Hepatitis E Virus Genotype Research Articles

Soluble ORF2 protein enhances HEV replication and induces long-lasting antibody response and protective immunity in vivo.

The HEV is a small positive-sense RNA virus that encodes a cytoplasmic form of the capsid protein (ORF2c), essential for virion structure, and a secreted glycosylated form (ORF2s) that accumulates at high titer in serum and can mask neutralizing epitopes. We explored the contribution of ORF2s to HEV replication and its role in generating antibodies against ORF2 in a nonhuman primate model. We used a recombinant HEV genotype 3 variant that does not express ORF2s due to the introduction of stop codons (ORF2s mut ). Rhesus macaques (RMs) were given intrahepatic injections of infectious wildtype HEV (ORF2s wt ) RNA or a variant lacking ORF2s expression (ORF2s mut ). The replication of the ORF2s mut virus was delayed by ~2 weeks compared with ORF2s wt , and peak titers were nearly tenfold lower. Reversions of the 3 mutations that blocked ORF2s expression were not detected in the ORF2s mut genomes, indicating genetic stability. However, serum antibodies against ORF2 were transiently detected in RMs infected with ORF2s mut , whereas they were long-lasting in RMs infected with ORF2s wt . Moreover, RMs infected with ORF2s mut were more susceptible to reinfection, as evidenced by the viral RNA detected in fecal samples and the expansion of HEV-specific CD8 + T cells. These findings indicate that ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody-mediated responses that protect against HEV re-exposure.

Q531L mutation in the capsid protein of hepatitis E virus genotype 1 causes infections in patients with altered immunity and immunosuppressive condition: Mechanism based on wet lab and in-silico findings

ObjectivesThe study aimed to observe the effects of Q531L mutation in the E2s domain of HEV ORF2 on the circulation of the virus among the immunocompetent patients, diabetic patients (immunosuppressive), and pregnant women (altered immunity). MethodsThirty two HEV-RNA samples from pregnant and non-pregnant females were subjected to real time (RT)-qPCR, conventional RT-PCR and Sanger sequencing of HEV ORF2 region covering immunodominant E2s domain (459–606 aa). Liver functions tests using serum samples of the patients were performed. In-silico prediction of CD8+ cytotoxic T lymphocytes (CTL) epitope covering the 531st residue of HEV genotype 1 ORF2/E2s was performed. ResultsThree out of 32 samples harbored a nonsynonymous substitution, resulting in HEV genotype 1 ORF2 531Gln>Leu. The patients with HEV 531Gln had significantly higher serum ALT levels than 531Leu (P = 0.001) and in addition, the former had 173-fold higher viral load than the latter (1,360,869.45 ± 5,298,899.45 vs 784 ± 507.73). The HLA-mutant peptide complexes had higher binding affinities than the HLA-wild type complexes, showing Cluspro2.0/PRODIGY docking scores(Kcal/mol)) of −724.6/−9.2 and −854.2/−9.8 versus −680.1/−8.2 and −757.7/−7.5, respectively. One of the three 531Leu patients was a non-pregnant woman with diabetes and the rest 2 were pregnant. ConclusionsThe hosts with immunosuppressive/altered immunity may provide conducive niches for HEV 531Leu.

Temporal changes in the genotypes of Paslahepevirus balayani in southern Spain and their possible link with changes in pig trade imports

IntroductionPaslahepevirus balayani (HEV) is an endemic zoonotic disease ranked as a major cause of acute hepatitis in Europe. Most infections occurring in Europe are due to the endemic several subtypes of genotype 3, through the consumption of raw or undercooked pork, observing a genotype geographical distribution pattern among countries Because of global changes in the pig and pork trading markets, subtype distribution might vary. We aimed to evaluate the temporal distribution of HEV genotypes in patients from southern Spain with acute hepatitis to determine whether these changes were related to the pig import trade during the study period between 2018 and 2022. MethodsProspective longitudinal study including patients with acute hepatitis from southern Spain between 2018 and 2022. HEV RNA and antibodies was tested in all patients. In patients with detectable HEV RNA, genotype was obtained. To determine the number of imported pigs and their origins, we checked the official data from the Spanish statistics on international trade of Spanish Minister of Industry during by country of origin during the same study period. ResultsA total of 659 patients with acute hepatitis were included in the study. Among them, 162 (24.5%) had at least one marker (IgM or RNA) of acute HEV infection. Among the 71 patients with detectable viral RNA, genotypes could be obtained for 58 (81.6%). The most prevalent HEV genotype was 3f (n = 48; 78.6%), showing a decreasing prevalence of over time, from 100% in 2018 to 70.6% in 2022. Since 2021, the emergence of other genotypes has been determined. A significant increase in the number of animals imported was observed since the beginning of the study. Denmark experienced a significant rise, from 0.03% in 2018 of total imports to 10.4% in 2022. ConclusionsHEV molecular diversity is changing in Spain, could be linked to changes in fattening pig import origin.

