response. Ribavirin is eliminated by the kidney. It can cause haemolytic anaemia in patients with impaired kidney function.
      • Kamar N.
      • Chatelut E.
      • Manolis E.
      • Lafont T.
      • Izopet J.
      • Rostaing L.
      Ribavirin pharmacokinetics in renal and liver transplant patients: evidence that it depends on renal function.
      Unfortunately, most transplant patients have a decreased glomerular filtration rate requiring ribavirin dose reduction. Consequently, a full ribavirin dose is rarely used in this population.
      The mode of action of ribavirin remains largely elusive. It has been shown that, similarly to mycophenolic acid, ribavirin inhibits HEV replication through depletion of guanosine triphosphate 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.
      In vitro, the combination of mycophenolic acid and ribavirin have an augmented anti-HEV effect,
      • 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.
      but this was not observed in treated patients.
      • 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.
      Furthermore, ribavirin may increase HEV mutagenesis which appears to be reversible.
      • Todt D.
      • Gisa A.
      • Radonic A.
      • Nitsche A.
      • Behrendt P.
      • Suneetha P.V.
      • et al.
      In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome.
      A G1634R mutation in viral polymerase has been associated with ribavirin treatment failure in organ transplant recipients.
      • Todt D.
      • Gisa A.
      • Radonic A.
      • Nitsche A.
      • Behrendt P.
      • Suneetha P.V.
      • et al.
      In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome.
      ,
      • 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.
      Reconstructing this mutation by reverse genetics showed increased viral replication, but did not provide HEV with resistance to ribavirin in cell culture.
      • 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.
      Overall, there is a lack of robust data to explain the precise mechanisms by which ribavirin inhibits HEV or drug resistance develops, which warrant further investigation. Finally, in ribavirin non-responders, there is currently no alternative expect for pegylated interferon-alpha that can be only used in liver transplant patients and non-transplant patients with chronic hepatitis E.
      • 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.
      In other transplant patients, pegylated interferon-alpha increases the risk of acute rejection.
      • Rostaing L.
      • Modesto A.
      • Baron E.
      • Cisterne J.M.
      • Chabannier M.H.
      • Durand D.
      Acute renal failure in kidney transplant patients treated with interferon alpha 2b for chronic hepatitis C.
      Thus, it is essential that the WHO continues to include ribavirin in the List of Essential Medicines, while the development of additional therapies is crucial.

