Type I and III interferons critically protect the host against viral infection. Previous studies showed that IFN responses are suppressed in cells infected by hepatitis E virus (HEV). Here, we studied the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), specialized producers of IFNs. We showed that pDCs co-cultured with HEV-replicating cells secreted IFN in a cell contact-dependent manner. This pDC response required the endosomal nucleic acid sensor TLR7 and adhesion molecules. IFNs secreted by pDCs reduced viral spread. Intriguingly, ORF2, the capsid protein of HEV, can be produced in various forms by the infected cells, and we wanted to study their role in anti-HEV immune response. During infection, a fraction of ORF2 localizes into the nucleus, and glycosylated forms of ORF2 are massively secreted by infected cells. We showed that glycosylated ORF2 potentiates the recognition of infected cells by pDCs, by regulating cell contacts. On the other hand, nuclear ORF2 triggers immune response by IRF3 activation. Together, our results suggest that pDCs may be essential to control HEV replication.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis globally. HEV infections can progress to chronic disease in immunocompromised individuals and may also cause extrahepatic complications. By contrast to other hepatitis viruses, HEV exhibits a broad tissue tropism, and certain genotypes have the ability to infect multiple species. The initial steps of surface attachment and host cell entry are critical steps in the viral infection cycle and serve as key determinants to establish an infection. This review summarizes the current understanding of HEV entry, focusing on molecular entry factors, such as viral receptors, and discusses differences between quasi-enveloped and non-enveloped HEV. We further cover recent developments of assay systems to study HEV entry and highlight experimental strategies to identify novel host components required for HEV entry. Advancing our understanding in these areas could help to guide the development of targeted antiviral strategies to block the early stages of HEV infection.

The contribution of innate immunity to clearance of viral infections of the liver, in particular sensing via Toll-like receptor 3 (TLR3), is incompletely understood. We aimed to identify the factors contributing to the TLR3 response in hepatocytes via CRISPR/Cas9 screening.

A genome-wide CRISPR/Cas9 screen on the TLR3 pathway was performed in two liver-derived cell lines, followed by siRNA knockdown validation. SiRNA knockdown and indisulam treatment were used to study the role of RNA-binding motif protein 39 (RBM39) in innate immunity upon poly(I:C) or cytokine treatment and viral infections. Transcriptome, proteome, and alternative splicing were studied via RNA sequencing and mass spectrometry upon depletion of RBM39.

Our CRISPR/Cas9 screen identified RBM39, which is highly expressed in hepatocytes, as an important regulator of the TLR3 pathway. Knockdown of RBM39 or treatment with indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion, along with multiple other cellular processes identified previously. RBM39 knockdown further restrained type I and type III IFN pathways by reducing the expression of individual receptor subunits and STAT1/2. The function of RBM39 was furthermore not restricted to hepatocytes.

We identified RBM39 as a regulatory factor of cell intrinsic innate immune signaling. Depletion of RBM39 impaired TLR3, RIG-I/MDA5, and IFN responses by affecting the basal expression of key pathway components.

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, and the only zoonotic hepatitis virus. HEV genotype 3 (HEV3) is associated with a range of clinical presentations including chronic infection in immunocompromised individuals in developed nations as well as sporadic cases of autochthonous HEV3 in Europe. Current in vitro models support low levels of HEV infection, hampering our understanding of viral pathogenesis and development of therapeutics. We developed modified culture methods for two widely used hepatoma cell lines, PLC-PRF-5 and Huh-7.5, and evaluated HEV infection. Simple epithelial-like polarity and differentiation formed in PLC-PRF-5 cells, evidenced by localisation of tight junction proteins occludin and zona-occludin 1 to intercellular junctions, and increased albumin production. Complex hepatocyte-like polarity was observed in Huh-7.5 cells, with tight junction proteins localised to shared internal bile canaliculi-like structures and retention of the fluorescent molecule, 5(6)-Carboxyfluorescein diacetate. Cells were infected with genotype 3 HEV, and enhanced infection and replication of HEV was observed using RT-qPCR and immunofluorescent labelling of HEV ORF2 and dsRNA. We describe robust, accessible models for HEV infection in vitro. These models will allow studies to further our understanding of this emerging zoonotic pathogen and develop therapeutic interventions.

