Molecularly generated rat hepatitis E virus strains from human and rat show efficient replication in a human hepatoma cell line.

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Whereas HEV genotypes 1-4 of species Paslahepevirus balayani are commonly found in humans, infections with ratHEV (species Rocahepevirus ratti) were previously considered to be restricted to rats. However, several cases of human ratHEV infections have been described recently. To investigate the zoonotic potential of this virus, a genomic clone was constructed here based on sequence data of ratHEV strain pt2, originally identified in a human patient with acute hepatitis from Hongkong. For comparison, genomic clones of ratHEV strain R63 from a rat and of HEV genotype 3 strain 47832mc from a human patient were used. After transfection of in vitro-transcribed RNA from the genomic clones into the human hepatoma cell line HuH-7-Lunet BLR, virus replication was shown for all strains by increasing genome copy numbers in cell culture supernatants. These cells developed persistent virus infections, and virus particles in the culture supernatant as well as viral antigen within the cells were demonstrated. All three generated virus strains successfully infected fresh HuH-7-Lunet BLR cells. In contrast, the human hepatoma cell lines HuH-7 and PLC/PRF/5 could only be infected with the genotype 3 strain and to a lesser extent with ratHEV strain R63. Infection of the rat-derived hepatoma cell lines clone 9, MH1C1 and H-4-II-E did not result in efficient virus replication for either strain. The results indicate that ratHEV strains from rats and humans can infect human hepatoma cells. The replication efficiency is strongly dependent on the cell line and virus strain. The investigated rat hepatoma cell lines could not be infected and other rat-derived cells should be tested in future to identify permissive cell lines from rats. The developed genomic clone can represent a useful tool for future research investigating pathogenicity and zoonotic potential of ratHEV.

Hepatitis E virus infects human testicular tissue and Sertoli cells.

Globally, hepatitis E virus (HEV) infections are prevalent. The finding of high viral loads and persistent viral shedding in ejaculate suggests that HEV replicates within the human male genital tract, but its target organ is unknown and appropriate models are lacking. We aimed to determine the HEV tropism in the human testis and its potential influence on male reproductive health. We conducted an ex vivo culture of human testis explants and in vitro culture of primary human Sertoli cells. Clinically derived HEV genotype 1 (HEV1) and HEV3 virions, as well as rat-derived HEV-C1, were used for inoculation. Transcriptomic analysis was performed on testis tissues collected from tacrolimus-treated rabbits with chronic HEV3 infection. Our findings reveal that HEV3, but not HEV1 or HEV-C1, can replicate in human testis explants and primary human Sertoli cells. Tacrolimus treatment significantly enhanced the replication efficiency of HEV3 in testis explants and enabled successful HEV1 infection in Sertoli cells. HEV3 infection disrupted the secretion of several soluble factors and altered the cytokine microenvironment within primary human Sertoli cells. Finally, intratesticular transcriptomic analysis of immunocompromised rabbits with chronic HEV infection indicated downregulation of genes associated with spermatogenesis. HEV can infect the human testicular tissues and Sertoli cells, with increased replication efficiency when exposed to tacrolimus treatment. These findings shed light on how HEV may persist in the ejaculate of patients with chronic hepatitis E and provide valuable ex vivo tools for studying countermeasures.

Heat stability of foodborne viruses - Findings, methodological challenges and current developments.

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.

Low Replication Efficiency of a Japanese Rabbit Hepatitis E Virus Strain in the Human Hepatocarcinoma Cell Line PLC/PRF/5.