      Future development

      Given that only a small proportion of patients with HEV require antiviral treatment, there is little economic incentive for the pharmaceutical industry to develop new antiviral drugs. Repurposing existing medications represents a realistic and attractive approach. Sofosbuvir, a potent nucleotide polymerase inhibitor of hepatitis C virus, was initially shown to effectively inhibit HEV in cell culture models.
      • 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.
      However, a follow-up experimental study
      • Wang W.
      • Hakim M.S.
      • Nair V.P.
      • de Ruiter P.E.
      • Huang F.
      • Sprengers D.
      • et al.
      Distinct antiviral potency of sofosbuvir against hepatitis C and E viruses.
      and subsequent off-label treatment in individual chronic HEV cases
      • van der Valk M.
      • Zaaijer H.L.
      • Kater A.P.
      • Schinkel J.
      Sofosbuvir shows antiviral activity in a patient with chronic hepatitis E virus infection.
      ,
      • Donnelly M.C.
      • Imlach S.N.
      • Abravanel F.
      • Ramalingam S.
      • Johannessen I.
      • Petrik J.
      • et al.
      Sofosbuvir and daclatasvir anti-viral therapy fails to clear HEV viremia and restore reactive T cells in a HEV/HCV Co-infected liver transplant recipient.
      yielded conflicting results. In rabbits acutely infected with genotype 3 or 4 HEV, high dose sofosbuvir appears to exert some degree of antiviral activity.
      • Liang Z.
      • Shu J.
      • He Q.
      • Zhang F.
      • Dai L.
      • Wang L.
      • et al.
      High dose sofosbuvir and sofosbuvir-plus-ribavirin therapy inhibit Hepatitis E Virus (HEV) replication in a rabbit model for acute HEV infection.
      Finally, a prospective, multicentre, phase II pilot trial has demonstrated that sofosbuvir exerts very mild inhibition on viral load in patients with chronic HEV, and none of the 9 treated patients was able to clear the infection,
      • Cornberg M.
      • Pischke S.
      • Muller T.
      • Behrendt P.
      • Piecha F.
      • Benckert J.
      • et al.
      Sofosbuvir monotherapy fails to achieve HEV RNA elimination in patients with chronic hepatitis E - The HepNet SofE pilot study.
      precluding future clinical applications of sofosbuvir as monotherapy for the treatment of chronic hepatitis E. An important lesson learned is that understanding the underlying mechanisms of the candidate drugs and thorough investigations in preclinical models are essential for increasing the success rate of drug repurposing. So far, several FDA-approved medications (e.g. azithromycin,
      • Miao Z.
      • Zhang R.
      • Yu P.
      • Li Y.
      • Pan Q.
      • Li Y.
      The macrolide antibiotic azithromycin potently inhibits hepatitis E virus in cell culture models.
      ivermectin,
      • Li Y.
      • Miao Z.
      • Li P.
      • Zhang R.
      • Kainov D.E.
      • Ma Z.
      • et al.
      Ivermectin effectively inhibits hepatitis E virus replication, requiring the host nuclear transport protein importin alpha1.
      niclosamide,
      • Li Y.
      • Li P.
      • He Q.
      • Zhang R.
      • Li Y.
      • Kamar N.
      • et al.
      Niclosamide inhibits hepatitis E virus through suppression of NF-kappaB signalling.
      and gemcitabine
      • Li Y.
      • Li P.
      • Li Y.
      • Zhang R.
      • Yu P.
      • Ma Z.
      • et al.
      Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation.
      ) and an essential micronutrient (Zinc
      • Kaushik N.
      • Subramani C.
      • Anang S.
      • Muthumohan R.
      • Shalimar
      • Nayak B.
      • et al.
      Zinc salts block hepatitis E virus replication by inhibiting the activity of viral RNA-dependent RNA polymerase.
      ) have been shown to inhibit HEV in vitro (Table 2). We recommend that clinical testing of these agents should be preceded by extensive preclinical studies especially in relevant animal models that are already available.
      Joint efforts from researchers, clinicians and health authorities are urgently required to further advance research and clinical management of chronic HEV infection.
      Table 2A list of candidate or experimental therapies that have been explored for the treatment of HEV infection in experimental models and/or patients.
      Regimen (ref.)Potential anti-HEV mechanismTestedRemark
      In vitroAnimal modelPatient
      Ribavirin
      • He Q.
      • Zhang F.
      • Shu J.
      • Li S.
      • Liang Z.
      • Du M.
      • et al.
      Immunocompromised rabbit model of chronic hepatitis E reveals liver fibrosis and distinct efficacy of different vaccination strategies.
      ,
      • Debing Y.
      • Mishra N.
      • Verbeken E.
      • Ramaekers K.
      • Dallmeier K.
      • Neyts J.
      A rat model for hepatitis E virus.
      ,
      • Li P.
      • Li Y.
      • Wang Y.
      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      ,
      • Kamar N.
      • Izopet J.
      • Tripon S.
      • Bismuth M.
      • Hillaire S.
      • Dumortier J.
      • et al.