Hepatitis E Virus RNA Detection from Hunted Wild Boars in Central Italy: an Epidemiological Investigation

Every year, foodborne pathogens, including the hepatitis E virus (HEV), cause thousands of infections in different continents. Final consumers become infected through the ingestion of contaminated animal origin foodstuffs. Generally, in industrialized countries, HEV genotype 3 is involved in sporadic outbreaks. Infections have been described, in Europe and Japan as consequence of pork products and contaminated wild boar’s primary or processed products (liver and muscle tissues) consumption. In Central Italy, hunting activities are largely practiced. In these small and rural communities, game meat and liver are ingested by hunters’ families or at local and traditional restaurants. Therefore, these food chains can be considered critical HEV reservoirs. In this study, 506 liver and diaphragm tissues were collected from hunted wild boars in the Southern Marche region (Central Italy) and were screened for HEV RNA detection. From the 10.87% of liver and 2.76% of muscle samples, HEV3 subtype c was discovered. The observed prevalence values resulted in line with previous investigations performed in other Central Italian regions, but higher than Northern ones (3.7% and 1.9% from liver tissue). Therefore, the obtained epidemiological data highlighted the wide occurrence of HEV RNA circulation in a low-investigated area. Basing on results, a One-health approach was adopted due to the sanitary relevance of this Public Health concern.

A Systematic Review and Meta-Analysis on Hepatitis E Virus Detection in Farmed Ruminants.

Swine are widely recognized as the main reservoir of zoonotic HEV; however, a growing body of data on the HEV prevalence in farmed ruminants of different species also points to a potential route for HEV transmission through ruminants and ruminant products and by-products. Definite information on the zoonotic potential of ruminants is still absent or unclear, compelling the necessity for increasing knowledge on this. The aim of the current study was to analyze the state-of-the-art in this research topic and provide a summary of HEV detection and characterization in farmed ruminants. A total of 1567 papers were retrieved from four search databases that resulted in 35 eligible papers after application of exclusion/inclusion criteria. Studies on HEV in farmed ruminants were mainly based on the detection of HEV RNA and were reported in Africa (n = 1), America (n = 3), Asia (n = 18) and Europe (n = 13), and focused on a variety of ruminants species, namely cow, goat, sheep, deer, buffalo and yak. The overall pooled prevalence of HEV was 0.02% (0.01-0.03, 95% CI). The subgroup pooled prevalence of HEV RNA was 0.01% (0.00-0.02, 95% CI) in cow milk, stool, serum, liver, intestinal, bile, blood, spleen and rectal swab samples; 0.09% (0.02-0.18, 95% CI) in goat serum, bile, stool, milk, liver, rectal swab and blood samples; 0.01% (0.00-0.04, 95% CI) in sheep stool, serum, milk, blood and liver samples. Most of the HEV genotypes found in farmed ruminants belonged to the zoonotic HEV-3 (subtypes 3a, 3c) and HEV-4 (subtype 4d, 4h), with Rocahepevirus also found. The wide HEV circulation observed in different farmed ruminants raises concerns for the possibility of HEV transmission through products from infected ruminants and alerts for the potential zoonotic route for HEV in ruminant products, such as meat and dairy products. Also, contact exposure to infected farmed animals could be a risk factor. Further research should be conducted in order to understand the circulation of HEV in these animals and its zoonotic potential, as there is currently a lack of data on this topic.

Open Reading Frame 4 Is Not Essential in the Replication and Infection of Genotype 1 Hepatitis E Virus

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837CRNA, T2885CRNA, and T2837C/T2885CRNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836TRNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Hepatitis E Virus in Finland: Epidemiology and Risk in Blood Donors and in the General Population.