Hepatitis E virus (HEV) is a major cause of acute and chronic hepatitis in humans. The HEV open reading frames (ORF1) encodes a large non-structural protein essential for viral replication, which contains several functional domains, including helicase and RNA-dependent RNA polymerase. A confusing aspect is that, while RNA viruses typically encode large polyproteins that rely on their enzymatic activity for processing into functional units, the processing of the ORF1 protein and the mechanisms involved remain unclear. The ORF1 plays a pivotal role in the viral life cycle, thus mutations in this region, especially those occurring under environmental pressures such as during antiviral drug treatment, could significantly affect viral replication and survival. Here, we summarize the recent advances in the functional domains, processing, and mutations of ORF1. Gaining a deeper understanding of HEV biology, particularly focusing on ORF1, could facilitate the development of new strategies to control HEV infections.

Zoonotic hepatitis E virus (HEV) infection is an emerging global public health concern, and understanding the dynamics of HEV transmission between animals and humans is crucial for public health. Animal models are critical to advancing the understanding of HEV pathogenesis, drug screening, vaccine development, and other related areas. Here, we provide an overview of recent studies investigating the cross-species transmission of HEV, and also delve into the current research and application of animal HEV infection models including non-human primates, rodents, pigs, and chickens, offering a comprehensive assessment of the advantages and disadvantages of each model. This review highlights the findings related to viral replication, shedding patterns, and immune response in these animal models, and discusses the implications for our understanding of HEV transmission to humans. These advancements in the field enhance our understanding of the biological traits and pathogenic mechanisms of HEV, offering robust support for the development of highly effective and targeted prevention and treatment strategies.

Hepatitis E virus (HEV) infections are one of the most common causes of acute viral hepatitis, annually causing over 3 million symptomatic cases and 70,000 deaths worldwide. Historically, HEV was described as a hepatotropic virus, but has recently been associated with various extrahepatic manifestations including neurological disorders such as Guillain-Barré syndrome and neuralgic amyotrophy. However, the underlying pathogenesis of these neurological diseases remains largely unknown. The aim of this study was to investigate extrahepatic HEV manifestations in a neuronal model system using human-induced primary neurons (iPNs). Renal epithelial cells from human urine were reprogrammed to induced pluripotent stem cells to generate neuronal progenitor cells, which were subsequently differentiated into iPNs over 21 d. These iPNs supported HEV infection preferentially in neurite-bearing cells. Transcriptional profiling of the neuronal development process as well as viral infection dynamics in iPNs uncovered a lack of antiviral innate immune responses to HEV infection with only an intrinsic expression of distinct interferon-regulated genes and signaling molecules. Viral open reading frame 2 encoded capsid protein could be visualized by volumetric three-dimensional reconstitution within the neurites, which were reduced in length in an HEV inoculation-dependent manner. In conclusion, this neuron-derived human model system provides a powerful tool for studying extrahepatic manifestations of HEV infection. It further indicates a potential mechanism of pathogenesis driven by the interaction between host and viral factors.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

HEV infection has become a global health concern. The study of HEV pathogenicity has been hindered by the lack of a suitable in vitro culture system. In the present research, we systematic demonstration of efficient replication of swine GT4 HEV in A549 cells, Huh-7 cells, and HepG2/C3A cell lines. The results of the immunofluorescence assay and immunofluorescence confocal assay showed that swine GT4 HEV is efficiently replicated in three cell lines at 72 h postinoculation. Meanwhile, we also detected the virus titer quantified were increased at 2-, 6,- and 11-days postinoculation. Moreover, we successfully observed HEV virus particles in the cell suspension at 6 days postinoculation. This finding holds significance for advancing in vitro HEV studies.

