Hepatitis E Virus Pathogenesis Research Articles

Hepatitis E virus: from the infected organism to the cellular response

HepatitisE virus (HEV) presents a worldwide distribution. In developing countries, hepatitisE, related to HEV1and HEV2, is a waterborne disease. In developed countries, hepatitisE is a zoonotic disease due to HEV3and HEV4. It is mainly transmitted through meat consumption from animal reservoirs such as pig, boar, deer and rabbit. New clinical forms include neurological manifestations that are now clearly associated with HEV3 infection. Recent studies showed that ORF1polyprotein was able to disrupt the innate immune response. It was also shown that ORF2protein exists at least in two forms: a free, glycosylated form and a non-glycosylated form, which assembles to form the capsid. Lastly, it was shown that ORF3protein, involved in the virus egress, acts as a viroporin. New culture systems and animal models have been developed recently, and will be very helpful to complete our understanding of HEV life cycle and pathogenesis.

Cross-species infection of mice by rabbit hepatitis E virus

Rabbits are recognized as a zoonotic reservoir of hepatitis E virus (HEV) for transmission to humans and other zoonotic reservoirs such as swine. The purpose of this study was to assess the ability of rabbit HEV to cross the species barrier to infect mice and also the usefulness of this animal to study HEV transmission and pathogenesis. In this study, uninfected BALB/c mice were experimentally inoculated with rabbit HEV either via gavage or through contact-exposure with infected mice. Rabbit HEV propagation in mice was evaluated by studying fecal virus shedding, viremia, seroconversion and microscopic liver lesions. Rabbit HEV could be detected in all mice infected by gavage, but only in some contact-exposed mice, with some animals exhibiting fecal virus shedding, seroconversion or viremia (one mouse only). Compared with inoculated mice, anti-rabbit HEV antibody titers and viral copy numbers in fecal and serum samples were lower in contact-exposed mice. Infected mice mainly exhibited phlebitis, hepatocyte swelling and necrosis. Microscopic liver lesion scores for inoculated and contact-exposed infected mice were higher than scores for negative controls. This study therefore demonstrates that rabbit HEV could infect BALB/c mice both though inoculation via gavage and through contact-exposure.

Hepatitis E Virus Genome Structure and Replication Strategy.

Hepatitis E virus (HEV) possesses many of the features of other positive-stranded RNA viruses but also adds HEV-specific nuances, making its virus-host interactions unique. Slow virus replication kinetics and fastidious growth conditions, coupled with the historical lack of an efficient cell culture system to propagate the virus, have left many gaps in our understanding of its structure and replication cycle. Recent advances in culturing selected strains of HEV and resolving the 3D structure of the viral capsid are filling in knowledge gaps, but HEV remains an extremely understudied pathogen. Many steps in the HEV life cycle and many aspects of HEV pathogenesis remain unknown, such as the host and viral factors that determine cross-species infection, the HEV-specific receptor(s) on host cells, what determines HEV chronicity and the ability to replicate in extrahepatic sites, and what regulates processing of the open reading frame 1 (ORF1) nonstructural polyprotein.

Increased oestradiol in hepatitis E virus-infected pregnant women promotes viral replication.

Hepatitis E virus (HEV) infection causes subclinical diseases, leading to high mortality (>25%) in pregnant women. HEV replication is aggressively escalated in pregnant women, especially in the third trimester of pregnancy. Oestrogen plays an important role in pregnancy. However, the pathogenesis of HEV in pregnant women or immunosuppressive pregnant women (such as HIV-infected or organ-transplanted pregnant women) remains unclear. We investigated the role of oestradiol in HEV infection in a cell culture system. HEV-infected pregnant women had significantly higher oestradiol levels compared with uninfected individuals. HEV infection was significantly increased in cells treated with analogues of oestradiol, diethylstilbestrol (DES) or 17β-oestradiol in a dose-dependent way. However, tamoxifen, an antagonist oestrogen, inhibited HEV replication. HEV infection inhibits oestrogen receptor (ER-α) expression. Immunofluorescence and co-immunoprecipitation assays indicated that ER-α interacted with the helicase of HEV ORF1 indirectly. More importantly, HEV infection was exacerbated in immunosuppressive cells treated with an inhibitor of PI3K-AKT-mTOR signal pathway (LY296004) and supplemented with pregnant women serum with high oestradiol simultaneously. These results strongly suggest that pregnant women with high oestradiol and/or immunosuppression will be vulnerable to HEV infection.