Autochthonous hepatitis E (HEV) cases have been increasingly recognized and reported in Europe, caused predominantly by the zoonotic HEV genotype 3. The clinical picture is highly variable, from asymptomatic to acute severe or prolonged hepatitis in immunocompromised patients. The main route of transmission to humans in Europe is the ingestion of undercooked pork meat. Transfusion-transmitted HEV infections have also been reported. The aim of the study was to determine the HEV epidemiology and risk in the Finnish blood donor population. A total of 23,137 samples from Finnish blood donors were screened for HEV RNA from individual samples and 1012 samples for HEV antibodies. Additionally, laboratory-confirmed hepatitis E cases in 2016-2022 were extracted from national surveillance data. The HEV RNA prevalence data was used to estimate the risk of transfusion transmission of HEV in the Finnish blood transfusion setting. Four HEV RNA-positive were found, resulting in 1:5784 (0.02%) RNA prevalence. All HEV RNA-positive samples were IgM-negative, and genotyped samples represented genotype HEV 3c. HEV IgG seroprevalence was 7.4%. From the HEV RNA rate found in this study and data on blood component usage in Finland in 2020, the risk estimate for a severe transfusion-transmitted HEV infection is 1:1,377,000 components or one in every 6-7 years. In conclusion, the results indicate that the risk of transfusion-transmitted HEV (HEV TTI) in Finland is low. However, continuous follow-up of the HEV epidemiology in relation to the transfusion risk landscape in Finland is necessary, as well as promoting awareness in the medical community of the small risk for HEV TTI, especially for immunocompromised patients.

Unravelling the Tripartite Interactions Among Hepatitis E Virus RNA, miR-140 and hnRNP K

In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation. Provision of mutant miR-140 oligos carrying same mutation as on mutant HEV successfully rescued mutant HEV replication. In vitro cell-based assays with modified oligos proved that host factor-miR-140 is a critical requirement for HEV replication. Biotinylated RNA pulldown and RNA immunoprecipitation assays proved that the predicted secondary RNA structure of the miR-140 binding site allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. We predicted the model from the obtained results that the miR-140 binding site can serve as a platform for recruitment of hnRNP K and other proteins of HEV replication complex only in the presence of miR-140.

Extrahepatic Replication Sites of Hepatitis E Virus (HEV)

Hepatitis E virus (HEV) is an emerging viral disease known to cause acute viral hepatitis globally. Various genotypes of HEV have been identified that produce genotype specific lesions depending on the HEV targeted population. Pregnant or immunosuppressed individuals develop significantly more severe hepatitis E in comparison to the general population. In the last 40 years, we discovered that the tropism of HEV is not restricted to the liver, and virus replication was demonstrated in multiple organs. Out of the 10 body systems described in humans, HEV produces lesions causing a broad range of extrahepatic clinical manifestations in each of them. Affected body systems include nervous and musculoskeletal, cardiovascular, digestive, endocrine, integumentary, renal, respiratory, immune, and reproductive systems producing systemic lesions. All extrahepatic signs are caused by either direct HEV replication in these tissues, or indirectly by various immune mediated mechanisms. Extrahepatic replication features of HEV allowed it to cross the placental barrier, blood–brain barrier (BBB), and blood–testis barrier (BTB) that do not typically grant entry to viruses in general. Thus, in this review, we summarized the extrahepatic replication sites of HEV, listed the body systems where HEV invaded, and described multiple animal models including immunocompetent and immunosuppressed that were used to study the extrahepatic replication sites of HEV.

Oral Zinc Supplementation in Chronically HEV-Infected Patients Not Responding to Ribavirin Monotherapy

Background: Chronic hepatitis E virus (HEV) infection may progress to end-stage liver disease in immunosuppressed individuals. Ribavirin therapy is efficient in most chronic HEV patients, but 10% remain without a sustained virological response (SVR). Objectives: We aimed to study whether zinc supplementation could represent a therapeutic approach in these patients. Methods: Antiviral properties of zinc salts were studied in vitro (subgenomic-replicon system), in vivo (rabbit model), and retrospectively in patients with chronic hepatitis E who did not achieve SVR under ribavirin monotherapy. Results: Zinc inhibited HEV genotype 3 replication in vitro. In a model of acute HEV infection in immunocompetent rabbits, zinc + ribavirin did not improve viral clearance compared to ribavirin monotherapy. In chronically HEV-infected patients not responding to ribavirin (n = 12), viral clearance was observed in 4/12 (33%) patients receiving additional zinc supplementation. Conclusions: Oral zinc, an inexpensive, harmless dietary supplement, could potentially represent a rescue treatment option for a few patients with chronic hepatitis E without SVR under ribavirin monotherapy. Further studies are needed to elucidate the role of zinc in HEV.