The hepatitis E virus (HEV) is infecting over 20 million people annually with a high morbidity especially in pregnant women and immune-suppressed individuals. While HEV genotype 1 (HEV-1) infects only humans, genotype 3 (HEV-3) is zoonotic and commonly transmitted from infected animals to humans. Whereas a few reverse genetics systems enabling targeted genome manipulations exist for HEV-3, those for HEV-1 are still very limited, mainly because of inefficient cell culture replication. Here, the generation of HEV-1 strain Sar55 and HEV-3 strain 47832mc by transfecting in vitro-transcribed and capped virus genomes into different cell lines was attempted. Culture supernatants of colon-derived colorectal adenocarcinoma cell line Caco-2 contained HEV-1 and HEV-3 capable of infecting Caco-2 cells. Density gradient centrifugation analyses of culture supernatants confirmed that HEV-1 particles were quasi-enveloped in analogy to HEV-3 and that non-virion-associated capsid protein was secreted from cells. Following transfection or infection of Caco-2 cells, HEV-1 consistently reached higher titers than HEV-3 in culture supernatants, but HEV-1 generated by transfection of Caco-2 cells was unable to efficiently infect hepatoma cell lines PLC/PRF/5 or HuH7-Lunet BLR. Taken together, our results indicate that HEV-1 is able to exert a complete replication cycle in Caco-2 cells. An efficient cell culture system for this genotype will be useful for studying species tropism, but further research is required to determine the significance of HEV-1 replication in colon-derived cells.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

Hepatitis E virus (HEV) infection is typically a self-limiting, acute illness that spreads through the gastrointestinal tract but replicates in the liver. However, chronic infections are possible in immunocompromised individuals. The HEV virion has two shapes: exosome-like membrane-associated quasi-enveloped virions (eHEV) found in circulating blood or in the supernatant of infected cell cultures and non-enveloped virions ("naked") found in infected hosts' feces and bile to mediate inter-host transmission. Although HEV is mainly spread via enteric routes, it is unclear how it penetrates the gut wall to reach the portal bloodstream. Both virion types are infectious, but they infect cells in different ways. To develop personalized treatment/prevention strategies and reduce HEV impact on public health, it is necessary to decipher the entry mechanism for both virion types using robust cell culture and animal models. The contemporary knowledge of the cell entry mechanism for these two HEV virions as possible therapeutic target candidates is summarized in this narrative review.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity.

Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is transmitted primarily through the fecal-oral route and can cause acute hepatitis in humans. Since HEV was identified as a zoonotic pathogen, different species of HEV strains have been globally identified from various hosts, leading to an expanding range of hosts. The HEV genome consists of a 5' noncoding region, three open reading frames (ORFs), and a 3' noncoding region. The ORF3 protein is the smallest but has many functions in HEV release and pathogenesis. In this review, we systematically summarize recent progress in understanding the functions of the HEV ORF3 protein in virion release, biogenesis of quasi-enveloped viruses, antigenicity, and host environmental regulation. This review will help us to understand HEV replication and pathogenesis mechanisms better.

Heat treatment of food represents an important measure to prevent pathogen transmission. Thus far, evaluation of heat treatment processes is mainly based on data from bacteria. However, foodborne viruses have gained increasing attention during the last decades. Here, the published literature on heat stability and inactivation of human norovirus (NoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) was reviewed. Data for surrogate viruses were not included. As stability assessment for foodborne viruses is often hampered by missing infectivity assays, an overview of applied methods is also presented. For NoV, molecular capsid integrity assays were mainly applied, but data from initial studies utilizing novel intestinal enteroid or zebrafish larvae assays are available now. However, these methods are still limited in applicability and sensitivity. For HAV, sufficient cell culture-based inactivation data are available, but almost exclusively for one single strain, thus limiting interpretation of the data for the wide range of field strains. For HEV, data are now available from studies using pig inoculation or cell culture. The results of the reviewed studies generally indicate that NoV, HAV and HEV possess a high heat stability. Heating at 70-72 °C for 2 min significantly reduces infectious titers, but often does not result in a >4 log10 decrease. However, heat stability greatly varied dependent on virus strain, matrix and heating regime. In addition, the applied method largely influenced the result, e.g. capsid integrity assays tend to result in higher measured stabilities than cell culture approaches. It can be concluded that the investigated foodborne viruses show a high heat stability, but can be inactivated by application of appropriate heating protocols. For HAV, suggestions for safe time/temperature combinations for specific foods can be derived from the published studies, with the limitation that they are mostly based on one strain only. Although significant improvement of infectivity assays for NoV and HEV have been made during the last years, further method development regarding sensitivity, robustness and broader applicability is important to generate more reliable heat inactivation data for these foodborne viruses in future.

The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.