Zoonotic Hepatitis E Virus: An Ignored Risk for Public Health.

Hepatitis E virus (HEV) is a quasi-enveloped, single-stranded positive-sense RNA virus. HEV belongs to the family Hepeviridae, a family comprised of highly diverse viruses originating from various species. Since confirmation of HEV’s zoonosis, HEV-induced hepatitis has been a public health concern both for developing and developed countries. Meanwhile, the demonstration of a broad host range for zoonotic HEV suggests the existence of a variety of transmission routes that could lead to human infection. Moreover, anti-HEV antibody serosurveillance worldwide demonstrates a higher than expected HEV prevalence rate that conflicts with the rarity and sporadic nature of reported acute hepatitis E cases. In recent years, chronic HEV infection, HEV-related acute hepatic failure, and extrahepatic manifestations caused by HEV infection have been frequently reported. These observations suggest a significant underestimation of the number and complexity of transmission routes previously predicted to cause HEV-related disease, with special emphasis on zoonotic HEV as a public health concern. Significant research has revealed details regarding the virology and infectivity of zoonotic HEV in both humans and animals. In this review, the discovery of HEV zoonosis, recent progress in our understanding of the zoonotic HEV host range, and classification of diverse HEV or HEV-like isolates from various hosts are reviewed in a historic context. Ultimately, this review focuses on current understanding of viral pathogenesis and cross-species transmission of zoonotic HEV. Moreover, host factors and viral determinants influencing HEV host tropism are discussed to provide new insights into HEV transmission and prevalence mechanisms.

Prevalence and genetic features of rabbit hepatitis E virus in Korea.

Hepatitis caused by hepatitis E virus (HEV) is a public health concern worldwide. HEV strains have been isolated from several animal species, some of which induce zoonosis. Recently, the isolation of HEV from rabbits was reported. Here, the partial capsid gene (320 bp) of HEV was detected in rabbit feces via reverse transcriptase-polymerase chain reaction (RT-PCR). Rabbit HEV was found in two of six rabbit farms and 17 of 264 rabbit fecal samples (6.4%). A phylogenetic analysis of the partial capsid gene classified the 17 HEV isolates into the putative rabbit HEV clade. A full genomic sequence, KOR-Rb-1, was obtained from one rabbit HEV isolate by 5' and 3' rapid amplification of cDNA ends-PCR and RT-PCR, and comprised 7275 bp excluding the 3' poly(A) tail. It shared 77.5-86.8%, 86.6%, and 80.2-84.3% nucleotide identities with rabbit HEV isolates from China, the US, and France, respectively. It also shared 72.3-73.0%, 71.4%, 76.7-78.3%, 72.8-73.3%, and 47.1-47.2% nucleotide identities with representative strains of HEV-1, HEV-2, HEV-3, HEV-4, and avian HEV, respectively. A full-genome phylogenetic analysis classified KOR-Rb-1 into the provisional rabbit HEV clade. This isolate could be used to study the pathogenesis and zoonotic potential of rabbit HEV.

Systematic identification of hepatitis E virus ORF2 interactome reveals that TMEM134 engages in ORF2-mediated NF-κB pathway

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Open reading frame 2 (ORF2) encodes the capsid protein of HEV, which is the main structural protein and may participate, together with the host factors, in viral entry and egress. However, it is still not clear which host proteins are involved in the interaction with ORF2 and what the functions of these ORF2-interacting proteins are. In this study, we have applied a split-ubiquitin yeast two-hybrid screening approach in combination with the pull-down and coimmunoprecipitation assays, identified and validated multiple interacting partners of ORF2 of genotype 1 and 4, which have diverse biological functions. Among these novel candidates that have not been previously reported, we have found that 20 of them are located in endoplasmic reticulum. TMEM134, which interacts and co-localizes with ORF2 in the endoplasmic reticulum, negatively regulates ORF2-mediated inhibition of the NF-κB signaling pathway. Our study for the first time has systematically mapped the ORF2 interactome in two genotypes of HEV, providing a new insight of understanding the virus-host interaction during the pathogenesis of HEV, and may offer potential therapeutic targets to intervene the HEV life cycle.