Absence of Hepatitis E Virus (HEV) in Italian Lagomorph Species Sampled between 2019 and 2021.

The zoonotic hepatitis E virus genotype 3 (HEV-3) causes most autochthonous human hepatitis E cases in Europe, which are due to the consumption of raw or undercooked food products of animal origin. Pigs and wild boars are considered the main reservoirs of this genotype, while rabbits are the reservoir of a distinct phylogenetic group named HEV-3ra, which is classified within the HEV-3 genotype but in a separate clade. Evidence for the zoonotic potential of HEV-3ra was suggested by its detection in immunocompromised patients in several European countries. HEV-3ra infection was found in farmed and feral rabbit populations worldwide and its circulation was reported in a few European countries, including Italy. Furthermore, Italy is one of the major rabbit meat producers and consumers across Europe, but only a few studies investigated the presence of HEV in this reservoir. The aim of this study was to assess the presence of HEV in 328 Italian hares and 59 farmed rabbits collected in 3 Italian macro-areas (North, North-Central, and South-Central), between 2019 and 2021. For this purpose, liver samples were used to detect HEV RNA using broad-range real-time RT-PCR and nested RT-PCR. Using 28 liver transudates from hares, the ELISA test for anti-HEV IgG detection was also performed. Neither HEV RNA nor anti-HEV antibodies were detected. Further studies will be conducted to assess the HEV presence in Italian lagomorphs to establish the role of this host and the possible risk of transmission for workers with occupational exposure, to pet owners and via food.

Molecular and serological investigation of Hepatitis E virus in pigs slaughtered in Northwestern Italy

BackgroundHepatitis E Virus (HEV) is recently considered an emerging public health concern. HEV genotypes 1 and 2 are widely distributed and pathogenic only for humans. In contrast, HEV, genotypes 3 and 4 are observed in swine, deer, wild boars and rabbits and can also be transmitted to humans. The presence of HEV in the liver, muscle, faeces, blood, and bile was detected by real-time RT-PCR in 156 pigs belonging to twenty different farms, ranging from 1 to 8 months of age. The phylogenetic analysis was performed on the viral strain present in the positive biological matrix, with the lowest Ct. HEV-IgG and HEV-IgM in the sera were analysed by two different ELISA kits.ResultsTwenty-one pigs, i.e., 13.46% of them (21/156, 95% CI: 8.53%-19.84%), tested positive for HEV in at least one biological matrix by real-time RT-PCR, while phylogenetic analysis revealed the presence of HEV subtypes 3f and 3c. Pig serums analysed by ELISA showed an overall prevalence of 26.92% (42/156, 95% CI: 20.14%-34.60%) for HEV-IgG, whereas the 28.95% (33/114, 95% CI: 20.84%-38.19%) of them tested negative resulted positive for the HEV-IgM.ConclusionsThe faeces are the biological matrix with the highest probability of detecting HEV. The best concordance value (Kappa Kohen index) and the highest positive correlation (Phi index) were observed for the correlation between bile and liver, even when the number of positive liver samples was lower than the positive bile samples. This finding may suggest that a higher probability of HEV occurs in the bile, when the virus is present in the liver, during the stages of infection. Finally, the presence of HEV in muscle was observed in 11 pigs, usually used for the preparation of some dishes, typical of the Italian tradition, based on raw or undercooked meat. Therefore, their consumption is a possible source of infection for final consumer.

Detection of Hepatitis E Virus Genotype 3 in Feces of Capybaras (Hydrochoeris hydrochaeris) in Brazil.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen associated with relevant public health issues. The aim of this study was to investigate HEV presence in free-living capybaras inhabiting urban parks in São Paulo state, Brazil. Molecular characterization of HEV positive samples was undertaken to elucidate the genetic diversity of the virus in these animals. A total of 337 fecal samples were screened for HEV using RT-qPCR and further confirmed by conventional nested RT-PCR. HEV genotype and subtype were determined using Sanger and next-generation sequencing. HEV was detected in one specimen (0.3%) and assigned as HEV-3f. The IAL-HEV_921 HEV-3f strain showed a close relationship to European swine, wild boar and human strains (90.7-93.2% nt), suggesting an interspecies transmission. Molecular epidemiology of HEV is poorly investigated in Brazil; subtype 3f has been reported in swine. This is the first report of HEV detected in capybara stool samples worldwide.

Survey of Zoonotic Diarrheagenic Protist and Hepatitis E Virus in Wild Boar (Sus scrofa) of Portugal.