Hepatitis E virus (HEV) is one of the leading causes of acute viral hepatitis. Transmission of HEV mainly occurs via the fecal-oral route (ingesting contaminated water or food) or by contact with infected animals and their raw meat products. Some animals, such as pigs, wild boars, sheep, goats, rabbits, camels, rats, etc., are natural reservoirs of HEV, which places people in close contact with them at increased risk of HEV disease. Although hepatitis E is a self-limiting infection, it could also lead to severe illness, particularly among pregnant women, or chronic infection in immunocompromised people. A growing number of studies point out that HEV can be classified as a re-emerging virus in developed countries. Preventative efforts are needed to reduce the incidence of acute and chronic hepatitis E in non-endemic and endemic countries. There is a recombinant HEV vaccine, but it is approved for use and commercially available only in China and Pakistan. However, further studies are needed to demonstrate the necessity of applying a preventive vaccine and to create conditions for reducing the spread of HEV. This review emphasizes the hepatitis E virus and its importance for public health in Europe, the methods of virus transmission and treatment, and summarizes the latest studies on HEV vaccine development.

Multiple mechanisms exist in a cell to cope with stress. Four independent stress-sensing kinases constitute the integrated stress response machinery of the mammalian cell, and they sense the stress signals and act by phosphorylating the eukaryotic initiation factor 2α (eIF2α) to arrest cellular translation. Eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4) is one of the four kinases and is activated under conditions of amino acid starvation, UV radiation, or RNA virus infection, resulting in shutdown of global translation. An earlier study in our laboratory constructed the protein interaction network of the hepatitis E virus (HEV) and identified eIF2AK4 as a host interaction partner of the genotype 1 (g1) HEV protease (PCP). Here, we report that PCP's association with the eIF2AK4 results in inhibition of self-association and concomitant loss of kinase activity of eIF2AK4. Site-directed mutagenesis of the 53rd phenylalanine residue of PCP abolishes its interaction with the eIF2AK4. Further, a genetically engineered HEV-expressing F53A mutant PCP shows poor replication efficiency. Collectively, these data identify an additional property of the g1-HEV PCP protein, through which it helps the virus in antagonizing eIF2AK4-mediated phosphorylation of the eIF2α, thus contributing to uninterrupted synthesis of viral proteins in the infected cells. IMPORTANCE Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. It causes chronic infection in organ transplant patients. Although the disease is self-limiting in normal individuals, it is associated with high mortality (~30%) in pregnant women. In an earlier study, we identified the interaction between the genotype 1 HEV protease (PCP) and cellular eukaryotic initiation factor 2 alpha kinase 4 (eIF2AK4). Since eIF2AK4 is a sensor of the cellular integrated stress response machinery, we evaluated the significance of the interaction between PCP and eIF2AK4. Here, we show that PCP competitively associates with and interferes with self-association of the eIF2AK4, thereby inhibiting its kinase activity. Lack of eIF2AK4 activity prevents phosphorylation-mediated inactivation of the cellular eIF2α, which is essential for initiation of cap-dependent translation. Thus, PCP behaves as a proviral factor, promoting uninterrupted synthesis of viral proteins in infected cells, which is crucial for survival and proliferation of the virus.

Hepatitis E virus is a major cause of acute hepatitis worldwide and can progress to chronicity in immunocompromised individuals. Various virus-host recombination events have been reported in the hypervariable region of the hepatitis E virus genome, but the patterns of assembly and selection remain unclear.

To gain further insight into viral evolution, we assessed the presence of low abundance variants in 16 samples from individuals with acute or chronic infection using a targeted next-generation sequencing approach.

In seven samples, different variants with insertions and/or deletions were identified. Among them, eight insertions originating either from human genes or from the hepatitis E virus genome. Five different deletions could be identified. The amino acid composition of sequences with insertions showed a higher frequency of lysine and a lower abundance of proline, and additionally acetylation and ubiquitination sites were more frequent than in hepatitis E virus wild-type sequences.

These findings suggest that the nucleotide composition of insertions and sites for post-translational modification may contribute to recombination events. Although the impact of low-level hepatitis E virus variants is uncertain, our results highlight the importance of a highly sensitive next-generation sequencing approach to capture the full diversity of hypervariable region.