A Japanese rabbit hepatitis E virus (HEV) strain, JP-59, has been identified in a feral rabbit. When this virus was transmitted to a Japanese white rabbit, it caused persistent HEV infection. The JP-59 strain shares an <87.5% nucleotide sequence identity with other rabbit HEV strains. Herein, to isolate JP-59 by cell culture, we used a 10% stool suspension recovered from a JP-59-infected Japanese white rabbit and contained 1.1 × 107 copies/mL of the viral RNA and using it to infect a human hepatocarcinoma cell line, PLC/PRF/5. No sign of virus replication was observed. Although long-term virus replication was observed in PLC/PRF/5 cells inoculated with the concentrated and purified JP-59 containing a high titer of viral RNA (5.1 × 108 copies/mL), the viral RNA of JP-59c that was recovered from the cell culture supernatants was <7.1 × 104 copies/mL during the experiment. The JP-59c strain did not infect PLC/PRF/5 cells, but its intravenous inoculation caused persistent infection in rabbits. The nucleotide sequence analyses of the virus genomes demonstrated that a total of 18 nucleotide changes accompanying three amino acid mutations occurred in the strain JP-59c compared to the original strain JP-59. These results indicate that a high viral RNA titer was required for JP-59 to infect PLC/PRF/5 cells, but its replication capability was extremely low. In addition, the ability of rabbit HEVs to multiply in PLC/PRF/5 cells varied depending on the rabbit HEV strains. The investigations of cell lines that are broadly susceptible to rabbit HEV and that allow the efficient propagation of the virus are thus needed.

Variable Outcomes of Hepatitis E Infections in Patients with Hemato-Oncologic Diseases.

INTRODUCTION: The hepatitis E virus (HEV) represents an important cause of viral hepatitis and could cause chronic infections in immunocompromised patients. However, data about immunocompromised patients other than solid organ transplant recipients are limited. METHODS: We identified patients from a laboratory database and retrospectively compiled and analyzed clinical as well as laboratory data in detail. RESULTS: Overall, 22 severely immunosuppressed patients, excluding solid organ transplant recipients, were identified. Four patients did not experience viral clearance (one without and three despite ribavirin therapy). Three patients acquired the infection after allogeneic hematopoietic stem cell transplantation (alloHSCT) and recovered spontaneously, whereas another patient, infected prior to alloHSCT, developed a chronic infection. Four patients failed to clear HEV, resulting in fatal liver failure in 2 patients. The CD4+ cell counts increased in all but 1 patient attaining a sustained virological response (SVR), as compared to patients with clinical failure. Severe immunoglobulin deficiency did not appear to obviate the control of HEV. Six of ten (60%) patients with and nine of 12 (75%) patients without ribavirin therapy achieved an SVR. CONCLUSIONS: Upfront ribavirin therapy does not appear mandatory in patients without CD4+ lymphopenia, but a prolonged HEV replication carries the risk of liver failure. Our data suggest that chronic HEV infections could cause T-cell exhaustion, which might be overruled with ribavirin therapy.

Survival and inactivation of hepatitis E virus on inanimate surfaces.

BACKGROUND: Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis, and mainly transmitted via faecal-oral contamination or consumption of contaminated food products. However, limited data on the surface stability and HEV sensitivity to chemical disinfectants are available. AIM: To establish an HEV-based carrier assay to evaluate its surface stability and the virucidal activity of nine surface disinfectants. METHODS: A recently developed robust HEV-3 cell culture system for an HEV-based carrier assay. FINDINGS: Alcohol-based disinfectants were insufficient to eliminate HEV infectivity, whereas disinfectants based on aldehyde, peracetic acid, oxygen, and/or quaternary ammonium inactivated HEV. CONCLUSION: These findings have strong implications for the recommendation of evidence-based hygiene guidelines to reduce HEV transmission.

Molecular docking and dynamic simulation analysis of Hepatitis E virus protease in complexing with the E64 inhibitor.

The unavailability of a suitable treatment for human Hepatitis E virus (HEV) infection necessitate the development of anti HEV drugs. The HEV papain-like cysteine proteases (HEV PCP) is a crucial target to prevent viral replication and progression. E64 is a known HEV PCP inhibitor; however, its molecular mechanism of inhibition is not yet known. Since the crystal structure of HEV PCP is not available, the primary focuses of the present study was to refine the predicted HEV PCP structural model by molecular dynamics (MD) simulation. Further, we performed a 200 ns MD simulation to understand the structural complexity of HEV PCP and the effect of E64 binding with HEV PCP. The E64 binding with active site residues Gln48, Thr51, Gln55, Cys52, Ser81, Gln 98, Cys 132, Arg158, His159, Asn 160 and Ala96 leads to reduced fluctuations in the residue at N-terminal (18-41) that include the CHC motif (26-28). However, most of the other non interacting residues, including the inter-domain linker region (46-87), showed increased fluctuations in the HEV PCP-E64 complex. The residue Asp21 and Ala96 are involved in the formation of interdomain interactions in the HEV PCP apo enzyme. While in the PCP-E64 complex, E64 binds to Ala96 and creates a steric hindrance to prevent interdomain interactions. Thus, the E64 binding reduces interdomain interactions and restrict domain movements in the HEV PCP-E64 complex. This information will be important for the chemically designing more effective derivatives of E64 developing HEV PCP specific inhibitors.Communicated by Ramaswamy H. Sarma.

Modulation of SOCS3 Levels via STAT3 and Estrogen-ERαp66 Signaling during Hepatitis E Virus Replication in Hepatocellular Carcinoma Cells.

Hepatitis E virus (HEV) infection usually results in a self-limiting acute disease; however, in infected pregnant women, it is associated with increased mortality and fulminant hepatic failure. Estrogen is known to be elevated during pregnancy, and estrogen signaling via classical estrogen receptor-ERα is known to regulate hepatocyte function and host innate immune response, including the STAT3 pathway. In this study, we investigated whether the estrogen classical signaling pathway via ERαp66 has any effect on STAT3 activation during HEV replication and HEV-induced IFN response. We first demonstrated that Huh7-S10-3 liver cells expressed the nonfunctional estrogen receptor ERαp36 isoform and lack the functional ERαp66 isoform. We further showed persistent phosphorylated-STAT3 levels in genotype 3 human HEV (Kernow P6 strain) RNA-transfected cells at later time points. In Huh7-S10-3 cells, estrogen at first-to-third trimester concentration (7.3 to 73 nM) did not significantly affect HEV replication; however, blocking of STAT3 activation led to a decrease in the HEV ORF2 protein level. Our mechanistic study revealed that STAT3 differentially regulates SOCS3 and type-III interferon (IFN) levels during HEV replication and the presence of estrogen-ERαp66 signaling stabilizes SOCS3 levels in vitro. We also demonstrate that HEV infection in pregnant and nonpregnant rabbits led to a significant increase in IFN response as measured by increased levels of IFN-stimulated-gene-15 (ISG15) mRNA levels irrespective of pregnancy status. Collectively, the results indicate that estrogen signaling and STAT3 regulate SOCS3 and IFN responses in vitro during HEV replication. The results have important implications for understanding HEV replication and HEV-induced innate immune response in pregnant women. IMPORTANCE Hepatitis E is usually a self-resolving acute disease; however, in pregnant women, HEV infection is associated with high mortality and fulminant hepatic failure. During pregnancy, estrogen levels are elevated, and in the liver, the estrogen receptor ERα is predominant and estrogen signaling is known to regulate hepatocyte metabolism and leptin-induced STAT3 levels. Viruses can module host innate immune response via STAT3. Therefore, in this study, we investigated whether STAT3 and estrogen-classical signaling via the ERαp66 pathway modulate HEV replication and HEV-induced innate immune response. We demonstrated that estrogen signaling did not affect HEV replication in human liver cells, but blocking of STAT3 activation reduced HEV capsid protein levels in human liver cells. We also showed that inhibition of STAT3 activation reduced SOCS3 levels, while the presence of the estrogen-ERαp66 signaling pathway stabilized SOCS3 levels. The results from this study will aid our understanding of the mechanism of HEV pathogenesis and immune response during pregnancy.

A ribavirin-induced ORF2 single-nucleotide variant produces defective hepatitis E virus particles with immune decoy function.

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.