Enteropathogenic parasites and viruses have been frequently reported in swine and can infect a wide range of mammals, including humans. Among the wide variety of parasites infecting swine, diarrhoeagenic protists are among those that cause significant morbidity. Hepatitis E virus (HEV) has also been reported both in domestic pigs and wild boar and is known to have an important public health significance. These agents share the fecal-oral transmission route, but data on their fecal shedding and circulation pathways are still lacking or incomplete. Hence, the aim of the present study was to characterize the presence of microeukaryotes and HEV in the wild boar of Portugal. Wild boar stool samples (n = 144) were obtained during the official hunting seasons (October to February) in 2018/2019, 2019/2020, and 2021/2022 and tested for Cryptosporidium spp., Balantioides coli, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi and HEV by molecular assays, followed by sequencing and phylogenetic analysis. We have detected Cryptosporidium scrofarum (1.4%, 95% CI: 0.2-4.9), B. coli (14.6%, 95% CI: 9.2-21.4), Blastocystis ST5 (29.2%, 95% CI: 21.9-37.2) and HEV genotype 3 (2.8%, 95% CI: 0.7-6.9; subgenotypes 3e and 3m). Co-infections were observed in thirteen animals where two were positive for both HEV and B. coli, one was positive for both C. scrofarum and Blastocystis ST5, and ten were positive for both B. coli and Blastocystis ST5. Giardia duodenalis and E. bieneusi were not detected in the surveyed wild boar population. As far as we know, this is the first report describing protist infections by Cryptosporidium spp., B. coli, and Blastocystis sp., as well as the first identification of the emerging HEV genotype 3m in wild boar of Portugal. The present work shows that potentially zoonotic protozoa and HEV are circulating in wild boar populations in Portugal. Awareness and epidemic-surveillance network implementation measures targeting wild boar are needed to prevent the spread of these pathogenic agents to humans.

Molecular epidemiological study of clinical cases of acute hepatitis E in Belarus

Relevance. The frequency of occurrence of anamnestic antibodies to the hepatitis E virus (HEV) in the general population of the Republic of Belarus is 7.3%, which is clearly not consistent with the low incidence of hepatitis E (HE). Most of primary HEV infections remain undiagnosed. The intensive epidemic process of HEV in the Belarusian population is hidden. Conducting epidemiological studies, including genotyping of HEV sequences isolated on the territory of the republic, makes it possible to more accurately characterize the sources of HEV infection and the mechanisms of its transmission.
 Aim molecular epidemiological study of two cases of acute hepatitis E detected in patients from Belarus.
 Materials and methods. During 20212022, samples of biological material were obtained from two patients undergoing treatment with an established diagnosis of acute hepatitis E. Serum samples were tested to detect antibodies to HEV using enzyme immunoassay, HEV RNA was detected in fecal samples using nested RT-PCR. The nucleotide sequence was determined by an automatic sequencer using the Sanger method. Analysis of nucleotide sequences, their genotyping, and calculation of evolutionary distances were performed using MEGA X software.
 Results. The HEV sequence isolated from a pregnant woman who had an epidemiological episode of alimentary contact with raw pork meat is clustered into a common phylogenetic clade with HEV sequence obtained from the patient from Belarus with a history of kidney transplantation and HEV sequences isolated from a domestic pigs. The HEV sequence isolated from a patient with a history of travel to Pakistan belongs to the HEV genotype 1 and joins a clade of HEV sequences isolated in Pakistan, India, Nepal and Mongolia.

A Randomized Large-Scale Cross-Sectional Serological Survey of Hepatitis E Virus Infection in Belgian Pig Farms.

Hepatitis E virus (HEV) is the causative agent of hepatitis E disease in humans. While sporadic HEV infections, which occur in industrialised countries and are typically due to HEV genotypes 3 or 4, are asymptomatic and self-limiting, a chronic form of the disease can lead to liver cirrhosis in immunocompromised individuals. Pigs share HEV 3 and 4 genotypes and are thus considered a major animal reservoir for human infection. A subset of animals has been shown to carry HEV particles at the age of slaughter, rendering raw or undercooked pig products potential vectors for human infection. To provide an overview of the current dissemination of HEV in Belgian pig herds, this study was designed as a randomized, robust, large-scale, cross-sectional, serological survey. HEV genotypes and subtypes recently circulating in Belgium (2020-2021) were investigated. Sample stratification as well as epidemiological investigation through the available demographic data of the sampled herds showed that HEV widely circulated in the Belgian pig population during this time and that a change in the circulating HEV strains may have occurred in the last decade. Herd size and type were identified as risk factors for HEV herd-seropositivity. Identifying farms at risk of being HEV-positive is an important step in controlling HEV spread and human infection.