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV's open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the 'pPCP' domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.

Viral hepatitis is an infection of human hepatocytes resulting in liver damage. Dual infection of two hepatotropic viruses affects disease outcomes. The hepatitis A virus (HAV) and hepatitis E virus (HEV) are two enterically transmitted viruses; they are single-stranded RNA viruses and have common modes of transmission. They are transmitted mainly by the fecal-oral route and ingestion of contaminated food, though the HAV has no animal reservoirs. The HAV and HEV cause acute self-limiting disease; however, the HEV, but not HAV, can progress to chronic and extrahepatic infections. The HAV/HEV dual infection was reported among acute hepatitis patients present in developing countries. The impact of the HAV/HEV on the prognosis for acute hepatitis is not completely understood. Studies showed that the HAV/HEV dual infection increased abnormalities in the liver leading to fulminant hepatic failure (FHF) with a higher mortality rate compared to infection with a single virus. On the other hand, other reports showed that the clinical symptoms of the HAV/HEV dual infection were comparable to symptoms associated with the HAV or HEV monoinfection. This review highlights the modes of transmission, the prevalence of the HAV/HEV dual infection in various countries and among several study subjects, the possible outcomes of this dual infection, potential model systems for studying this dual infection, and methods of prevention of this dual infection and its associated complications.

The hepatitis E caused by the virus HEV of genotypes HEV-3 and HEV-4 is a zoonotic foodborne disease spread worldwide. HEV is currently classified into eight different genotypes (HEV-1-8). Genotypes HEV-3 and HEV-4 are zoonotic and are further divided into subtypes. Most of the information on HEV replication remains unknown due to the lack of an efficient cell cultivation system. Over the last couple of years, several protocols for HEV cultivation have been developed on different cell lines; even if they were troublesome, long, and scarcely reproducible, they offered the opportunity to study the replicative cycle of the virus. In the present study, we aimed to obtain a protocol ready to use viral stock in serum free medium that can be used with reduced time of growth and without any purification steps. The employed method allowed isolation and cell adaptation of four swine HEV-3 strains, belonging to three different subtypes. Phylogenetic analyses conducted on partial genome sequences of in vitro isolated strains did not reveal any insertion in the hypervariable region (HVR) of the genomes. A limited number of mutations was acquired in the genome during the virus growth in the partial sequences of Methyltransferase (Met) and ORF2 coding genes.

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 (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.

Hepatitis E virus (HEV) infects over 20 million people worldwide per year, leading to 30,000-40,000 deaths. In most cases HEV infection in a self-limited, acute illness. However, chronic infections could occur in immunocompromised individuals. Due to scarcity of robust cell culture models in vitro and genetic tractable animal models in vivo, the details of HEV life cycle, as well as its interaction with host cells still remain elusive, which dampens antivirals discovery. In this chapter, we present an update in the HEV infectious cycle steps: entry, genome replication/subgenomic RNA transcription, assembly, and release. Moreover, we discussed the future prospective on HEV research and illustrates important questions urgently to be addressed.

Hepatitis E virus, a leading cause of acute hepatitis worldwide, has been recognized as non-enveloped virus since its discovery in the 1980s. However, the recent identification of lipid membrane-associated form termed as "quasi-enveloped" HEV has changed this long-held notion. Both naked HEV and quasi-enveloped HEV play important roles in the pathogenesis of hepatitis E. However, the biogenesis and the mechanisms underlying the composition, biogenesis regulation, and functions of the novel quasi-enveloped virions remain enigmatic. In this chapter, we highlight the most recent discoveries on the dual life cycle of these two different types of virions, and further discuss the implication of the quasi-envelopment in our understanding of the molecular biology of HEV.

Despite the advances in hepatitis E virus (HEV) cell infection models' development, HEV infection efficacy in these cell models is still low, which hampers the further study of molecular mechanism of HEV infection and replication and even the interaction between HEV and host. Along with the advances in the technology for liver organoids generation, major efforts will be made to develop liver organoids for HEV infection. Here, we summarize the entire new and impressive cell culture system of liver organoids and discuss their potential application in HEV infection and pathogenesis. Liver organoids can be generated from tissue-resident cells isolated from biopsies of adult tissues or from iPSCs/ESCs differentiation, which can expand the large-scale experiments such as antiviral drug screening. Different types of liver cells working together can recapitulate the liver organ maintaining the physiological and biochemical microenvironments to support cell morphogenesis, migration, and response to viral infections. Efforts to optimize the protocols for liver organoids generation will speed up the research for HEV infection and pathogenesis and even the antiviral drug identification and evaluation.