Molecular Biology and Infection of Hepatitis E Virus

Hepatitis E virus (HEV) is a viral pathogen transmitted primarily via fecal-oral route. In humans, HEV mainly causes acute hepatitis and is responsible for large outbreaks of hepatitis across the world. The case fatality rate of HEV-induced hepatitis ranges from 0.5 to 3% in young adults and up to 30% in infected pregnant women. HEV strains infecting humans are classified into four genotypes. HEV strains from genotypes 3 and 4 are zoonotic, whereas those from genotypes 1 and 2 have no known animal reservoirs. Recently, notable progress has been accomplished for better understanding of HEV biology and infection, such as chronic HEV infection, in vitro cell culture system, quasi-enveloped HEV virions, functions of the HEV proteins, mechanism of HEV antagonizing host innate immunity, HEV pathogenesis and vaccine development. However, further investigation on the cross-species HEV infection, host tropism, vaccine efficacy, and HEV-specific antiviral strategy is still needed. This review mainly focuses on molecular biology and infection of HEV and offers perspective new insight of this enigmatic virus.

A novel porcine cell culture based protocol for the propagation of hepatitis E virus

ObjectiveTo present a comprehensive protocol for the processing of hepatitis E virus (HEV) infected samples and propagation of the virus in primary cell cultures. MethodsHepatitis E was extracted from porcine liver and faecal samples following standard protocols. The virus was then allowed to attach in the presence of trypsin to primary cells that included porcine and bovine intestinal epithelial cells and macrophages over a period of up to 3 h. The virus was propagated by rotational passaging through the cell cultures. Propagation was confirmed by immunoblotting. ResultsWe developed a comprehensive protocol to propagate HEV in porcine cell model that includes (i) rotational culturing of the virus between porcine cell types, (ii) pre-incubation of infected cells for 210 min, (iii) use of a semi-complete cell culture medium supplemented with trypsin (0.33 µg/mL) and (iv) the use of simple immunoblot technique to detect the amplified virus based on the open reading frame 2/3. ConclusionsThis protocol opens doors towards systematic analysis of the mechanisms that underlie the pathogenesis of HEV in vitro. Using our protocol, one can complete the propagation process within 6 to 9 d.

The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1.

Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity.

Serum Levels of Soluble CD26, A Novel Prognostic Marker for Hepatitis E Infection.

Background:Even without treatment, most acute hepatitis E virus (HEV) infected patients resolve HEV but sometimes the disease leads to acute liver failure, chronic infection, or extrahepatic symptoms. The mechanisms of HEV pathogenesis appear to be substantially immune mediated. However, the immune responses to HEV are not precisely identified.Objectives:This study aimed to evaluate the Th1/Th2 ratio by investigating serum soluble markers from Th1 and Th2 cells in acute HEV infected patients.Patients and Methods:This case-control study included 35 acute HEV infected patients and 35 age and gender matched anti-HEV negative healthy controls. The serum levels of Interferon (IFN)-γ, IL-4, soluble CD26 (sCD26) and sCD30 were determined by the enzyme-linked immunosorbent assay.Results:The results showed a significant difference in IFN-γ and sCD26 (P < 0.0001 and P = 0.001) yet not IL-4 and sCD30 (P = 0.354 and P = 0.159) between acute HEV patients and controls, respectively. There was a positive direct correlation between serum levels of sCD26 and IFN-γ in acute HEV patients (r = 0.64, P = 0.001). In addition, the ratio of sCD26/sCD30 in the acute HEV group was more than two folds higher than in the HEV negative controls.Conclusions:Acute HEV infection shows a pattern of Th1-type immune response, and the direct significant positive correlation between the serum level of sCD26 and IFN-γ in acute HEV infected patients, suggests that the trend of sCD26 levels is a valuable marker for predicting hepatic inflammation in hepatitis E.

Identification and fine mapping of nuclear and nucleolar localization signals within the human ribosomal protein S17.

Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.

The lysine residues within the human ribosomal protein S17 sequence naturally inserted into the viral nonstructural protein of a unique strain of hepatitis E virus are important for enhanced virus replication.