Hepatitis E Virus: Isolation, Propagation, and Quantification

Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver disease. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and propagation of hepatitis E virus in cultured cells from clinical HEV specimens Support Protocol 1: Quantification of HEV RNA by RT-qPCR Basic Protocol 2: Recovery of HEV from infectious cDNA clones and purification of the virus Support Protocol 2: Quantification of HEV live particles by infectivity assay.

Prevalence of Hepatitis E Virus (HEV) in Feral and Farmed Wild Boars in Xinjiang, Northwest China.

Hepatitis E virus (HEV) causes infections in humans and a wide range of animal hosts. Wild boar is an important natural reservoir of HEV genotypes 3−6 (HEV-3−HEV-6), but comparative analysis of HEV infections in both feral and farmed wild boars remains limited. In this study, samples from 599 wild boars were collected during 2017−2020, including 121 feral wild boars (collected 121 fecal, 121 serum, and 89 liver samples) and 478 farmed wild boars (collected 478 fecal and 478 serum samples). The presence of anti-HEV IgG antibodies were detected by the HEV-IgG enzyme-linked immunosorbent assay (ELISA) kit. HEV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), targeting the partial ORF1 genes from fecal and liver samples, and the obtained genes were further genotyped by phylogenetic analysis. The results showed that 76.2% (95% CI 72.1−79.9) of farmed wild boars tested anti-HEV IgG seropositive, higher than that in feral wild boars (42.1%, 95% CI 33.2−51.5, p < 0.001). HEV seropositivity increased with age. Wild boar HEV infection presented a significant geographical difference (p < 0.001), but not between sex (p = 0.656) and age (p = 0.347). HEV RNA in fecal samples was detected in 13 (2.2%, 95% CI 1.2−3.7) out of 599 wild boars: 0.8% (95% CI 0.0−4.5, 1/121) of feral wild boars and 2.5% (95% CI 1.3−4.3, 12/478) of farmed wild boars. Phylogenetic analysis showed that all these viruses belonged to genotype HEV-4, and further grouped into sub-genotypes HEV-4a, HEV-4d, and HEV-4h, of which HEV-4a was first discovered in the wild boar populations in China. Our results suggested that farms could be a setting for amplification of HEV. The risk of HEV zoonotic transmission via rearing and consumption of farmed wild boars should be further assessed.

Low mortality despite temporary liver dysfunction in severe courses of acute hepatitis E

HepatitisE virus (HEV) infection can cause severe viral hepatitis and eventually liver failure. We aim to provide novel data on the epidemiology and the course of HEV infections from Q1/2008 to Q3/2018 at the Vienna General Hospital. Of the 88,945 people tested, we identified HEV-IgM positive (+) or HEV-PCR (+) patients and retrospectively collated information on the course of infection from patient records. Among 151 HEV-IgM or PCR (+) (median age 51years, 45.8% female), 7 (4.6%) had non-severe acute HEV infection (ALT ≥ 2-5-fold upper limit of normal, ULN), 11 (7.3%) had severe HEV infection without liver dysfunction (LD) (ALT > 5-fold ULN), and 9 (6.0%) with LD (ikterus or bilirubin > 5 mg/dL, OR coagulopathy or INR > 1.5, OR encephalopathy or ammonia > 100 µmol/L). HEV-RNA-PCR was performed in 58/190 (30.5%) HEV-IgM (+) patients and was positive in 19 (30.6%). Rates of HEV IgM/PCR positivity remained stable over the observation period. The HEV genotype (GT) was GT‑1 in 71.4% (n = 5) and GT‑3 in 28.6% (n = 2). Travel history was recorded for 9/20 (45.0%) of severe HEV and 12/20 (60.0%) patients with severe HEV infection were hospitalized. One patient with pre-existing liver disease and concomitant EBV infection required intensive care. No patient required transplantation and the 30-day mortality was 3/151 (1.9%). Despite the increased testing rates, the absolute number of diagnosed HEV infections at Vienna General Hospital remained constant between 2008 to 2018. Although approximately half of the patients with severe acute HEV infection required hospitalization, admissions to the intensive care unit (ICU) and short-term mortality were low.

From the Editor’s Desk…

From the Editor’s Desk…