Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells.

Introduction: Hepatitis E virus (HEV) is a common cause of enterically transmitted acute hepatitis worldwide. The virus is transmitted by the fecal-oral route via the consumption of contaminated water supplies and is also a zoonotic foodborne pathogen. Swine are the main reservoir of zoonotic HEV. In humans, HEV infection is usually asymptomatic or causes acute hepatitis that is self-limited. However, fulminant hepatic failure and chronic cases of HEV infection can occur in some patients. In contrast, HEV infection in pigs remains asymptomatic, although the virus replicates efficiently, suggesting that swine are able to control the virus pathogenesis. Upon viral infection, IFN is secreted and activates cellular pathways leading to the expression of many IFN-stimulated genes (ISGs). ISGs can restrict the replication of specific viruses and establish an antiviral state within infected and neighboring cells. Methods: In this study, we used PCR arrays to determine the expression level of up to 168 ISGs and other IFN-related genes in the liver tissues of pigs infected with zoonotic HEV-3c and HEV-3f and in human bipotent liver HepaRG cells persistently infected with HEV-3f. Results and discussion: The expression of 12 and 25 ISGs was found to be up-regulated in infected swine livers and HepaRG cells, respectively. The expression of CXCL10, IFIT2, MX2, OASL and OAS2 was up-regulated in both species. Increased expression of IFI16 mRNA was also found in swine liver tissues. This study contributes to the identification of potential ISGs that could play a role in the control or persistence of HEV infection.

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

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

Micro RNAs-The Small Big Players in Hepatitis E Virus Infection: A Comprehensive Review.

The molecular mechanism of hepatitis E virus (HEV) pathology is still unclear. The micro RNAs (miRNAs), of host or viral origin, interfere with virus replication and host environment in order to create an appropriate condition for the production of mature HEV progeny. Understanding the biogenesis and the interference of miRNAs with HEV will help to revile the mechanism of viral pathogenesis.

Two mutations in the ORF1 of genotype 1 hepatitis E virus enhance virus replication and may associate with fulminant hepatic failure.

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

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Characterization of highly expressed novel hub genes in hepatitis E virus chronicity in rabbits: a bioinformatics and experimental analysis.

BACKGROUND: Hepatitis E virus (HEV), which is the leading cause of acute viral hepatitis worldwide, usually causes self-limited infections in common individuals. However, it can lead to chronic infection in immunocompromised individuals and its mechanisms remain unclear. Rabbits are the natural host of HEV, and chronic HEV infections have been observed in rabbits. Therefore, we aimed to investigate potential key genes in HEV chronicity process in rabbits. In this study, both bioinformatics and experimental analysis were performed to deepen the understanding of hub genes in HEV chronic infection in rabbits. RESULTS: Ninety-four candidate differentially expressed genes (DEGs) and the pathways they enriched were identified to be related with HEV chronicity. A total of 10 hub genes were found by protein-protein interaction (PPI) network construction. Rabbits of group P (n = 4) which showed symptoms of chronic HEV infection were selected to be compared with HEV negative rabbits (group N, n = 6). By detecting the identified hub genes in groups P and N by real-time PCR, we found that the expressions of MX1, OAS2 and IFI44 were significantly higher in group P (P < 0.05). CONCLUSIONS: In this work, we presented that MX1, OAS2 and IFI44 were significantly upregulated in HEV chronic infected rabbits, indicating that they may be involved in the pathogenesis of HEV chronicity.

Advanced sequencing approaches detected insertions of viral and human origin in the viral genome of chronic hepatitis E virus patients.

The awareness of hepatitis E virus (HEV) increased significantly in the last decade due to its unexpectedly high prevalence in high-income countries. There, infections with HEV-genotype 3 (HEV-3) are predominant which can progress to chronicity in immunocompromised individuals. Persistent infection and antiviral therapy can select HEV-3 variants; however, the spectrum and occurrence of HEV-3 variants is underreported. To gain in-depth insights into the viral population and to perform detailed characterization of viral genomes, we used a new approach combining long-range PCR with next-generation and third-generation sequencing which allowed near full-length sequencing of HEV-3 genomes. Furthermore, we developed a targeted ultra-deep sequencing approach to assess the dynamics of clinically relevant mutations in the RdRp-region and to detect insertions in the HVR-domain in the HEV genomes. Using this new approach, we not only identified several insertions of human (AHNAK, RPL18) and viral origin (RdRp-derived) in the HVR-region isolated from an exemplary sample but detected a variant containing two different insertions simultaneously (AHNAK- and RdRp-derived). This finding is the first HEV-variant recognized as such showing various insertions in the HVR-domain. Thus, this molecular approach will add incrementally to our current knowledge of the HEV-genome organization and pathogenesis in chronic hepatitis E.

