Hepatitis E Virus Particles Research Articles

Quasi-enveloped hepatitis virus assembly and release.

Hepatitis A virus (HAV) and hepatitis E virus (HEV) infections are the main causes for acute hepatitis worldwide. Both viruses had long been considered as nonenveloped viruses. However, recent work has uncovered that both viruses circulate in the bloodstream as membrane-cloaked, "quasi-enveloped" particles that are, surprisingly, infectious and likely the only form mediating virus spread within the host. The discovery of quasi-enveloped HAV and HEV particles has fundamentally changed the traditional view on the life cycle and pathogenesis of these viruses. However, because HAV and HEV are phylogenetically unrelated and their capsid assembly processes are quite distinct, it is not clear whether they use similar or different mechanisms for envelopment and exit. This review provides an overview of the current knowledge about the assembly and exit processes of HAV and HEV and perspectives for future studies.

Updates in Hepatitis E virus (HEV) field; lessons learned from human liver chimeric mice.

Hepatitis E virus (HEV) is the most common cause of viral hepatitis globally, and it is an emerging pathogen in developed countries. In vivo studies of HEV have long been hindered due to the lack of an efficient small animal model. Recently, human liver chimeric mice were described as an elegant model to study chronic HEV infection. HEV infection was established in mice with humanized liver that were challenged with stool preparations containing HEV genotype (gt)1 and/or gt3. An increase in viral load and the level of HEV Ag in mouse samples were markers of active infection. Plasma-derived HEV preparations were less infectious. The kinetics of HEV ORF2 Ag during HEV infection and its impact on HEV diagnosis were described in this model. In addition, the nature of HEV particles and HEV ORF2 Ag were characterized. Moreover, humanized mice were used to study the impact of HEV infection on the hepatic innate transcriptome and evaluation of anti-HEV therapies. This review highlights recent advances in the HEV field gathered from well-established experimental mouse models, with an emphasis on this model as a tool for elucidating the course of HEV infection, the study of the HEV life cycle, the interaction of the virus with the host, and the evaluation of new anti-HEV therapies.

New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

Development of a viability PCR assay for the analysis of Hepatitis E virus in food matrices

Background: Hepatitis E virus (HEV) infection is considered an emerging problem in developed countries, where foodborne transmission plays a key role. As for other viruses, HEV detection in food matrices is based on molecular methods (PCR) that cannot discriminate between infectious and non-infectious virus. Viability PCR (v-PCR) is based on the use of intercalating dyes that penetrate through damaged viral capsid and block PCR amplification of their genome, therefore differentiating damaged virions from intact viruses. Aim of this study was the development of a v-PCR protocol for HEV in food matrices. Methods: A v-PCR protocol was optimized using a standardized amount of in vitro synthesized HEV RNA and ethidium bromide monoazide (EMA). Different EMA treatment conditions (incubation time from 15 to 120 minutes and concentrations from 20 to 320 microM) were tested. Efficiency of the treatment in removing free RNA was assessed by real-time RT(q)PCR. Control reactions were included to assess inhibition effects on PCR detection. The optimized v-PCR protocol was applied on HEV deactivated by heating (90 °C for 2 minutes) and on a food matrix (bivalve shellfish) experimentally contaminated with both intact and thermally treated virus. Results: Results showed that a 30 minute treatment with an EMA concentration of 200 microM provided a reduction of free HEV RNA ≥98% with little or no inhibition in the subsequent PCR reactions. Results were confirmed in the assays on thermally treated virus, while a lower reduction (∼60%) was obtained in experimentally contaminated shellfish, possibly in relation to dispersion of EMA activity onto nucleic acids resulting from the food matrix. Conclusions: The developed EMA-PCR protocol allows a better estimation of intact HEV particles compared to the standard real-time PCR protocol and, following optimization, can be applied for the detection of viable HEV in food matrices of animal origin. Key messages: - The analysis of foodborne viruses relies on molecular methods that may overestimate risk as a result of detection of free nucleic acids or damaged viral particles. - Viabilty PCR is a promising approach to differentiate infectious and non-infectious viruses in order to improve the assessment of the risk associated to viral contamination in foods.

Stem Cell-Derived Culture Models of Hepatitis E Virus Infection.

Similar to other hepatotropic viruses, hepatitis E virus (HEV) has been notoriously difficult to propagate in cell culture, limiting studies to unravel its biology. Recently, major advances have been made by passaging primary HEV isolates and selecting variants that replicate efficiently in carcinoma cells. These adaptations, however, can alter HEV biology. We have explored human embryonic or induced pluripotent stem cell (hESC/iPSC)-derived hepatocyte-like cells (HLCs) as an alternative to conventional hepatoma and hepatocyte cell culture systems for HEV studies. HLCs are permissive for nonadapted HEV isolate genotypes (gt)1-4 replication and can be readily genetically manipulated. HLCs, therefore, enable studies of pan-genotype HEV biology and will serve as a platform for testing anti-HEV treatments. Finally, we discuss how hepatocyte polarity is likely an important factor in the maturation and spread of infectious HEV particles.

SAT-386 - Hepatitis E virus lifecycle and identification of 3 forms of the ORF2 capsid protein

BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantai enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway.

Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles-now considered "quasi-enveloped particles"-are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses.IMPORTANCE Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles present in feces and bile are nonenveloped, while those in circulating blood and culture supernatants are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help to elucidate the entry mechanisms and receptors for HEV infection in the future. This is the first report to characterize the detailed morphological features of membrane-associated HEV particles.

Hepatitis E Virus Lifecycle and Identification of 3 Forms of the ORF2 Capsid Protein

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed invitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. We developed an efficient cell culture system and isolated HEV particles that were infectious invitro and invivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

Characterization of the lipid envelope of exosome encapsulated HEV particles protected from the immune response

The hepatitis E virus (HEV) is the most common cause of acute hepatitis worldwide. Although HEV is a small, naked RNA virus, HEV particles become associated with lipids in the blood of infected patients and in the supernatant of culture systems. The egress of these particles from cells implies the exocytosis pathway but the question of the role of the resulting HEV RNA containing exosomes and the nature of the lipids they contain has not been fully addressed.We determined the lipid proportions of exosomes from uninfected and HEV-infected cells and their role in HEV spreading. We cultured a suitable HEV strain on HepG2/C3A cells and analyzed the population of exosomes containing HEV RNA using lipidomics methods and electron microscopy. We also quantified HEV infectivity using an infectivity endpoint method based on HEV RNA quantification to calculate the tissue culture infectious dose 50.Exosomes produced by HEV-infected HepG2/C3A cells contained encapsidated HEV RNA. These HEV RNA-containing exosomes were infectious but ten times less than stools. HEV from stools, but not exosome-associated HEV from culture supernatant, was neutralized by anti-HEV antibodies in a dose-dependent manner. HEV infection did not influence the morphology or lipid proportions of the bulk of exosomes. These exosomes contained significantly more cholesterol, phosphatidylserine, sphingomyelin and ceramides than the parent cells, but less phosphoinositides and polyunsaturated fatty acids.Exosomes play a major role in HEV egress but HEV infection does not modify the characteristics of the bulk of exosomes produced by infected cells. PS and cholesterol enriched in these vesicles could then be critical for HEV entry. HEV particles in exosomes are protected from the immune response which could lead to the wide circulation of HEV in its host.

Hepatitis E Virus Infects Neurons and Brains.

Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.

Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles.

Eight murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the C-terminal 15-amino-acid portion of the ORF3 protein of rat hepatitis E virus (ratHEV) were produced and characterized. Immunofluorescence assays using the anti-ratHEV ORF3 MAbs revealed the accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmids or inoculated with cell-culture-generated ratHEV strains. Anti-ORF3 MAbs could capture ratHEV particles in culture supernatant and serum following treatment with 0.5% deoxycholate, but not those without prior detergent treatment or fecal ratHEV particles. Following treatment with 0.5% deoxycholate and 0.5% trypsin, the buoyant density of ratHEV particles in culture supernatant with ORF3 protein on the surface shifted from 1.15g/cm3 to 1.26g/cm3 in a sucrose gradient; the resulting particles were capturable by an anti-ORF2 MAb but not by an anti-ORF3 MAb. This indicates that the ORF3 protein (at least its C-terminal portion) is incorporated into the enveloped ratHEV virions released from infected cells but that it is not found in the virions in the feces, supporting the hypothesis that the ratHEV ORF3 protein is associated with the egress of virions from infected cells, similar to human HEV, despite the fact that the ratHEV ORF3 protein lacks a PSAP amino acid motif.

Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system

Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101–644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV.

Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies.

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

Rat hepatitis E virus derived from wild rats (Rattus rattus) propagates efficiently in human hepatoma cell lines

Although rat hepatitis E virus (HEV) has been identified in wild rats, no cell culture systems for this virus have been established. A recent report suggesting the presence of antibodies against rat HEV in human sera encouraged us to cultivate rat HEV in human cells. When liver homogenates obtained from wild rats (Rattus rattus) in Indonesia were inoculated onto human hepatocarcinoma cells, the rat HEV replicated efficiently in PLC/PRF/5, HuH-7 and HepG2 cells, irrespective of its genetic group (G1–G3). The rat HEV particles released from cultured cells harbored lipid-associated membranes on their surface that were depleted by treatment with detergent and protease, with the buoyant density in sucrose shifting from 1.15–1.16g/ml to 1.27–1.28g/ml. A Northern blotting analysis revealed genomic RNA of 7.0kb and subgenomic RNA of 2.0kb in the infected cells. The subgenomic RNA of G1–G3 each possessed the extreme 5′-end sequence of GUAGC (nt 4933–4937), downstream of the highly conserved sequence of GAAUAACA (nt 4916–4923). The establishment of culture systems for rat HEV would allow for extended studies of the mechanisms of viral replication and functional roles of HEV proteins. Further investigation is required to clarify the zoonotic potential of rat HEV.