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. HEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the hypervariable region (HVR) within the HEV genome, allowing for cell culture adaptation and expansion of the host range, have been reported. We utilized these cell culture-adapted HEV strains to assess how the HVR may be involved in virus replication and host range. We provide evidence that insertion of the RPS17 sequence in HEV likely confers nuclear trafficking capabilities to the nonstructural protein of the virus and that lysine residues within the RPS17 insertion are important for enhanced replication of the virus. These data will help to elucidate the mechanism of cross-species infection of HEV in the future.

Toll like receptors in self-recovering hepatitis E patients with or without pregnancy

Hepatitis E virus (HEV) causes high mortality among pregnant women. Pathogenesis of HEV, especially during pregnancy, is poorly understood. Our aim was to assess the role of Toll-like-receptors (TLRs) in hepatitis E patients with pregnancy (Antenatal care, ANC) or without pregnancy (non-ANC). The patient categories included acute-phase, non-ANC (n=46) and ANC patients (2nd/3rd trimesters, n=13) and non-ANC patients (n=31) during convalescence. Controls included apparently healthy non-ANC (n=30) and ANC subjects in the first (n=10) and later (2nd/3rd, n=20) trimesters. TLR2/TLR3/TLR4/TLR7/TLR8 levels were determined by flow-cytometry. Cytokine responses induced by TLR-specific-ligands-stimulated-PBMCs from ANC/non-ANC-patients and TLR-signaling-molecules (non-ANC-patients) were measured. PBMCs were used to assess gene expression levels by TaqMan-Low-Density-Array.Compared to the temporal activation of TLR4/TLR7/TLR8 at protein and mRNA levels, the ANC-patients and controls exhibited reduced TLRs indicative of impaired TLR response. Stimulation of PBMCs with TLR-specific ligands led to the induction of type-I interferons, IFNβ by the non-ANC group and IFNα by the ANC category. Involvement of MyD88-independent (TLR3/TLR4) and MyD88-dependent (TLR4/TLR7/TLR8) pathways and association of TLR4/TLR7/TLR8 with recovery was documented in the non-ANC-patients. Except for robust type-I-interferon response, HEV infection could not modulate pregnancy-related diminished immune response. The results have implications in the understanding of HEV pathogenesis.

Evidence of MAPK-JNK1/2 activation by hepatitis E virus ORF3 protein in cultured hepatoma cells.

Hepatitis E virus (HEV) has recently emerged to cause chronic infection in some immunosuppressed individuals, including extrahepatic manifestations in acute and chronic patients. Mammalian MAPK–JNK1/2 is expressed in hepatocytes, which is known to be involved in anti-apoptotic signaling pathway for the establishment of persistent infection. Though in vitro modulation of cellular MAPK–ERK cascade by HEV-ORF3 protein is suggested to have a role in host pathobiology, activation of the JNK module has not been studied so far. In this report, we have shown for the first time, evidence of MAPK–JNK1/2 activation by HEV-ORF3, using viral replicon as well as expression vector in human hepatoma cells. Phospho-ELISA based relative quantitaion has demonstrated ~54% and ~66% phosphorylation of JNK1/2 in replicon-RNA and ORF3-vector DNA transfected cells, respectively. Our finding however, suggests further molecular studies to validate a role of JNK1/2 in HEV pathogenesis.

Hepatitis E virus inhibits type I interferon induction by ORF1 products.

Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-β) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. Importance: Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host IFNs, though the viral genome was sequenced more than 2 decades ago. This is the first report of identification of the potential IFN antagonists encoded by HEV. By screening all the domains in the open reading frame 1 (ORF1) polyprotein, we identified two IFN antagonists and performed further research to determine how and at which step in the IFN induction pathway they antagonize host IFN induction. Our work provides valuable information about HEV-cell interaction and pathogenesis.

Open reading frame 3 of genotype 1 hepatitis E virus inhibits nuclear factor-κappa B signaling induced by tumor necrosis factor-α in human A549 lung epithelial cells.

Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3) on TNF-α-induced nucleus factor-κappa B (NF-κB) signaling. Human lung epithelial cells (A549) were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-α and the nucleus translocation of the p65 NF-κB subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA), and the suppression of NF-κB target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKBα. We focused on the gene of negative regulation of NF-κB, represented by TNF-α-induced protein 3 (TNFAIP3, also known as A20). Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-κB. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6), involved in unfolded protein response (UPR), resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1), a major upstream activator of IKB kinase compounds (IKKs). Consequently, the phosphorylation of IKBα and the nucleus translocation of p65 are blocked, which contributes to diminished NF-κB DNA-binding activation and NF-κB-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-κB through UPR in early stage, and subsequently inhibit TNF-α-induced NF-κB signaling in late phase so as to create a favorable virus replication environment.