Cell culture is an important research method in virology. Although many attempts have been conducted to culture HEV in cells, only a few cell culture systems were considered to be efficient enough for usage. Concentration of virus stocks, host cells, and medium components affects the culture efficiency and the genetic mutations during HEV passage were found to be associated with the increased virulence in cell culture. As an alternative method for traditional cell culture, the infectious cDNA clones were constructed. The viral thermal stability, factors that impact the host range, post-translation of viral proteins, and function of different viral proteins were studied using the infectious cDNA clones. HEV cell culture studies on progeny virus showed that the viruses secreted from host cells have an envelope and its formation was associated with pORF3. This result explained the phenomenon that virus could infect host cells in the presence of anti-HEV antibodies.

Comparative analysis of the genomic sequences of multiple hepatitis E virus (HEV) isolates has revealed extensive genomic diversity among them. Recently, a variety of genetically distinct HEV variants have also been isolated and identified from large numbers of animal species, including birds, rabbits, rats, ferrets, bats, cutthroat trout, and camels, among others. Furthermore, it has been reported that recombination in HEV genomes takes place in animals and in human patients. Also, chronic HEV infection in immunocompromised individuals has revealed the presence of viral strains carrying insertions from human genes. This paper reviews current knowledge on the genomic variability and evolution of HEV.

The intimate relationship between virus and host cell can result in highly adapted viruses that are restricted to a single host. However, some viruses have the ability to infect multiple host species. Remarkably, hepatitis E viruses (HEV) comprise genotypes that are either 'single-host' or 'multi-host' genotypes, a trait that raises fundamental questions: Why do different genotypes differ in their host range, despite a high degree of genomic similarity? What are the underlying molecular determinants that shape species barriers? Here, we review the current knowledge of viral and host determinants that may affect the evolutionary trajectories of HEV. We also provide a perspective on techniques and methods that address open questions of HEV host range and adaptation.

Hepatitis E virus (HEV) infection rarely causes icteric hepatitis, yet 10%-40% of adult Americans have serological evidence of previous infection. The aim of this study was to investigate the incidence, presentation, and outcome of acute and previous HEV infection in a large cohort of patients with suspected drug-induced liver injury (DILI).

Serum samples from 2012 patients enrolled in the DILI Network were tested for anti-HEV immunoglobulin G (IgG). Those with detectable anti-HEV IgG underwent testing for anti-HEV IgM; those with detectable anti-HEV immunoglobulin m (IgM) were tested for HEV RNA.

Anti-HEV IgG was detected in 407 (20%) patients and associated with increasing subject age and earlier year of enrollment. The median age of seropositive subjects was more than a decade higher than seronegative subjects (59.8 vs 48.7 years). The overall prevalence of anti-HEV declined from 22% (2004-2011) to 18% (2012-2019), suggestive of a cohort effect. The frequency of acute hepatitis E (median ALT = 1231 IU/L) also decreased from 3% (2004-2008) to 1.2% (2009-2013) to 0.6% (2014-2019). These results suggest that acute HEV infection is usually subclinical and was much more frequent in this cohort before 2004.

Acute HEV infection accounts for less than 1% of suspected American DILI cases and is more frequent in older men. Previous HEV infection is also most commonly seen in older individuals. Clinicians should consider testing for unsuspected acute HEV infection in older adult patients with acute hepatocellular DILI and jaundice.

