Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with Hepatitis E virus (HEV) is established as an etiological agent of the epidemic and sporadic forms of waterborne hepatitis (6, 25). The first well-characterized HEV epidemic was reported in Delhi, India, in 1955 (35). Several other epidemics have since been described in developing countries on nearly every continent (19,21). HEV has a positive-strand polyadenylated RNA genome ϳ7.2 kb in length (27) containing three open reading frames (ORFs). Nonstructural ORF1 (5Ј end) codes for a polyprotein of 1,693 amino acids (pORF1) and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicase, and an RNAdependent RNA polymerase (RdRp). The second ORF (3Ј end) codes for the major viral capsid protein of 660 amino acid (pORF2), while the third and smallest ORF (ORF3) codes for a 123-amino-acid-long polypeptide (pORF3) whose function is unknown (32). Apart from its coding region, the viral genome has 27-and 68-nucleotide (nt)-long noncoding regions at its 5Ј and 3Ј ends, respectively (32). The genome sequences of HEV have been reported from different geographical isolates and show a high degree of homology at both the nucleotide and amino acid levels (3,4,12,32,33). Expression of structural proteins pORF2 and pORF3 in prokaryotic and eukaryotic systems has been reported by different investigators (7,11,14,23,29). Earlier, we have expressed and characterized pORF2 and pORF3 in animal cells. pORF2 is an 88-kDa glycoprotein which is expressed intracellularly, as well as on the cell surface (14, 37). The ORF3 protein (pORF3) is a 13.5-kDa phosphoprotein which is phosphorylated by the cellular mitogen-activated protein kinase and associates with the cytoskeleton (36). We have also recently expressed the ORF1 polyprotein in both prokaryotic and eukaryotic systems (2).In the absence of a reliable in vitro culture system...

Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world. Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels. The approximately 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein. The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far. In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein. The nonstructural polyprotein of HEV was expressed as a 186 kDa protein. No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation.

We investigated the virus-host interaction for hepatitis E virus (HEV) by performing competitive binding assays using in vitro assembled virus-like particles (VLPs). We used Escherichia coli expressed native capsid protein (pORF2) and its mutants with an attached Gly((5))-Ala (linker) reporter [enhanced green fluorescent protein (EGFP)/firefly luciferase (Fluc)]. Transmission electron microscopy and nanoparticle tracking showed near uniform particles of approximately 30-35 nm in diameter for pORF2 VLPs and 60-100 nm for reporter-linked VLPs. Binding of reporter-linked full-length (1-660aa) and N-terminal truncated (Δ1-112aa) pORF2 VLPs to Huh7 cell surfaces was found to be specific with 1.92 ± 0.065 × 10(5) sites per cell. Saturation binding indicated an equilibrium dissociation constant (K(d)) of 121.1 ± 23.83 and 123.8 ± 16.15 nm for pORF2-linker-EGFP and pORF2-linker-Fluc VLPs respectively. A similar binding pattern was observed for Δ1-112aa pORF2-linker-EGFP and Δ1-112aa pORF2-linker-Fluc VLPs with K(d) values of 123.6 ± 10.60 and 135.6 ± 16.19 nm respectively. The affinity (log K(i)) of pORF2 binding on Huh7 cells in the presence of EGFP-tagged and Fluc-tagged pORF2 VLPs was found to be approximately 2.0. However, no VLP formation or binding was observed with refolded C-terminal truncated (Δ458-660aa) pORF2. We investigated HEV internalization using fluorescent VLPs (EGFP-VLPs), which showed vesicle-mediated uptake starting at 5 min post-incubation. The uptake of VLPs could be stopped by inhibitors for clathrin-dependent endocytosis, but not by caveosome inhibitors. No binding and uptake of EGFP-VLPs were observed on non-hepatic cell lines (HeLa and SiHa). These findings suggest that HEV attaches to the host cell via a specific high affinity receptor and enters the cytoplasm by clathrin-mediated endocytosis.

We studied epidemics of viral hepatitis occurring at three different places in India. One was a combined epidemic due to hepatitis E virus (HEV) and hepatitis A virus (HAV) infections. In this epidemic, HAV affected children below 10 years of age, whereas HEV infected the young adult population. HEV was transmitted to rhesus monkeys (Macaca mulata) and confirmed by the polymerase chain reaction (PCR) on bile from the animals. Fecal material from acutely infected patients in one of the epidemics was also found positive for HEV RNA by PCR. This may help in confirming the nature of future epidemics. The bile and liver from experimental animals can be used as a source of material for further virological and molecular biological studies of HEV.

Hepatitis E virus infection (HEV) is a major cause of acute viral hepatitis in the developing world. The immunopathology of HEV infections has not yet been elucidated. The virus is noncytopathic, and therefore, liver injury may be attributed to immune-mediated damage by cytotoxic T cells and natural killer cells. Therefore, we studied the nature of immune cells involved in HEV-induced liver damage using immunohistochemistry in liver biopsies taken from patients with HEV-induced acute liver failure and demonstrated a significant infiltration of activated CD8(+) T cells containing granzymes. These findings suggest the possible involvement of cytotoxic T cells in disease pathogenesis during HEV infection.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a nonenveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440-610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the~35 kDa methyltransferase,~35 kDa replicase and 56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.

Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of approximately 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.

Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV.