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.

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.

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.

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.

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.

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.

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.

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichia pastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus that bas been classified in the family of Caliciviridae. The virus appears to be a polya-denylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoding amino acids（aa） 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS 115. SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P pastoris. Under optimized conditions （culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h）, the yield of soluble p293 was approximately 80 mg/L. We also observed p293 secretory expressed in P. pastoris to be 30 nm viral like particles by using electron microscopy. These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as a useful antigen for both diagnostic and vaccine purposes.

Mutational analysis of hepatitis E virus ORF1 'Y-domain' : Effects on RNA replication and virion infectivity

AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.RESULTSIn silico analysis identified a potential palmitoylation-site (C336C337) and an α-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.CONCLUSIONThis is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.

Detection and characterization of Hepatitis E virus from commercial rabbit livers in Hebei, China

Rabbit hepatitis E virus(HEV)has been reported for years and is thought to have the potential for zoonotic transmission from rabbits to humans.As reported,HEV genotype 3(gt3)is the most prevalent form of HEV in rabbits.To determine the prevalence of HEV in commercial rabbit livers,176 liver samples were collected from an abattoir in Hebei Province,China.Three(1.7%)samples tested positive for RNA of HEV-0RF2(open reading frames-2).Sequence analysis showed that the three isolates shared high identities with each other(94.08-98.85%).Further analysis showed that all the rabbit strains clustered together in the branch of HEV gt3.Further study by immunohistochemistry(IHC)assays showed that 131(74.4%)liver samples were positive for HEV ORF2 protein.Pathological changes including cell degeneration,inflammatory cell infiltration and bile duct epithelial cell hyperplasia were observed under microscopy.These findings indicated the presence of HEV in commercial livers of rabbits.Additional studies should be conducted to investigate the infectivity of rabbit HEV(rHEV)and the potential risks of zoonotic transmission of rHEV from rabbits to human beings.

Cloning and expression of cDNAs from hepatitis E virus structural gene

OBJECTIVE: To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection. METHODS: A 492 base cDNA was collected from 5'-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E. coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E. RESULTS: The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100. CONCLUSION: The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

Epidemiology of hepatitis E virus in Iran

Iran is known as an endemic country for hepatitis E virus(HEV) infection, while there are variations in the epidemiology of HEV infection throughout the country. The available epidemiological studies in different regions of Iran show HEV seroprevalence of 1.1%-14.2% among general population, 4.5%-14.3% among blood donors, 6.1%-22.8% among injecting drug users, 6.3%-28.3% among hemodialysis patients, 1.6%-11.3% among patients infected with other hepatitis viruses, 27.5% among patients with chronic liver disease, 30.8% among kidney transplant recipient patients, and 10%-16.4% among human immunodeficiency virus-infected patients. These variations reflect differences in the status of public health and hygiene, risk factors, and routes of transmission in different regions and groups. Therefore, it is necessary to review the epidemiology of HEV infection to determine the most prevalent risk factors and routes of transmission, and to evaluate the effectiveness of preventive strategies employed in the public health services of the country. Moreover, the other epidemiological aspects of HEV, including the genotypic pattern, extra hepatic manifestations, and incidence of chronic infection need to be investigated among Iranian population to expand the current knowledge on the epidemiology of HEV and to clarify the real burden of HEV infection. Therefore, this review was performed to provide a general overview regarding the epidemiology of HEV in Iran.

Insights into hepatitis E virus epidemiology in Croatia

BACKGROUND Hepatitis E virus(HEV)is an emerging virus of global health concern.The seroprevalence rates differ greatly according to geographic region and population group.AIM To analyze the seroprevalence of HEV in exposed(animal-related professions)and nonexposed populations,as well as solid organ and hematopoietic stem cell transplant patients.METHODS Forestry workers(n=93),hunters(n=74),and veterinarians(n=151)represented the exposed population.The general population(n=126)and pregnant women(n=118)constituted the control group.Transplant patients included liver transplant recipients(LTRs)(n=83),kidney transplant recipients(KTRs)(n=43),and hematopoietic stem cell transplant recipients(HSCRs)(n=39).HEV immunoglobulin G antibodies were detected using the enzyme-linked immunosorbent assay and confirmed by the immunoblot test.RESULTS The HEV seroprevalence significantly differed between groups:Veterinarians 15.2%,hunters 14.9%,forestry workers 6.5%,general population 7.1%,and pregnant women 1.7%.In transplant patients,the seropositivity was highest in LTRs(19.3%),while in KTRs and HSCRs,the seroprevalence was similar to the general population(6.9%and 5.1%,respectively).A significant increase in seropositivity with age was observed from 2.9%in individuals less than 30 years to 23.5%in those older than 60 years.Sociodemographic characteristics(sex,educational level,area of residence,and number of household members),eating habits(game meat,offal,and pork products consumption),and environmental and housing conditions(drinking water supply,type of water drainage/sewer,waste disposal,domestic animals)were not associated with HEV seropositivity.However,individuals who reported a pet ownership were more often seropositive compared to those who did not have pet animals(12.5%vs 7.0%).CONCLUSION The results of this study showed that individuals in professional contact with animals and LTRs are at higher risk for HEV infection.In addition,age is a significant risk factor for HEV seropositivity.

Seroprevalence of Hepatitis E Virus in Human and Pigs in Pig Farms from Guangdong Province,Southern China

Hepatitis E is caused by hepatitis E virus （HEV）, which has been classified into four genotypes. Genotypes 3 and 4 are regarded as zoonotic pathogens. Accumulating researches indicate that genotype 4 is the main HEV strain circulating in China, and there are high levels of seropositive pigs and human in some provinces of China. In this study, serum samples from pigs and from human occupationally exposed to pigs were obtained from pig farms in Guangdong Province, in subtropical southern China, in order to investigate for the first time the prevalence of anti-HEV immunoglobulin G （IgG） in the region. Antibodies against HEV were detected by Enzyme-Linked Immunosorbent Assay （ELISA） using a commercially marketed kit. The results showed that high numbers of pigs （74/94; 78.7%） and human （50/94; 53.2%） from three pig farms in Guangdong Province were positive for anti-HEV IgG. The correlation coefficient relating the prevalence in pigs and human on different farms was 0.920. The seropositive rate in males （human） was 48.8% （20/41） and that in females was 47.7% （9/19）, which showed no statistically significant difference. These data indicated that there was a high prevalence of anti-HEV antibodies in pigs and in human with occupational exposure to pigs. The risk of infection with HEV in both human and pigs in Guangdong Province appeared to be age-dependent, to a certain extent. This study provided basic data for further researches on HEV and was a reminder that more attention should be paid to HEV infection both in pigs and workers on pig farms in the study region.

Hepatitis E virus in professionally exposed: A reason for concern?

The zoonotic risk of hepatitis E virus(HEV)is well established.The HEV seroprevalence rates vary according to geographical region,assays used,and study cohorts.HEV infection is still underdiagnosed,implying the need to evaluate the disease's burden in the general population and specific risk groups,such as professionally exposed.Close contact with various animal reservoirs such as pigs,rabbits,sheep,dogs,wild boars,and deer has been associated with higher anti-HEV seroprevalence as a part of occupational exposure.While exact transmission routes remain to be determined,some general preventive measures such as proper hand hygiene,the usage of personal protective equipment,and the thermal processing of food before consumption should be followed.A“One-Health”multisectoral approach should be implemented to achieve optimal health and well-being outcomes,recognizing the interconnections between humans,animals,plants,and their shared environment,in which a vaccine against the zoonotic genotypes 3 and 4 and swine vaccination should be considered as a possible public health measure.This opinion review comprehensively addresses the HEV burden of professional exposure for butchers,slaughterhouse workers,veterinarians,farmers,hunters,and forestry workers delineates the current limits of protective work measures,and tackles future directions.