Nationwide survey of hepatitis E virus infection among wildlife in Japan.

In Japan, hepatitis E virus (HEV) causes hepatitis in humans through the consumption of raw or undercooked meat, including game meat. In the present study, nationwide surveillance of HEV infection among a total of 5,557 wild animals, including 15 species, was conducted in Japan. The prevalence of anti-HEV antibodies in wild boar was 12.4%, with higher positive rates in big boars (over 50 kg, 18.4%) than in small individuals (less than 30 kg, 5.3%). Furthermore, HEV RNA was more frequently detected in piglets than in older boars. Interestingly, the detection of HEV among wildlife by ELISA and RT-PCR suggested that HEV infection in Sika deer was a very rare event, and that there was no HEV infection among wild animals except for wild boar, Sika deer and Japanese monkeys. In conclusion, wild boar, especially piglets, are at high risk of HEV infection, while other wild animals showed less risk or no risk of HEV transmission.

Hepatitis E virus-encoded microRNA promotes viral replication by inhibiting type I interferon.

MicroRNAs (miRNAs), the non-coding RNAs of ~22 nucleotides (nt) in length, play a vital role in regulating viral replication. Hepatitis E virus (HEV), a single-stranded RNA virus, is a predominant pathogen of acute hepatitis worldwide. Virus-encoded miRNAs regulate the viral life cycle and escape from the host innate immune system. However, it is rarely known about HEV-encoded miRNA (HEV-miR-A6). In the present study, HEV-miR-A6 was screened by microarray, and further identified in vivo and in vitro. HEV-miR-A6 originated from the methylase (MeT) of HEV open reading frame 1 (ORF1) and was highly conserved in eight HEV genotypes. HEV-miR-A6 expression was growing during HEV replication, and significantly increased in acute hepatitis E patients than convalescence patients. Furthermore, HEV-miR-A6 was specifically detected in liver, spleen, kidney and colon by in situ hybridization. To identify the specificity of HEV-miR-A6, its mutants (HEV-miR-A6M1 and HEV-miR-A6M2) were constructed to change the stem-loop structure. Interestingly, over-expression of HEV-miR-A6 or HEV-miR-A6M1 significantly facilitated viral replication, while HEV-miR-A6M2, another mutant completely changed the stem-loop structure was invalid. SIRP-α, a candidate target gene of HEV-miR-A6, was activated when HEV-miR-A6 over-expressed to inhibit the phosphorylation of IRF3, and subsequently suppressed the expression of type I interferon ß (IFN-ß). The promotion of viral replication by HEV-miR-A6 further identified in vivo. Significant suppression of IFN-ß production in the serum of HEV-infected mice pre-treated with HEV-miR-A6 was observed. In summary, HEV-miR-A6 activates SIRP-α to promote viral replication by inhibition of IFN-ß expression.

Interplay between Hepatitis E Virus and Host Cell Pattern Recognition Receptors.

Hepatitis E virus (HEV) usually causes self-limiting acute hepatitis, but the disease can become chronic in immunocompromised individuals. HEV infection in pregnant women is reported to cause up to 30% mortality, especially in the third trimester. Additionally, extrahepatic manifestations like neuronal and renal diseases and pancreatitis are also reported during the course of HEV infection. The mechanism of HEV pathogenesis remains poorly understood. Innate immunity is the first line of defense triggered within minutes to hours after the first pathogenic insult. Growing evidence based on reverse genetics systems, in vitro cell culture models, and representative studies in animal models including non-human primates, has implicated the role of the host's innate immune response during HEV infection. HEV persists in presence of interferons (IFNs) plausibly by evading cellular antiviral defense. This review summarizes our current understanding of recognizing HEV-associated molecular patterns by host cell Pattern Recognition Receptors (PRRs) in eliciting innate immune response during HEV infection as well as mechanisms of virus-mediated immune evasion.

Generation of a Bactrian camel hepatitis E virus by a reverse genetics system.

Bactrian camel hepatitis E virus (HEV) is a novel HEV belonging to genotype 8 (HEV-8) in the Orthohepevirus A species of the genus Hepevirus in the family Hepeviridae. HEV-8 cross-transmits to cynomolgus monkeys and has a potential risk for zoonotic infection. Until now, neither a cell-culture system to grow the virus nor a reverse genetics system to generate the virus has been developed. To generate replication-competent HEV-8 and to establish a cell-culture system, we synthesized capped genomic HEV-8 RNAs by in vitro transcription and used them to transfect into PLC/PRF/5 cells. A HEV-8 strain, HEV-8M2, was recovered from the capped HEV-8 RNA-transfected cell-culture supernatants and subsequently passaged in the cells, demonstrating that PLC/PRF/5 cells were capable of supporting the replication of the HEV-8, and that a cell-culture system for HEV-8 was successfully established. In addition to PLC/PRF/5 cells, A549 and Caco-2 cells appeared to be competent for the replication, but HepG2 C3/A, Vero, Hela S3, HEp-2C, 293T and GL37 cells were incompetent. The HEV-8M2 strain was capable of infecting cynomolgus monkeys by an intravenous inoculation, indicating that HEV-8 was infectious and again carried a risk for zoonotic infection. In contrast, HEV-8 did not infect nude rats and BALB/c nude mice, suggesting that the reservoir of HEV-8 was limited. In addition, the replication of the HEV-8M2 strain was efficiently abrogated by ribavirin but not by favipiravir, suggesting that ribavirin is a drug candidate for therapeutic treatment of HEV-8-induced hepatitis. The infectious HEV-8 produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of type E hepatitis.

Progesterone-Mediated Enhancement of Hepatitis E Virus Replication in Human Liver Cells.

Progesterone is crucial for the maintenance of pregnancy. During pregnancy hepatitis E virus (HEV) infection is associated with increased fulminant hepatic failure and mortality rates. In this study, we determined whether progesterone modulates HEV replication and HEV-induced innate cytokine response in Huh7-S10-3 human liver cells. We first demonstrated that Huh7-S10-3 liver cells expressed SH3-domain-containing progesterone receptor membrane component (PGRMC)1/2 receptors involved in the progesterone nonclassical signaling pathway, while the classical progesterone receptor isoforms progesterone receptor-A and -B protein levels were undetectable. We showed that the genotype 3 HEV (strain P6) induced mRNA expression of type III interferon (IFN-λ1), but not other innate cytokines in Huh7-S10-3 cells. Pretreatment with progesterone at concentrations of 80 nM, 160 nM, or 480 nM, which are the physiological concentrations typically seen in the first- to third-trimester during pregnancy, significantly increased HEV replication in Huh7-S10-3 cells. However, pretreatment of cells with progesterone (80 nM) did not affect the level of HEV-induced IFN-λ1 mRNA expression. We further showed that loss of PGRMC1/2 receptors by small interfering RNA (siRNA) knockdown leads to an increase in HEV-induced IFN-λ1 expression levels at early time points via the extracellular signal-regulated kinase pathway and thus resulted in a reduced level of HEV replication. Collectively, the results indicated that progesterone-mediated modulation of HEV replication in human liver cells is plausibly through SH3-domain containing proteins such as PGRMC1/2, but not likely through immunomodulation of HEV-induced interferon response in liver cells. The results have important implications in understanding the underlying mechanisms of high mortality and fulminant hepatitis in HEV-infected pregnant women. IMPORTANCE Hepatitis E is usually a self-limiting acute disease; however, during pregnancy, a severe form of fulminant hepatic failure and high mortality rate are associated with hepatitis E virus (HEV) infection. Increased levels of progesterone and HEV RNA are observed in pregnant women with fulminant hepatic failures. Since progesterone is crucial for maintenance of pregnancy, we investigated the potential role of progesterone in HEV replication and disease pathogenesis. We demonstrated that progesterone at a concentration seen during pregnancy enhances HEV replication in human liver cells, but did not modulate HEV-induced interferon response in human liver cells. We also showed that loss of the progesterone nonclassical receptor, progesterone receptor membrane component (PGRMC)1/2, leads to a reduced level of HEV replication and an increased level of HEV-induced type III interferon (IFN-λ1) mRNA expression via the extracellular signal-regulated kinase pathway. The results from this study will aid our understanding of the underlying mechanism of pathogenesis and HEV-associated severe disease during pregnancy.

