Animal models of hepatitis E infection: Advances and challenges

Hepatitis E virus(HEV)is one of the leading causes of acute viral hepatitis worldwide.Although most of HEV infections are asymptomatic,some patients will develop the symptoms,especially pregnant women,the elderly,and patients with preexisting liver diseases,who often experience anorexia,nausea,vom-iting,malaise,abdominal pain,and jaundice.HEV infection may become chronic in immunosuppressed individuals.In addition,HEV infection can also cause several extrahepatic manifestations.HEV exists in a wide range of hosts in nature and can be transmitted across species.Hence,animals susceptible to HEV can be used as models.The establishment of animal models is of great significance for studying HEV transmission,clinical symptoms,extrahepatic manifestations,and therapeutic strategies,which will help us understand the pathogenesis,prevention,and treatment of hepatitis E.This review summarized the animal models of HEV,including pigs,monkeys,rabbits,mice,rats,and other animals.For each animal species,we provided a concise summary of the HEV genotypes that they can be infected with,the cross-species transmission pathways,as well as their role in studying extrahepatic manifestations,prevention,and treatment of HEV infection.The advantages and disadvantages of these animal models were also emphasized.This review offers new perspectives to enhance the current understanding of the research landscape surrounding HEV animal models.

Current perspectives of viral hepatitis

Viral hepatitis represents a major danger to public health,and is a globally leading cause of death.The five liver-specific viruses:Hepatitis A virus,hepatitis B virus,hepatitis C virus,hepatitis D virus,and hepatitis E virus,each have their own unique epidemiology,structural biology,transmission,endemic patterns,risk of liver complications,and response to antiviral therapies.There remain few options for treatment,in spite of the increasing prevalence of viral-hepatitiscaused liver disease.Furthermore,chronic viral hepatitis is a leading worldwide cause of both liver-related morbidity and mortality,even though effective treatments are available that could reduce or prevent most patients’complications.In 2016,the World Health Organization released its plan to eliminate viral hepatitis as a public health threat by the year 2030,along with a discussion of current gaps and prospects for both regional and global eradication of viral hepatitis.Today,treatment is sufficiently able to prevent the disease from reaching advanced phases.However,future therapies must be extremely safe,and should ideally limit the period of treatment necessary.A better understanding of pathogenesis will prove beneficial in the development of potential treatment strategies targeting infections by viral hepatitis.This review aims to summarize the current state of knowledge on each type of viral hepatitis,together with major innovations.

Pathogenesis and clinical features of severe hepatitis E virus infection

The hepatitis E virus(HEV),a member of the Hepeviridae family,is a small,non-enveloped icosahedral virus divided into eight distinct genotypes(HEV-1 to HEV-8).Only genotypes 1 to 4 are known to cause diseases in humans.Genotypes 1 and 2 commonly spread via fecal-oral transmission,often through the consum-ption of contaminated water.Genotypes 3 and 4 are known to infect pigs,deer,and wild boars,often transferring to humans through inadequately cooked meat.Acute hepatitis caused by HEV in healthy individuals is mostly asymptomatic or associated with minor symptoms,such as jaundice.However,in immunosup-pressed individuals,the disease can progress to chronic hepatitis and even escalate to cirrhosis.For pregnant women,an HEV infection can cause fulminant liver failure,with a potential mortality rate of 25%.Mortality rates also rise amongst cirrhotic patients when they contract an acute HEV infection,which can even trigger acute-on-chronic liver failure if layered onto pre-existing chronic liver disease.As the prevalence of HEV infection continues to rise worldwide,highlighting the particular risks associated with severe HEV infection is of major medical interest.This text offers a brief summary of the characteristics of hepatitis developed by patient groups at an elevated risk of severe HEV infection.

Hepatitis E virus infections

Hepatitis E virus(HEV)infection is now endemic worldwide.Most patients with acute infection recover uneventfully.Outbreaks and sporadic cases,particularly in high-risk individuals are emerging increasingly.The patients with risk factors like pregnancy and pre-existing chronic liver disease,present with or progress rapidly to severe disease.Immuno-suppression in post-transplant patients is an additional risk factor.Standardized FDA-approved diagnostic tests are the need of the hour.Further studies are needed to establish guideline-based treatment regimen and outbreak preparedness for HEV to decrease global morbidity,mortality,and healthcare burden.Policies for screening donors and transplant cases are requi-red.

Partial nucleotide sequencing of hepatitis E viruses detected in sera of patients with hepatitis E from 14 cities in China

OBJECTIVE: To investigate the genotypes of hepatitis E viruses (HEV) detected in sera of patients from different regions of China. METHODS: The partial genome (nt6461-6860, nt5994-6294) of open reading frame 2 (ORF2) of 45 HEV strains detected from 14 cities of China was amplified and sequenced using polymerase chain reaction (PCR) and direct sequencing. RESULTS: Forty-one of 45 strains (91%) share the same genotype with HEV Burma strain (B), with nucleotide identities higher than 98% with the representative HEV Chinese strain. Only 4 HEV strains are significantly divergent from the 3 prototype strains of HEV, with nucleotide identities of 77%-80% with HEV Burmese/Chinese strain, 74%-76% with Mexican strain and 74%-77% with the newly discovered HEV US/swine strain, respectively. Phylogenetic analysis suggests that these 4 strains may represent 2 different subtypes that belong to a novel genotype of HEV, which is significantly divergent from the prototype Mexico, Burmese and US/swine strains. CONCLUSION: Among patients with hepatitis E in China, most are infected by the Chinese prototype HEV, and only a small part by the new genotype HEV.

Similarities,differences,and possible interactions between hepatitis E and hepatitis C viruses:Relevance for research and clinical practice

Hepatitis E virus(HEV)and hepatitis C virus(HCV)are both RNA viruses with a tropism for liver parenchyma but are also capable of extrahepatic manifestations.Hepatitis E is usually a viral acute fecal-oral transmitted and self-limiting disease presenting with malaise,jaundice,nausea and vomiting.Rarely,HEV causes a chronic infection in immunocompromised persons and severe fulminant hepatitis in pregnant women.Parenteral HCV infection is typically asymptomatic for decades until chronic complications,such as cirrhosis and cancer,occur.Despite being two very different viruses in terms of phylogenetic and clinical presentations,HEV and HCV show many similarities regarding possible transmission through organ transplantation and blood transfusion,pathogenesis(production of antinuclear antibodies and cryoglobulins)and response to treatment with some direct-acting antiviral drugs.Although both HEV and HCV are well studied individually,there is a lack of knowledge about coinfection and its consequences.The aim of this review is to analyze current literature by evaluating original articles and case reports and to hypothesize some interactions that can be useful for research and clinical practice.

Hepatitis E virus: Epidemiology, diagnosis, clinical manifestations, and treatment

The hepatitis E virus(HEV)is the fifth known form of viral hepatitis and was first recognized as the cause of an epidemic of unexplained acute hepatitis in the early 1980s.Globally,it is one of the most frequent causes of acute viral hepatitis.The majority of HEV infections are asymptomatic and lead to the spontaneous clearance of the virus.Among the eight different genotypes identified to date,HEV genotype 1(HEV1),HEV2,HEV3,and HEV4 are the most frequent genotypes causing infections in humans.HEV1 and HEV2 are prevalent in developing regions and able to result in large-scale outbreaks originating from contaminated water supplies.They are also responsible for severe hepatitis in pregnant patients and infants.In contrast,HEV3 and HEV4 are zoonotic,and the transmission of these genotypes to humans occurs mainly through the fecal contamination of water and consumption of contaminated meat from infected animals.Their main reservoir is the pig,and they are mostly encountered in developed countries.The major risk groups for HEV infection and its ensuing adverse consequences are pregnant women,infants,older people,immunocompromised individuals,patients with underlying chronic liver diseases,and workers that come into close contact with HEV-infected animals.In the clinical perspective,HEV infections have diverse clinical manifestations including acute and self-limiting hepatitis,acute-on-chronic liver disease,chronic hepatitis,cirrhosis,and liver failure.Although HEV mainly results in acute selflimiting infection,chronic HEV infection may occur among immunocompromised patients(e.g.,solid-organ transplant recipients).Additionally,HEV-associated extrahepatic manifestations involving various organs have been reported in the last decade,although the causal link for many of them still needs to be proven.Ribavirin and interferon-alpha are the most widely used agents for the treatment of HEV infections with a certain level of success.However,ribavirin is contraindicated in pregnant patients,and interferon-alpha cannot be used in most transplant recipients.Therefore,there is an urgent need for novel antiviral compounds that are safe and effective particularly for patients having contraindications for ribavirin or interferon-alpha and infected by the ribavirinresistant HEV.In this review article,a literature search using PubMed and MEDLINE databases was performed,up to March 2020.Only the articles published in English were reviewed.

Viral hepatitis update: Progress and perspectives

Viral hepatitis,secondary to infection with hepatitis A,B,C,D,and E viruses,are a major public health problem and an important cause of morbidity and mortality.Despite the huge medical advances achieved in recent years,there are still points of conflict concerning the pathogenesis,immune response,development of new and more effective vaccines,therapies,and treatment.This review focuses on the most important research topics that deal with issues that are currently being solved,those that remain to be solved,and future research directions.For hepatitis A virus we will address epidemiology,molecular surveillance,new susceptible populations as well as environmental and food detections.In the case of hepatitis B virus,we will discuss host factors related to disease,diagnosis,therapy,and vaccine improvement.On hepatitis C virus,we will focus on pathogenesis,immune response,direct action antivirals treatment in the context of solid organ transplantation,issues related to hepatocellular carcinoma development,direct action antivirals resistance due to selection of resistanceassociated variants,and vaccination.Regarding hepatitis D virus,we describe diagnostic methodology,pathogenesis,and therapy.Finally,for hepatitis E virus,we will address epidemiology(including new emerging species),diagnosis,clinical aspects,treatment,the development of a vaccine,and environmental surveillance.

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

AIM： To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3＇-portion of hepatitis E virus （HEV） open reading frame 2 （ORF2） in plastids of tobacco （Nicotiana tabacum cv. SR1）, to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS： Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS： Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 μg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION： HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.

Significance of serum IgA in patients with acute hepatitis E virus infection

AIM： To study the significance of serum anti-hepatitis E virus （HEV） IgA in patients with hepatitis E. METHODS： A new method was established to assay anti-HEY IgA, which could be detected in the middle phase of the infection. We compared anti-HEV IgA assay with anti-HEV IgM and anti-HEV IgG assay in sera from 60 patients with positive HEV-RNA. RESULTS： The 60 patients with positive HEV-RNA had both anti-HEV IgA and anti-HEV IgM and 410 patients with negative HEV-RNA were used as control. Periodic serum samples obtained from 60 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, anti-HEV IgA and anti-HEV IgG. Their HEV-RNA was detectable in the serum until 20 ±11 d. We used anti-HEV IgM and anti-HEV IgA assay to detect HEV infection and positive results were found in 90 ± 15 d and 120 ±23 d respectively, the positive rate of anti-HEV IgA was higher than that of anti-HEV IgM and HEV-RNA （P 〈0.05）. CONCLUSION： The duration of anti-HEV IgA in serum is longer than that of anti-HEV IgM, and anti-HEV IgA assay is a good method to detect HEV infection.

Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice

AIM： To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS： The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 ug. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS： After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 （3.1＋0.49） was higher than that in the control group （0.787±0.12, P〈0.01）. None in the control group had a detectable level of anti-HEV IgG. CONCLUSION： DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.

Transfusion-transmitted hepatitis E:What we know so far?