Detection and Localization of Rabbit Hepatitis E Virus and Antigen in Systemic Tissues from Experimentally Intraperitoneally Infected Rabbits

Rabbit hepatitis E virus (HEV) is a novel genotype of HEV, and is considered to pose a risk of zoonotic transmission. Research into the systemic distribution of rabbit HEV in rabbits during different periods of infection has rarely been reported. To better understand this virus, we infected rabbits with second-passage rabbit HEV via an intraperitoneal route. After inoculation, the infection showed two types, temporary and constant infection. The detection of HEV RNA in the feces varied with time, and serum antigen correlated with fecal HEV RNA. Viremia only appeared 72 days after inoculation. The rabbits remained antibody negative throughout the experimental period. When HEV was localized, several organs besides the liver were HEV RNA positive. Tissue antigen was observed immunohistochemically in the different cells of various organs, especially in parts of the small intestine and the characteristic rabbit gut-associated lymphoid tissue. These data provide valuable information for future research into the pathogenesis of HEV.

Hepatitis E Virus: Foodborne, Waterborne and Zoonotic Transmission

Hepatitis E virus (HEV) is responsible for epidemics and endemics of acute hepatitis in humans, mainly through waterborne, foodborne, and zoonotic transmission routes. HEV is a single-stranded, positive-sense RNA virus classified in the family Hepeviridae and encompasses four known Genotypes (1–4), at least two new putative genotypes of mammalian HEV, and one floating genus of avian HEV. Genotypes 1 and 2 HEVs only affect humans, while Genotypes 3 and 4 are zoonotic and responsible for sporadic and autochthonous infections in both humans and several other animal species worldwide. HEV has an ever-expanding host range and has been identified in numerous animal species. Swine serve as a reservoir species for HEV transmission to humans; however, it is likely that other animal species may also act as reservoirs. HEV poses an important public health concern with cases of the disease definitively linked to handling of infected pigs, consumption of raw and undercooked animal meats, and animal manure contamination of drinking or irrigation water. Infectious HEV has been identified in numerous sources of concern including animal feces, sewage water, inadequately-treated water, contaminated shellfish and produce, as well as animal meats. Many aspects of HEV pathogenesis, replication, and immunological responses remain unknown, as HEV is an extremely understudied but important human pathogen. This article reviews the current understanding of HEV transmission routes with emphasis on food and environmental sources and the prevalence of HEV in animal species with zoonotic potential in humans.

Cutthroat trout virus as a surrogate in vitro infection model for testing inhibitors of hepatitis E virus replication

Hepatitis E virus (HEV) is one of the most important causes of acute hepatitis worldwide. Although most infections are self-limiting, mortality is particularly high in pregnant women. Chronic infections can occur in transplant and other immune-compromised patients. Successful treatment of chronic hepatitis E has been reported with ribavirin and pegylated interferon-alpha, however severe side effects were observed. We employed the cutthroat trout virus (CTV), a non-pathogenic fish virus with remarkable similarities to HEV, as a potential surrogate for HEV and established an antiviral assay against this virus using the Chinook salmon embryo (CHSE-214) cell line. Ribavirin and the respective trout interferon were found to efficiently inhibit CTV replication. Other known broad-spectrum inhibitors of RNA virus replication such as the nucleoside analog 2′-C-methylcytidine resulted only in a moderate antiviral activity. In its natural fish host, CTV levels largely fluctuate during the reproductive cycle with the virus detected mainly during spawning. We wondered whether this aspect of CTV infection may serve as a surrogate model for the peculiar pathogenesis of HEV in pregnant women. To that end the effect of three sex steroids on in vitro CTV replication was evaluated. Whereas progesterone resulted in marked inhibition of virus replication, testosterone and 17β-estradiol stimulated viral growth. Our data thus indicate that CTV may serve as a surrogate model for HEV, both for antiviral experiments and studies on the replication biology of the Hepeviridae.