The membrane on the surface of hepatitis E virus particles is derived from the intracellular membrane and contains trans-Golgi network protein 2

Our previous studies demonstrated that hepatitis E virus (HEV) requires the multivesicular body (MVB) pathway to release virus particles, suggesting that HEV utilizes the cellular ESCRT machinery in the cytoplasm, not at the cell surface, to be released from infected cells. In this study, we generated a murine monoclonal antibody (mAb) against the membrane-associated HEV particles to examine whether the membrane is derived from intracellular vesicles or the cell surface. An established mAb, TA1708, was found to capture the membrane-associated HEV particles, but not the membrane-dissociated particles or fecal HEV, in an immunocapture RT-PCR assay. Furthermore, digitonin treatment confirmed that the membrane on the surface of cell-culture-generated HEV particles was a lipid membrane. Double immunofluorescence staining revealed that mAb TA1708 specifically recognizes trans-Golgi network protein 2 (TGOLN2), an intracellular antigen derived from the trans-Golgi network. Supporting these findings, HEV particles with lipid membranes and ORF3 proteins on their surface were found abundantly in the lysates of HEV-infected cells. These results indicate that HEV forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and that the released HEV particles with a lipid membrane retain the antigenicity of TGOLN2 on their surface.

Culture systems for hepatitis E virus

The lack of an efficient cell culture system for hepatitis E virus (HEV) has greatly hampered detailed analyses of this virus. The first efficient cell culture systems for HEV that were developed were capable of secreting infectious HEV progenies in high titers into culture media, using PLC/PRF/5 cells derived from human hepatocellular carcinoma and A549 cells derived from human lung cancer as host cells. The success achieved with the original genotype 3 JE03-1760F strain has now been extended to various HEV strains in fecal and serum samples obtained from hepatitis E patients and to HEV strains in fecal and serum samples and liver tissues obtained from pigs and wild boar across species barriers. In addition, infectious HEV cDNA clones of the wild-type JE03-1760F strain and its variants have been engineered. Cell culture-generated HEV particles and those in circulating blood were found to be associated with lipids and open reading frame 3 (ORF3) protein, thereby likely contributing to the assembly and release of HEV from infected cells both in vivo and in vitro. The ORF3 protein interacts with the tumor susceptibility gene 101, a critical cellular protein required for the budding of enveloped viruses, through the Pro, Ser, Ala, and Pro (PSAP) motif in infected cells; ORF3 is co-localized with multivesicular bodies (MVBs) in the cytoplasm of infected cells, thus suggesting that HEV requires the MVB pathway for the egress of virus particles. This article reviews the development of efficient cell culture systems for a wide variety of infectious HEV strains obtained from humans, pigs, and wild boar, and also provides details of a new model for virion egress.

Pathogenesis and Treatment of Hepatitis E Virus Infection

Hepatitis E has been considered to be a travel-associated, acute, self-limiting liver disease that causes fulminant hepatic failure in specific high-risk groups only. However, hepatitis E virus (HEV) infection can also be acquired in industrialized countries-HEV genotype 3 infection is a zoonosis, with pigs and rodents serving as animal reservoirs. In recent years, cases of chronic HEV infection that were associated with progressive liver disease have been described in several cohorts of immunocompromised individuals, including recipients of organ transplants. The topic of hepatitis E is therefore re-emerging and has raised the following important questions: what is the risk for HEV infection in Western countries (eg, from eating uncooked meat)? How frequently does chronic hepatitis E develop among human immunodeficiency virus-infected patients and recipients of organ transplants? What are the treatment options? What is the current status of vaccine development? What do we know about the pathogenesis of HEV infection, and why does it have a more severe course in pregnant women? This review summarizes the current knowledge on the pathogenesis and treatment of HEV infection.

Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses

Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.

Efficient cell culture systems for hepatitis E virus strains in feces and circulating blood

Attempts have been made to propagate hepatitis E virus (HEV) in primary hepatocyte culture and various other cultured cells. However, the replication ability of HEV recovered from culture media remains extremely low. Recently, efficient culture systems have been established in PLC/PRF/5 (hepatocellular carcinoma) and A549 (lung cancer) cell lines for HEV strains of genotypes 3 and 4 in our laboratory. They originated in fecal extracts from patients containing HEV RNA in extremely high-titers (10(7) copies/ml), and named the JE03-1760F (genotype 3) and HE-JF5/15F (genotype 4) strains, respectively. HEV RNA in culture supernatants reached 10(8) copies/ml in titer, and were transmitted successively through many passages. An infectious HEV cDNA clone (pJE03-1760F/wt) was constructed that has replication activity comparable to that of the wild-type JE03-1760F in feces. The ORF3 protein is indispensable for shedding HEV particles from cells in the reverse genetics system. HEV recovered from culture media, as well as circulating HEV, possess ORF3 proteins on the surface and are covered with cellular membranes, and therefore, ORF2 epitopes are buried in these particles. In contrast, HEV excreted into feces are naked nucleocapsids without a lipid layer or surface expression of the ORF3 protein. HEV in sera of patients with acute hepatitis E can infect and replicate in PLC/PRF/5 and A549 cells, with efficiency comparable to the circulating HEV RNA levels. High-efficiency cell culture systems for infectious viruses, thus developed, are expected to open up a new era and resolve many mysteries in the epidemiology, molecular biology, and treatment of HEV.