The different replication between nonenveloped and quasi-enveloped hepatitis E virus.

Hepatitis E virus (HEV) is the major pathogen of viral hepatitis. However, the understanding of the HEV life cycle is limited. In the present study, cells were separately infected with nonenveloped HEV (derived from feces or bile) or quasi-enveloped HEV (derived from the cell culture after serial passages, eHEV) and observed by confocal fluorescence microscopy to investigate the life cycle of HEV. HEV finished its binding and entry into host cells at first 6 h postinoculation (hpi). Cells inoculated with eHEV showed less infectivity than cells inoculated with nonenveloped HEV. Newly synthesized progeny virions were released into the supernatant of cell cultures from 48 hpi. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis results showed that the supernatant's progeny viruses were infectious even after five serial passages. These results show the significant difference between nonenveloped HEV and eHEV, which will provide novel insights into the HEV replication cycle. The efficient cell culture of HEV will promote the development of anti-HEV drugs and vaccines.

The Foodborne Transmission of Hepatitis E Virus to Humans.

Globally, Hepatitis E virus (HEV) causes over 20 million cases worldwide. HEV is an emerging and endemic pathogen within economically developed countries, chiefly resulting from infections with genotype 3 (G3) HEV. G3 HEV is known to be a zoonotic pathogen, with a broad host range. The primary source of HEV within more economically developed countries is considered to be pigs, and consumption of pork products is a significant risk factor and known transmission route for the virus to humans. However, other foods have also been implicated in the transmission of HEV to humans. This review consolidates the information available regarding transmission of HEV and looks to identify gaps where further research is required to better understand how HEV is transmitted to humans through food.

Advances in Hepatitis E Virus Biology and Pathogenesis.

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.

Ectopic Expression of Genotype 1 Hepatitis E Virus ORF4 Increases Genotype 3 HEV Viral Replication in Cell Culture.

Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.

Autoimmune liver disease-associated serologic profiling in Chinese patients with acute hepatitis E virus infection.

The association between hepatitis E virus (HEV) and autoimmune liver diseases has been well-researched; however, the focus has been on autoimmune hepatitis (AIH) and not primary biliary cholangitis (PBC). Therefore, we aimed to investigate the prevalence and evolution of AIH- and PBC-related autoantibodies in Chinese patients with HEV infection. In this retrospective study, 164 patients with acute HEV were included, specifically those whose liver autoantibody results were available and who had no pre-existing liver disease at the time of HEV diagnosis. Positive liver autoimmune serology was present in 69 (42.1%) patients and 21 (12.8%) had at least two autoantibodies at diagnosis. Greater age and alkaline phosphatase levels were independent risk factors for autoantibody positivity. Follow-up serologic tests, which were available for 27 of the 69 autoantibody-positive patients, showed that although antinuclear antibodies disappeared in 11/20 (55.0%) and antimitochondrial antibodies disappeared in 4/5 (80%) patients, 16 still remained positive for autoantibodies and two of them even developed new PBC-related antibodies, as described below. One patient developed a rim-like ANA pattern, accompanied by an enhancement of anti-gp210 positivity; and the other was diagnosed as PBC, based on chronic elevation of cholestatic enzymes and presentation with de novo AMA-M2, 18 months after HEV clearance. In conclusion, AIH- and PBC-related autoantibodies are frequently present during acute HEV infection, indicating that HEV should be excluded before diagnosing AIH and/or PBC. Importantly, some cases maintained or developed autoantibodies after viral clearance, and one patient subsequently developed PBC, highlighting that these individuals warrant long-term follow-up.