      Ribavirin for chronic hepatitis E virus infection in transplant recipients.
      ,
      • 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.
      ,
      • 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.
      Targeting nucleotide synthesisCell lines; Organoids; Primary hepatocytes; Primary intestinal cells; Stem cell-derived HLCsHuman liver chimeric mice; Athymic nude rats; RabbitsChronic HEV patientsFirst-line treatment for SOT recipients with chronic hepatitis E
      Interferon-alpha
      • Li P.
      • Li Y.
      • Wang Y.
      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      ,
      • Haagsma E.B.
      • Riezebos-Brilman A.
      • van den Berg A.P.
      • Porte R.J.
      • Niesters H.G.
      Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2b.
      ,
      • van de Garde M.D.B.
      • Pas S.D.
      • van Oord G.W.
      • Gama L.
      • Choi Y.
      • de Man R.A.
      • et al.
      Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice.
      Activating innate antiviral responseCell lines; OrganoidsHuman liver chimeric miceChronic HEV patientsOnly used for a few cases of chronic HEV; risk of causing transplant rejection
      Sofosbuvir
      • Cornberg M.
      • Pischke S.
      • Muller T.
      • Behrendt P.
      • Piecha F.
      • Benckert J.
      • et al.
      Sofosbuvir monotherapy fails to achieve HEV RNA elimination in patients with chronic hepatitis E - The HepNet SofE pilot 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.
      ,
      • Wang W.
      • Hakim M.S.
      • Nair V.P.
      • de Ruiter P.E.
      • Huang F.
      • Sprengers D.
      • et al.
      Distinct antiviral potency of sofosbuvir against hepatitis C and E viruses.
      ,
      • Liang Z.
      • Shu J.
      • He Q.
      • Zhang F.
      • Dai L.
      • Wang L.
      • et al.
      High dose sofosbuvir and sofosbuvir-plus-ribavirin therapy inhibit Hepatitis E Virus (HEV) replication in a rabbit model for acute HEV infection.
      Viral RdRp inhibitorCell lines; Stem cell-derived HLCsRabbitsChronic HEV patientsA phase II trial demonstrated failure to clear HEV in patients with chronic infection
      Interferon-lambda
      • Sari G.
      • Mulders C.E.
      • Zhu J.
      • van Oord G.W.
      • Feng Z.
      • Kreeft-Voermans J.J.C.
      • et al.
      Treatment induced clearance of hepatitis E viruses by interferon-lambda in liver-humanized mice.
      Activating innate antiviral responseCell linesHuman liver chimeric miceInterferon-lambda compared to interferon-alpha has the potential to spare patients from systemic side effects
      Mycophenolic acid
      • 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.
      ,
      • Li P.
      • Li Y.
      • Wang Y.
      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      Targeting nucleotide synthesisCell lines; OrganoidsChronic HEV patientsUsed as immunosuppressant in transplant recipients with chronic hepatitis E, but the anti-HEV activity in patients remains unclear
      Gemcitabine
      • Li Y.
      • Li P.
      • Li Y.
      • Zhang R.
      • Yu P.
      • Ma Z.
      • et al.
      Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation.
      Targeting nucleotide synthesis; activating innate antiviral responseCell lines; OrganoidsA widely used anti-cancer drug
      Brequinar
      • Li P.
      • Li Y.
      • Wang Y.
      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      ,
      • Wang Y.
      • Wang W.
      • Xu L.
      • Zhou X.
      • Shokrollahi E.
      • Felczak K.
      • et al.
      Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis E virus replication.
      Targeting nucleotide synthesisCell lines; OrganoidsDHODH inhibitor of pyrimidine nucleotide biosynthesis pathway
      Leflunomide
      • Wang Y.
      • Wang W.
      • Xu L.
      • Zhou X.
      • Shokrollahi E.
      • Felczak K.
      • et al.
      Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis E virus replication.
      Targeting nucleotide synthesisCell linesDHODH inhibitor of pyrimidine nucleotide biosynthesis pathway
      6-azauracil
      • Wang Y.
      • Wang W.
      • Xu L.
      • Zhou X.
      • Shokrollahi E.
      • Felczak K.
      • et al.
      Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis E virus replication.
      Targeting nucleotide synthesisCell linesODCase inhibitor of pyrimidine nucleotide biosynthesis pathway
      Homoharringtonine
      • Li P.
      • Li Y.
      • Wang Y.
      • Liu J.
      • Lavrijsen M.
      • Li Y.
      • et al.
      Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.
      Cell lines; OrganoidsFDA-approved medication for treating chronic myeloid leukaemia
      Silvestrol
      • Todt D.
      • Moeller N.
      • Praditya D.
      • Kinast V.
      • Friesland M.
      • Engelmann M.
      • et al.
      The natural compound silvestrol inhibits hepatitis E virus (HEV) replication in vitro and in vivo.
      ,
      • Glitscher M.
      • Himmelsbach K.
      • Woytinek K.
      • Johne R.
      • Reuter A.
      • Spiric J.