Hepatitis E virus (HEV) infection in pregnant women has a high incidence of developing fulminant hepatic failure (FHF) with significant mortality. Multiple amino acid changes in genotype 1 HEV (HEV-1) are reportedly linked to FHF clinical cases, but experimental confirmation of the roles of these changes in FHF is lacking. By utilizing the HEV-1 indicator replicon and infectious clone, we generated 11 HEV-1 single mutants, each with an individual mutation, and investigated the effect of these mutations on HEV replication and infection in human liver cells. We demonstrated that most of the mutations actually impaired HEV-1 replication efficiency compared with the wild type (WT), likely due to altered physicochemical properties and structural conformations. However, two mutations, A317T and V1120I, significantly increased HEV-1 replication. Notably, these two mutations simultaneously occurred in 100% of 21 HEV-1 variants from patients with FHF in Bangladesh. We further created an HEV-1 A317T/V1120I double mutant and found that it greatly enhanced HEV replication, which may explain the rapid viral replication and severe disease. Furthermore, we tested the effect of these FHF-associated mutations on genotype 3 HEV (HEV-3) replication and found that all the mutants had a reduced level of replication ability and infectivity, which is not unexpected due to distinct infection patterns between HEV-1 and HEV-3. Additionally, we demonstrated that these FHF-associated mutations do not appear to alter their sensitivity to ribavirin (RBV), suggesting that ribavirin remains a viable option for antiviral therapy for patients with FHF. The results have important implications for understanding the mechanism of HEV-1-associated FHF.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.

Hepatitis E virus (HEV) infections are a leading cause of acute viral hepatitis in humans and pose a considerable threat to public health. Current standard of care treatment is limited to the off-label use of nucleoside-analog ribavirin (RBV) and PEGylated interferon-α, both of which are associated with significant side effects and provide limited efficacy. In the past few years, a promising natural product compound class of eukaryotic initiation factor 4A (eIF4A) inhibitors (translation initiation inhibitors), called rocaglates, were identified as antiviral agents against RNA virus infections. In the present study, we evaluated a total of 205 synthetic rocaglate derivatives from the BU-CMD compound library for their antiviral properties against HEV. At least eleven compounds showed inhibitory activities against the HEV genotype 3 (HEV-3) subgenomic replicon below 30 nM (EC₅₀ value) as determined by Gaussia luciferase assay. Three amidino-rocaglates (ADRs) (CMLD012073, CMLD012118, and CMLD012612) possessed antiviral activity against HEV with EC₅₀ values between 1 and 9 nM. In addition, these three selected compounds inhibited subgenomic replicons of different genotypes (HEV-1 [Sar55], wild boar HEV-3 [83-2] and human HEV-3 [p6]) in a dose-dependent manner and at low nanomolar concentrations. Furthermore, tested ADRs tend to be better tolerated in primary hepatocytes than hepatoma cancer cell lines and combination treatment of CMLD012118 with RBV and interferon-α (IFN-α) showed that CMLD012118 acts additive to RBV and IFN-α treatment. In conclusion, our results indicate that ADRs, especially CMLD012073, CMLD012118, and CMLD012612 may prove to be potential therapeutic candidates for the treatment of HEV infections and may contribute to the discovery of pan-genotypic inhibitors in the future.

As a zoonotic pathogen, the hepatitis E virus (HEV) leads to numerous infections in humans with different clinical manifestations. Especially genotype 3, as causative agent of a foodborne zoonosis, is transmitted to humans by ingestion of undercooked or raw meat containing liver from HEV-infected animals. Although the virus' prevalence and dissemination in hosts like wild boar and pig have been well characterized, HEV is greatly understudied on a molecular level and reliable cell culture models are lacking. For this reason, the present study concentrated on the isolation and subsequent characterization of porcine HEV from tissue samples derived from wild boar and domestic pigs: 222 wild boars hunted in Northern Germany were investigated for the presence of HEV RNA with a detection rate of 5.9%. Three additional HEV-positive wild boar liver samples as well as an HEV-positive spleen and a positive kidney from domestic pigs were included. After inoculation of positive samples onto the human hepatoma cell line PLC/PRF/5, cells were grown for several weeks. Successful isolation was confirmed by RT-qPCR, virus passage, immunofluorescence staining and titration. Overall, 15 strains from a total of 18 RNA-positive organ samples could be obtained and viral loads >10⁹  RNA copies/ml were measured in cell culture supernatants. Accordingly, 83.3% of the HEV RNA-positive samples contained infectious hepatitis E viral particles and therefore must be considered as a potential source for human infections. Phylogenetic analyses revealed that all isolated strains belong to genotype 3. Further genetic characterization showed a high degree of sequence variability, but no sequence insertions, in the hypervariable region within the open reading frame 1.