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.

A tightly clustered hepatitis E virus genotype 1a is associated with endemic and outbreak infections in Bangladesh.

BACKGROUND: Hepatitis E virus (HEV) infection is endemic in Bangladesh and there are occasional outbreaks. The molecular characteristics and pathogenesis of endemic and outbreak HEV strains are poorly understood. We compared the genetic relatedness and virulence associated mutations of endemic HEV strains with outbreak strains. METHODS: We analyzed systematically collected serum samples from HEV immunoglobulin M (IgM) positive patients attended at Bangabandhu Sheikh Mujib Medical University, Dhaka from August 2013 to June 2015. HEV RNA positive samples were subjected to whole genome sequencing. Genotype and subtype of the strains were determined by phylogenetic analysis. Virulence associated mutations e.g. acute viral hepatitis (AVH), fulminant hepatic failure (FHF), chronic hepatitis, ribavirin treatment failure (RTF), B and T cell neutralization epitopes were determined. RESULTS: 92 HEV immunoglobulin M (IgM) antibody positive plasma samples (43 in 2013-2014 and 49 in 2014-2015) were studied. 77.1% (70/92) of the samples were HEV RNA positive. A 279 bp open reading frame (ORF) 2 and ORF 3 sequence was obtained from 54.2% (38/70) of the strains. Of these 38 strains, whole genome sequence (WGS) was obtained from 21 strains. In phylogenetic analysis of 38 (279 bp) sequence all HEV sequences belonged to genotype 1 and subtype 1a. Further phylogenetic analysis of 21 HEV WGS, Bangladeshi HEV sequences clustered with genotype 1a sequences from neighboring countries. Within genotype 1a cluster, Bangladesh HEV strains formed a separate cluster with the 2010 HEV outbreak strains from northern Bangladesh. 80.9 to 100% of the strains had A317T, T735I, L1120I, L1110F, P259S, V1479I, G1634K mutations associates AVH, FHF and RTF. Mutations in T cell recognition epitope T3, T5, T7 was observed in 76.1%, 100% and 100% of the strains respectively. CONCLUSION: Strains of HEV genotype 1a are dominant in Bangladesh and are associated with endemic and outbreak of HEV infection. HEV isolates in Bangladesh have high prevalence of virulence associated mutations and mutation which alters antigenicity to B and T cell epitopes.

A surveillance study of hepatitis E virus infection in household cats.

Hepatitis E virus (HEV) typically causes self-limiting acute viral hepatitis, however chronic infection and extrahepatic manifestations have increasingly become a significant health problem. Domestic pigs and wild boars are the main reservoirs of HEV genotype 3 and genotype 4 for human infections in industrialized countries, although molecular and serological evidence suggest that several additional animal species may act as HEV hosts. In this study, by assessing serologically and molecularly the sera of 324 household cats from Apulia region (Italy), HEV antibodies were detected with an overall prevalence of 3.1%. Viral RNA was not detected in the sera of the animals using both HEV-specific assays and a pan-hepevirus broadly reactive set of primers for Hepeviridae. These findings document a low seroprevalence to HEV in cats in the investigated geographical setting. The exact nature of the HEV-like strains circulating in feline population remains to be established.

Expression Profiles of Exosomal MicroRNAs from HEV- and HCV-Infected Blood Donors and Patients: A Pilot Study.

Exosomes seem to play an important role in hepatits C virus (HCV) and hepatitis E virus (HEV) infection by shielding their cargo from the host immune responses, with microRNAs being key exosomal components. Little is known about their involvement in a mixed HCV/HEV infection or at the early stages of infection, such as in asymptomatic blood donors (BDs). To obtain preliminary data, we have compared the exosomal microRNA expression profiles in four each of HCV RNA-positive, HEV RNA-positive and negative blood donors and four patients, one of whom was a rare patient with HCV/HEV co-infection. Exosomes were purified from sera by a combination of a precipitation and density gradient centrifugation and exosomal microRNA was analysed using Taqman array cards. Out of 33 deregulated miRNAs, miR-885-5p and miR-365 were upregulated in HCV BDs, miR-627-5p was downregulated in HCV BD and miR-221 was downregulated in HCV patients and BDs. In HEV infection, miR-526b appeared specifically downregulated. Six miRNAs (miR-628-3p, miR-194, miR-151-3p, miR-512-3p, miR-335 and miR-590) indicated a potential involvement in both infections. First time preliminary data on pre- and post-antiviral treatment exosomal microRNA profiles of the HEV/HCV co-infected patient revealed a pool of 77 upregulated and 43 downregulated miRNAs to be further investigated for their potential roles in these viral infections.

Uncovering the Roles of miR-214 in Hepatitis E Virus Replication.

Viral pathogenesis is a complex event and its regulation involve dynamic interactions with various host factors, of which microRNAs are the key players. In the current study, we have identified the functional importance of an interplay between hepatitis E virus (HEV) and miR-214. Computational analysis indicated that miR-214 binding site is significantly conserved among HEV and related RNA viruses. Intact miR-214 binding site is imperative for HEV replication. miR-214 is an essential host factor for HEV replication. Herein, we demonstrate that miR-214 interacts directly with HEV RNA to enhance HEV replication and HEV genome translation. Augmented translation results in increased levels of HEV ORF2, which is a factor responsible for upregulation of miR-214. HEV usurps host cellular machinery for improving viral fitness and elevates miR-214 expression for amplifying the expression of proviral host factor intracellular active thrombin. This is because miR-214 represses the expression of the negative regulator of thrombin, i.e., protein C. Another viral factor, HEV ORF3, also contributes to the enhancement of intracellular active thrombin. Furthermore, miR-214 directly targets antiviral host factor 2'-5'-oligoadenylate synthetase. Conclusively, we identified a novel mechanism of positive regulation of HEV replication. miR-214 interacts directly with HEV genome and fine-tunes host factors expression. This results in outweighing the proviral factors on the proviral-antiviral axis probably for generating virus supportive environment.

Clinical significance of post-liver transplant hepatitis E seropositivity in high prevalence area of hepatitis E genotype 3: a prospective study.

High hepatitis E (HEV) seroprevalence has been reported in the general population and in post-liver transplant (LT) cases in several regions, including Thailand, with genotype 3 being a predominant genotype. We hypothesized that HEV might persist at a subclinical level and might pose clinical risks in the post-LT period. We performed a cross-sectional study with 108 post-LT patients and found an IgG seroprevalence of 55.6%. Subsequently, 91 cases without clinical evidence of HEV-related hepatitis were enrolled in 1 year of prospective follow-up to determine clinical status, serologies and serum/feces HEV RNA every 4 months. HEV RNA was detected, indicating subclinical infections in patients with or without seropositivity, with an annual incidence of 7.7%. Our results suggest that subclinical HEV infection exists among LT patients in this high-prevalence area. Thus, clinicians should be aware of the possibility of disease reemergence and HEV viral transmission in LT patients.

A simplified qPCR method revealing tRNAome remodeling upon infection by genotype 3 hepatitis E virus.

The landscape of tRNA-viral codons regulates viral adaption at the translational level, presumably through adapting to host codon usage or modulating the host tRNAome. We found that the major zoonotic genotype of hepatitis E virus (HEV) has not adapted to host codon usage, prompting exploration of the effects of HEV infection on the host tRNAome. However, tRNAome quantification is largely impeded by the extremely short sequences of tRNAs and redundancy of tRNA genes. Here, we present a length-extension and stepwise simplified qPCR method that utilizes a universal DNA/RNA hybrid tRNA adaptor and degenerate primers. Using this novel methodology, we observe that HEV infection dramatically reprograms the hepatic tRNAome, which is likely to facilitate translation of viral RNAs. This tRNAome quantification method bears broad implications for future tRNA research and possibly tRNA-based diagnostics.

Specific circulating microRNAs during hepatitis E infection can serve as indicator for chronic hepatitis E.