      • et al.
      Inhibition of hepatitis E virus spread by the natural compound silvestrol.
      Affecting the translation of HEV RNA; involving the host factor Major Vault ProteinCell linesHuman liver chimeric miceA natural compound extracted from the plant species Aglaia foveolata; a potent and selective inhibitor of the eukaryotic initiation factor 4A
      Zinc
      • Kaushik N.
      • Subramani C.
      • Anang S.
      • Muthumohan R.
      • Shalimar
      • Nayak B.
      • et al.
      Zinc salts block hepatitis E virus replication by inhibiting the activity of viral RNA-dependent RNA polymerase.
      May inhibit HEV RdRpCell linesAn essential micronutrient
      Deptropine
      • Qu C.
      • Li Y.
      • Li Y.
      • Yu P.
      • Li P.
      • Donkers J.M.
      • et al.
      FDA-drug screening identifies deptropine inhibiting hepatitis E virus involving the NF-kappaB-RIPK1-caspase axis.
      involving the NF-κB-RIPK1-caspase axisCell linesA classical histamine H1 receptor antagonist for treating asthmatic symptoms; but nowadays rarely used in the clinic
      Ivermectin
      • Li Y.
      • Miao Z.
      • Li P.
      • Zhang R.
      • Kainov D.E.
      • Ma Z.
      • et al.
      Ivermectin effectively inhibits hepatitis E virus replication, requiring the host nuclear transport protein importin alpha1.
      Involving the host nuclear transport protein importin α1Cell linesA widely used anti-parasitic drug
      Azithromycin
      • Miao Z.
      • Zhang R.
      • Yu P.
      • Li Y.
      • Pan Q.
      • Li Y.
      The macrolide antibiotic azithromycin potently inhibits hepatitis E virus in cell culture models.
      Cell linesTreating a number of bacterial infections; an FDA pregnancy category B drug
      Ciprofloxacin
      • Nishiyama T.
      • Kobayashi T.
      • Jirintai S.
      • Kii I.
      • Nagashima S.
      • Prathiwi Primadharsini P.
      • et al.
      Screening of novel drugs for inhibiting hepatitis E virus replication.
      Cell linesA fluoroquinolone antibiotic used to treat a number of bacterial infections
      GPC-N114
      • Netzler N.E.
      • Enosi Tuipulotu D.
      • Vasudevan S.G.
      • Mackenzie J.M.
      • White P.A.
      Antiviral candidates for treating hepatitis E virus infection.
      Viral RdRp inhibitorCell lines
      NITD008
      • Netzler N.E.
      • Enosi Tuipulotu D.
      • Vasudevan S.G.
      • Mackenzie J.M.
      • White P.A.
      Antiviral candidates for treating hepatitis E virus infection.
      Viral RdRp inhibitorCell lines
      2’-C-methylcytidine
      • Qu C.
      • Xu L.
      • Yin Y.
      • Peppelenbosch M.P.
      • Pan Q.
      • Wang W.
      Nucleoside analogue 2'-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin.
      Nucleoside analogueCell linesThe effect could be reverted by cytidine triphosphate, but not guanosine triphosphate
      Amodiaquine
      • Tietcheu Galani B.R.
      • Ayissi Owona V.B.
      • Guemmogne Temdie R.J.
      • Metzger K.
      • Atsama Amougou M.
      • Djamen Chuisseu P.D.
      • et al.
      In silico and in vitro screening of licensed antimalarial drugs for repurposing as inhibitors of hepatitis E virus.
      Potential binding to RdRp indicated by molecular dockingCell linesAn antimalarial drug; cation for interpreting the specificity as accompanied by clear cytotoxicity
      Isocotoin
      • Nimgaonkar I.
      • Archer N.F.
      • Becher I.
      • Shahrad M.
      • LeDesma R.A.
      • Mateus A.
      • et al.
      Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90.
      Through interference with HSP90Cell linesIdentified through compound screening
      TCR-based adoptive T cell therapy
      • Soon C.F.
      • Behrendt P.
      • Todt D.
      • Manns M.P.
      • Wedemeyer H.
      • Sallberg Chen M.
      • et al.
      Defining virus-specific CD8+ TCR repertoires for therapeutic regeneration of T cells against chronic hepatitis E.
      Killing HEV infected target cellsPatient derived T cells (TCR-engineered)FDA approved T-cell immunotherapies for treating cancer; may be an alternative approach for treating chronic HEV infection
      Monoclonal antibody
      • Gu Y.
      • Tang X.
      • Zhang X.
      • Song C.
      • Zheng M.
      • Wang K.
      • et al.
      Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody.
      Neutralising HEVCell linesMonkeysExerting cross-genotype neutralising activity; blocking HEV infection in cell culture and preventing hepatitis E disease in animals
      RNAi
      • Zhang C.
      • Freistaedter A.
      • Schmelas C.
      • Gunkel M.
      • Dao Thi V.L.
      • Grimm D.
      An RNA interference/adeno-associated virus vector-based combinatorial gene therapy approach against hepatitis E virus.
      Targeting HEV genomeCell lines; Stem cell-derived HLCsCombination of multiple siRNA targets to counteract viral escape through mutations