Hepatitis E virus(HEV)is a major cause of viral hepatitis globally.There is growing concern about transfusion-transmitted HEV(TT-HEV)as an emerging global health problem.HEV can potentially result in chronic infection in immunocompromised patients,leading to a higher risk of liver cirrhosis and even death.Between 0.0013%and 0.281%of asymptomatic blood donors around the world have HEV viremia,and 0.27%to 60.5%have anti-HEV immunoglobulin G.HEV is infectious even at very low blood concentrations of the virus.Immunosuppressed patients who develop persistent hepatitis E infection should have their immunosuppressant regimen reduced;ribavirin may be considered as treatment.Pegylated interferon can be considered in those who are refractory or intolerant to ribavirin.Sofosbuvir,a nucleotide analog,showed modest antiviral activity in some clinical studies but sustained viral response was not achieved.Therefore,rescue treatment remains an unmet need.The need for HEV screening of all blood donations remains controversial.Universal screening has been adopted in some countries after consideration of risk and resource availability.Various pathogen reduction methods have also been proposed to reduce the risk of TT-HEV.Future studies are needed to define the incidence of transmission through transfusion,their clinical features,outcomes and prognosis.

Genetic characteristics and pathogenicity of human hepatitis E virus in Nanjing,China

AIM： To investigate the genetic characteristics and pathogenicity of hepatitis E virus （HEV） and assess the potential risk factors for sporadic hepatitis E.

Neurological manifestations of hepatitis E virus infection:An overview

Hepatitis E virus(HEV)is an important cause of repeated waterborne outbreaks of acute hepatitis.Recently,several extrahepatic manifestations(EHMs)have been described in patients with HEV infection.Of these,neurological disorders are the most common EHM associated with HEV.The involvement of both the peripheral nervous system and central nervous system can occur together or in isolation.Patients can present with normal liver function tests,which can often be misleading for physicians.There is a paucity of data on HEV-related neurological manifestations;and these data are mostly described as case reports and case series.In this review,we analyzed data of 163 reported cases of HEV-related neurological disorders.The mechanisms of pathogenesis,clinico-demographic profile,and outcomes of the HEV-related neurological disorders are described in this article.Nerve root and plexus disorder were found to be the most commonly reported disease,followed by meningoencephalitis.

Hepatitis E virus is a leading cause of acute-on-chronic liver disease:experience from a tertiary centre in Bangladesh

BACKGROUND:Acute-on-chronic liver failure(ACLF) is common in Bangladesh.Acute viral E hepatitis is sporadically encountered in this country each year,with a rising incidence in the rainy season.This study aimed to identify the etiology of ACLF in Bangladesh. METHODS:In this retrospective study,69 ACLF patients were included.They presented to our department at the Bangabandhu Sheikh Mujib Medical University in Dhaka.History of diseases was recorded and appropriate investigations were conducted in all patients. RESULTS:Acute hepatitis E virus(HEV)infection was positive in 21.7%(15/69)of the patients,while 14.5% (10/69)had septicemia.Upper gastrointestinal tract hemorrhage was seen in 4.3%of the patients(3/69),while another 4.3%(3/69)had a positive history for alcohol or drugs.None of the patients tested positive for hepatitis A virus infection and no evidence of hepatitis B virus flare was found in any patient.No specific cause for ACLF could be identified. CONCLUSIONS:Acute HEV infection is a leading cause of ACLF in Bangladesh.Many patients were thought to have decompensation of cirrhosis,but subsequently were recognized as having ACLF by a retrospective review according to the definition of the Asian Pacific Association for the Study of the Liver Working Party Meeting on ACLF in New Delhi in early 2008.

Update on the management and treatment of viral hepatitis

This review aims to summarize the current evidence on the treatment of viral hepatitis,focusing on its clinical management.Also,future treatment options and areas of potential research interest are detailed.PubMed and Scopus databases were searched for primary studies published within the last ten years.Keywords included hepatitis A virus,hepatitis B virus(HBV),hepatitis C virus,hepatitis D virus(HDV),hepatitis E virus,and treatment.Outcomes reported in the studies were summarized,tabulated,and synthesized.Significant advances in viral hepatitis treatment were accomplished,such as the advent of curative therapies for hepatitis C and the development and improvement of hepatitis A,hepatitis B,and hepatitis E vaccination.Drugs that cure hepatitis B,going beyond viral suppression,are so far unavailable;however,targeted antiviral drugs against HBV(immunomodulatory therapies and gene silencing technologies)are promising approaches to eradicating the virus.Ultimately,high vaccination coverage and large-scale test-and-treat programmes with high screening rates may eliminate viral hepatitis and mitigate their burden on health systems.The development of curative hepatitis C treatment renewed the enthusiasm for curing hepatitis B,albeit further investigation is required.Novel therapeutic options targeting HDV life cycle are currently under clinical investigation.