Hepatitis E virus (HEV) genotypes 3 and 4 (HEV-3, HEV-4) infections are an emerging public health issue in industrialized countries. HEV-3 and -4 are usually self-limiting but can progress to chronic hepatitis E in immunocompromised individuals. The molecular mechanisms involved in persistent infections are poorly understood. Micro RNAs (miRNAs) can regulate viral pathogenesis and can serve as novel disease biomarkers. We aimed to explore the modulation of serum miRNAs in patients with acute (AHE) and chronic (CHE) hepatitis E. Both AHE- and CHE-patients exhibited high viral loads (median 3.23E + 05 IU/mL and 2.11E + 06 IU/mL, respectively) with HEV-3c being the predominant HEV-genotype. Expression analysis of liver-specific serum miRNAs was performed using real-time PCR. miR-99a-5p, miR-122-5p, and miR-125b-5p were upregulated in AHE (4.70-5.28 fold) and CHE patients (2.28-6.34 fold), compared to HEV-negative controls. Notably, miR-192-5p was increased 2.57 fold while miR-125b-5p was decreased 0.35 fold in CHE but not in AHE patients. Furthermore, decreased miR-122-5p expression significantly correlates with reduced liver transaminases in CHE patients. To our knowledge, this marks the first investigation concerning the regulation of circulating liver-specific miRNAs in acute and chronic HEV infections. We found that miR-125b-5p, miR-192-5p, and miR-99a-5p may prove useful in the diagnosis of chronic hepatitis E.

The expression patterns of immune response genes in the Peripheral Blood Mononuclear cells of pregnant women presenting with subclinical or clinical HEV infection are different and trimester-dependent: A whole transcriptome analysis.

Hepatitis E is an enteric disease highly prevalent in the developing countries. The basis for high mortality among pregnant hepatitis E patients remains unclear. Importantly, a large proportion of infected pregnant women present with subclinical infection as well. In order to understand the possible mechanisms influencing clinical presentation of hepatitis E in pregnant women, we explored a system biology approach. For this, PBMCs from various categories were subjected to RNAseq analysis. These included non-pregnant (NPR, acute and convalescent phases) and pregnant (PR, 2nd and 3rd trimesters, acute phase and subclinical HEV infections) patients and corresponding healthy controls. The current study deals with immune response genes. In contrast to exclusive up-regulation of nonspecific, early immune response transcripts in the NPR patients, the PR patients exhibited broader and heightened expression of genes associated with innate as well as adaptive T and B cell responses. The study identified for the first time (1) inverse relationship of immunoglobulin (Ig) genes overexpression and (2) association of differential expression of S100 series genes with disease presentation. The data suggests possible involvement of TLR4 and NOD1 in pregnant patients and alpha defensins in all patient categories suggesting a role in protection. Induction of IFNγ gene was not detected during the acute phase irrespective of pregnancy. Association of response to vitamin D, transcripts related to NK/NKT and regulatory T cells during subclinical infection are noteworthy. The data obtained here could be correlated with several studies reported earlier in hepatitis E patients suggesting utility of PBMCs as an alternate specimen. The extensive, informative data provided here for the first time should form basis for future studies that will help in understanding pathogenesis of fulminant hepatitis E.

IFN-λ3 as a host immune response in acute hepatitis E virus infection.

BACKGROUND AND AIM: Hepatitis E virus (HEV) is mainly transmitted orally, either waterborne or zoonotic foodborne. Intestinal viruses such as rotavirus are known to induce type III interferon (IFN) in the gastrointestinal (GI) tract where type III IFN dominantly functions in comparison with type I IFN. Therefore, the aim of this study is to investigate the significance of type III IFN (IFN-λ3) in acute hepatitis E. METHODS: IFN-λ3 and HEV RNA levels in the sera of patients with acute HEV infection and in the supernatant of HEV-inoculated cells were measured, using an in-house high-sensitivity method and reverse transcription-polymerase chain reaction, respectively. RESULTS: High serum IFN-λ3 levels were found in the early phase of acute HEV infection, which normalized after resolution. Interestingly, serum IFN-λ3 levels correlated well with serum HEV RNA titers in the same sera, both of which showed the peak before the robust increase of transaminases. In vitro experiments demonstrated that HEV replicated well in the cells with little IFN-λ3 induction (Caco-2, A549) and recombinant IFN-λ3 inhibited HEV replication in a dose-dependent manner. In contrast, in HT-29 cells, a colon cancer cell line, HEV poorly replicated and induced IFN-λ3 in a titer-dependent manner. CONCLUSIONS: These clinical and experimental observations suggest that HEV induced IFN-λ3 as a host innate immune response, which may play a protective role against HEV.