      DHODH, dihydroorotate dehydrogenase; HEV, hepatitis E virus; HLCs, hepatocyte-like cells; Hsp90, heat shock protein 90; ODCase, orotidine-5'-monophosphate decarboxylase; RdRp, RNA-dependent RNA polymerase; RIPK1, receptor-interacting serine/threonine-protein kinase 1; RNAi, RNA interference; siRNA, small-interfering RNA; SOT, solid organ transplant; TCR, T-cell receptor.
      Various strategies
      • Velavan T.P.
      • Pallerla S.R.
      • Johne R.
      • Todt D.
      • Steinmann E.
      • Schemmerer M.
      • et al.
      Hepatitis E: an update on One Health and clinical medicine.
      • Haagsma E.B.
      • Riezebos-Brilman A.
      • van den Berg A.P.
      • Porte R.J.
      • Niesters H.G.
      Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2b.
      • van de Garde M.D.B.
      • Pas S.D.
      • van Oord G.W.
      • Gama L.
      • Choi Y.
      • de Man R.A.
      • et al.
      Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice.
      • Wang Y.
      • Wang W.
      • Xu L.
      • Zhou X.
      • Shokrollahi E.
      • Felczak K.
      • et al.
      Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis E virus replication.
      • Sari G.
      • Mulders C.E.
      • Zhu J.
      • van Oord G.W.
      • Feng Z.
      • Kreeft-Voermans J.J.C.
      • et al.
      Treatment induced clearance of hepatitis E viruses by interferon-lambda in liver-humanized mice.
      • Nishiyama T.
      • Kobayashi T.
      • Jirintai S.
      • Kii I.
      • Nagashima S.
      • Prathiwi Primadharsini P.
      • et al.
      Screening of novel drugs for inhibiting hepatitis E virus replication.
      • Netzler N.E.
      • Enosi Tuipulotu D.
      • Vasudevan S.G.
      • Mackenzie J.M.
      • White P.A.
      Antiviral candidates for treating hepatitis E virus infection.
      • Qu C.
      • Xu L.
      • Yin Y.
      • Peppelenbosch M.P.
      • Pan Q.
      • Wang W.
      Nucleoside analogue 2'-C-methylcytidine inhibits hepatitis E virus replication but antagonizes ribavirin.
      including screening of drug libraries,
      • Qu C.
      • Li Y.
      • Li Y.
      • Yu P.
      • Li P.
      • Donkers J.M.
      • et al.
      FDA-drug screening identifies deptropine inhibiting hepatitis E virus involving the NF-kappaB-RIPK1-caspase axis.
      exploration of preclinical
      • Nimgaonkar I.
      • Archer N.F.
      • Becher I.
      • Shahrad M.
      • LeDesma R.A.
      • Mateus A.
      • et al.
      Isocotoin suppresses hepatitis E virus replication through inhibition of heat shock protein 90.
      or natural
      • Todt D.
      • Moeller N.
      • Praditya D.
      • Kinast V.
      • Friesland M.
      • Engelmann M.
      • et al.
      The natural compound silvestrol inhibits hepatitis E virus (HEV) replication in vitro and in vivo.
      ,
      • Glitscher M.
      • Himmelsbach K.
      • Woytinek K.
      • Johne R.
      • Reuter A.
      • Spiric J.
      • et al.
      Inhibition of hepatitis E virus spread by the natural compound silvestrol.
      compounds as well as in silico docking
      • Tietcheu Galani B.R.
      • Ayissi Owona V.B.
      • Guemmogne Temdie R.J.
      • Metzger K.
      • Atsama Amougou M.
      • Djamen Chuisseu P.D.
      • et al.
      In silico and in vitro screening of licensed antimalarial drugs for repurposing as inhibitors of hepatitis E virus.
      have been dedicated to the quest to find novel HEV inhibitors (Table 2). When transferred into T cells obtained from patients with chronic HEV, engineered T-cell receptors have been shown to recognise virus-specific epitopes and mediate killing of the target cells in cell culture,
      • Soon C.F.
      • Behrendt P.
      • Todt D.
      • Manns M.P.
      • Wedemeyer H.
      • Sallberg Chen M.
      • et al.
      Defining virus-specific CD8+ TCR repertoires for therapeutic regeneration of T cells against chronic hepatitis E.
      indicating that T cell-based immunotherapy could be used to treat chronic HEV infection. Important lessons have been learned from the COVID-19 pandemic, which has highlighted the promise of monoclonal antibodies for the treatment of viral infections. Several monoclonal antibodies targeting the HEV capsid and exerting cross-genotype neutralising activity have been developed. These antibodies are capable of blocking HEV infection in cell culture and preventing hepatitis E disease in animals.
      • Gu Y.
      • Tang X.
      • Zhang X.
      • Song C.
      • Zheng M.
      • Wang K.
      • et al.
      Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody.
      RNA interference-based gene therapy targeting the HEV genome has been shown to persistently inhibit viral replication in vitro.
      • Zhang C.
      • Freistaedter A.
      • Schmelas C.
      • Gunkel M.
      • Dao Thi V.L.
      • Grimm D.
      An RNA interference/adeno-associated virus vector-based combinatorial gene therapy approach against hepatitis E virus.
      A common challenge for these experimental therapies is the lack of sustainable strategies that promote development towards clinical translation. We recommend that researchers located in Europe look for opportunities from the European Innovation Council to further develop their experimental anti-HEV therapies.