Open reading frame 3 protein of hepatitis E virus:Multi-function protein with endless potential

Hepatitis E virus (HEV), a fecal-orally transmitted foodborne viral pathogen,causes acute hepatitis in humans and is responsible for hepatitis E outbreaksworldwide. Since the identification of HEV as a zoonotic agent, this virus has beenisolated from a variety of hosts with an ever-expanding host range. HEV-openreading frame (ORF) 3, the smallest ORF in HEV genomes, initially had beenperceived as an unremarkable HEV accessory protein. However, as novel HEVORF3function has been discovered that is related to the existence of a putativethird virion structural form, referred to as “quasi-enveloped” HEV particles, HEVis challenging the conventional virion structure-based classification scheme,which assigns all viruses to two groups, “enveloped” or “non-enveloped”. In thisreview, we systematically describe recent progress that has identified multiplepathogenic roles of HEV-ORF3, including roles in HEV virion release, biogenesisof quasi-enveloped virus, regulation of the host innate immune response, andinterference with host signaling pathways. In addition, implications of HEVORF3-associated quasi-enveloped virions are discussed to guide futuredevelopment of improved vaccines against zoonotic HEV infection.

Seroprevalence and evolutionary dynamics of genotype 4 hepatitis E virus in Shandong Province,China

AIM: To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus (HEV) and assess the ancestor of HEVs in China’s Shandong Province.

Hepatitis E virus in patients with acute severe liver injury

AIM: To examine the incidence of hepatitis E(HepE) in individuals with acute liver injury severe enough to warrant treatment at a transplant unit.METHODS: Hepatitis E virus(HEV) is an emerging pathogen in developed countries causing severe illness, particularly in immunocompromised patients or those with underlying chronic liver disease. HepE infection isoften under diagnosed, as clinicians can be reluctant to test patients who have not travelled to regions traditionally considered hyperendemic for HepE. There are few data regarding the significance of HEV in patients with very severe acute liver injury in developed countries. Eighty patients with acute severe liver injury attending the Scottish Liver Transplant unit were tested for HEV and anti-HEV IgG and IgM. Severe acute liver injury was defined as a sudden deterioration in liver function confirmed by abnormal liver function tests and coagulopathy or presence of hepatic encephalopathy. Eighty percent of these patients were diagnosed with paracetomol overdose. No patients had a history of chronic or decompensated chronic liver disease at time of sampling. IgG positive samples were quantified against the World Health Organization anti-HEV IgG standard. Samples were screened for HEV viral RNA by quantitative reverse transcription polymerase chain reaction.RESULTS: Four cases of hepatitis E were identified. Three of the four cases were only diagnosed on retrospective testing and were initially erroneously ascribed to drug-induced liver injury and decompensated chronic liver disease, with the cause of the decompensation uncertain. One case was caused by HEV genotype 1 in a traveller returning from Asia, the other three were autochthonous and diagnosed on retrospective testing. In two of these cases(where RNA was detected) HEV was found to be genotype 3, the most prevalent genotype in developed countries. Three patients survived, two of whom had been misdiagnosed as having drug induced liver injury. The fourth patient died from sepsis and liver failure precipitated as a result of hepatitis E infection and previously undiagnosed cirrhosis. Histopathology data to date is limited to mainly that seen for endemic HepE. All patients, with the exception of patient 1, demonstrated characteristics of HepE infection, as seen in previously described locally acquired cases.CONCLUSION: In patients with acute severe liver injury, HEV testing should be part of the initial diagnostic investigation algorithm irrespective of suspected initial diagnosis, age or travel history.