      Conclusion

      HEV was initially thought to cause acute infection only, but the discovery of chronic hepatitis E has profoundly changed our understanding of the virus and the disease. Over the past decade, we have gained substantial insight into the epidemiology, pathophysiological mechanisms and clinical features of chronic HEV infection. Reducing immunosuppression or initiating ribavirin monotherapy has been recommended as first-line treatment for chronically infected organ transplant recipients.
      European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L
      EASL Clinical Practice Guidelines on hepatitis E virus infection.
      As emphasized above, many knowledge gaps remain and major challenges in translating knowledge into disease prevention and improving clinical outcomes for patients with chronic HEV have yet to be overcome (Box 1). Closing these gaps in turn requires joint efforts from fundamental, translational and clinical researchers as well as health authorities to further advance HEV research and patient care. Finally, the WHO has launched an initiative to eliminate viral hepatitis by 2030, but the burden of hepatitis E has not yet been recognised. Without tackling HEV, achieving the goal of eliminating viral hepatitis is impossible.

      Abbreviations

      HEV, hepatitis E virus; HCC, hepatocellular carcinoma; MMF, mycophenolate mofetil; NLRP3, NOD-like receptor family pyrin domain-containing 3; SVR, sustained virological response; WHO, World Health Organization.

      Financial support

      Q.P. is supported by a VIDI grant (No. 91719300 ) from the Netherlands Organisation for Scientific Research (NWO) and a Young Investigator Grant ( 10140 ) from the Dutch Cancer Society .

      Authors’ contributions

      Z.M. and Q. P. were responsible for conceptualizing and drafting the manuscript. R.A.M. and N.K. were responsible for discussion and critically revising the manuscript. All authors approved the manuscript.

      Conflicts of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      The authors wish to thank Dr. Jose Debes (Department of Medicine, University of Minnesota, USA) for his critical editing of the manuscript.

      Supplementary data

      The following are the supplementary data to this article:

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