The processing of polyprotein(s) to form structural and non-structural components remains an enigma due to the non-existence of an efficient and robust Hepatitis E Virus (HEV) culture system. We used the BacMam approach to construct an HEV replication model in which the HEV genome was cloned in the BacMam vector under the CMV promoter. The recombinant BacMam was used to infect Huh7 cells to transfer the HEV genome. HEV replication was authenticated by the presence of RNAs of both the polarity (+) and (-) and formation of hybrid RNA, a replication intermediate. The presence of genes for Papain-like Cysteine Protease (PCP), methyltransferase (MeT), RNA dependent RNA polymerase (RdRp), and ORF2 was confirmed by PCR amplification. Further, the infectious nature of the culture system was established as evidenced by the cross-infection of uninfected cells using the cell lysate from the infected cells. The HEV replication model was validated by detection of the ORF1 (Open Reading Frame1) encoded proteins, identified by Western blotting and Immunofluorescence by using epitope-specific antibodies against each protein. Consequently, discrete bands of 18, 35, 37, and 56 kDa corresponding to PCP, MeT, RdRp, and ORF2, respectively, were seen. Besides demonstrating the presence of non-structural enzymes of HEV along with ORF2, activity of a key enzyme, HEV-methyltransferase has also been observed. A 20% decrease in the replicative forms of RNA could be seen in presence of 100 μM Ribavirin after 48 h of treatment. The inhibition gradually increased from 0 to 24 to 48 h post-treatment. Summarily, infectious HEV culture system has been established, which could demonstrate the presence of HEV replicative RNA forms, the structural and non-structural proteins and the methyltransferase in its active form. The system may also be used to study the mechanism of action of Ribavirin in inhibiting HEV replication and develop a therapy.

Virus-like particles (VLPs) are potential containers for delivery of therapeutic agents at the nanoscale. In this study, the capsid protein of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was expressed in a baculovirus insect cell system. The 37-kDa recombinant protein containing the hexahistidine residues (His Tag) at N-terminal was purified using immobilized metal affinity chromatography (IMAC) and assembled into VLPs with a diameter of 23 ± 3 nm analyzed by transmission electron microscopy. We also verified that disassembly/reassembly of IHHNV-VLPs was controlled in the presence and absence of DTT. The efficiency of IHHNV-VLPs to encapsulate plasmid DNA was about 48.2%, and the VLPs encapsulating the pcDNA3.1(+)-EGFP plasmid DNA could recognize the primary shrimp hemocytes and deliver the loaded plasmid into cells by detection of expressed enhanced green fluorescent protein (EGFP). These results implied that the IHHNV-VLPs might be a good candidate for packaging and delivery of expressible plasmid DNA, and may produce an antiviral product in shrimp cells for gene therapy.

The hepatitis E virus- (HEV-) helicase as a novel drug-target was evaluated. While cell culture model was used for mutational characterization of helicase, in silico protein modeling and virtual screening were employed to identify helicase inhibitors. None of the saturation mutant replicons significantly affected RNA replication. Notably, mutants encompassing the Walker motifs replicated as wild-type, showing indispensability of nucleotides conservation in viability compared to known criticality of amino acids. A 3D modeling of HEV-helicase and screening of a compound dataset identified ten most promising inhibitors with drug likeness, notably, JFD02650, RDR03130, and HTS11136 that interacted with Walker A residues Gly975, Gly978, Ser979, and Gly980. Our model building and virtual identification of novel helicase inhibitors warrant further studies towards developing anti-HEV drugs.

To investigate the role of non-structural open reading frame 1 "Y-domain" sequences in the hepatitis E virus (HEV) life cycle.

Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions' infectivity was assayed on naïve HepG2/C3A cells.

In silico analysis identified a potential palmitoylation-site (C336C337) and an α-helix segment (L410Y411S412W413L414F415E416) in the HEV Y-domain. Molecular characterization of C336A, C337A and W413A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.

This is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.

Comparative analysis of the genomic sequences of multiple hepatitis E virus (HEV) isolates has revealed extensive genomic diversity among them. Recently, a variety of genetically distinct HEV variants have also been isolated and identified from large numbers of animal species, including birds, rabbits, rats, ferrets, bats, cutthroat trout, and camels, among others. Furthermore, it has been reported that recombination in HEV genomes takes place in animals and in human patients. Also, chronic HEV infection in immunocompromised individuals has revealed the presence of viral strains carrying insertions from human genes. This paper reviews the current knowledge on the genomic variability and evolution of HEV.

The hepatitis E virus (HEV) ORF1 gene encodes the non-structural polyprotein wherein the 'X-domain' still remains poorly defined. Cellular X-domain associated macrodomain protein/ADP-ribose-1″-monophosphatase (Appr-1″-pase) activities are also reported in coronaviruses (CoV), including identification of its homologs in alpha and rubella viruses. The present study investigated the role(s) of X-domain residues in HEV replication cycle. In silico analysis showed a high degree of evolutionary conservation of X-domain (a.a. 785-942) a.a. positions wherein the N-terminus residues 'Asn806, Asn809, His812, Gly815, Gly816, and Gly817' formed a potential catalytic-site homolog of CoVAppr-1″-pase. To experimentally test this prediction, X-domain 'active-site' residues were subjected to mutational analysis using the HEV-SAR55 replicon (pSK-GFP). FACS analysis of mutant RNA transfected S10-3 cells showed that Gly816Ala and Gly817Ala constructs completely abrogated HEV replication, similar to their Gly816Val and Gly817Val counterparts. However, 'Gly815Ala' mutant replicated very poorly in contrast to 'Gly815Val' that completely abolished GFP synthesis. Furthermore, while 'Asn806Ala' mutant retained RNA replication, the 'Asn809Ala' and His812Leu mutants showed non-viability. Notably, in a sequential-nucleotide mutation analysis, the dispensability of X-domain in HEV replication at transcriptional level has already been demonstrated (Parvez, 2013b). Taken together, the present data strongly argue for an essential role of X-domain residues (Asn809, His812, Gly816 and Gly817) at post-translational level, indicating its involvement in viral replication. In conclusion, the speculated regulatory role of ORF1 X-domain in HEV replication cycle critically depends on the 'Asn, Asn, His, Gly, Gly, Gly' segment/secondary structure. Nevertheless, further biochemical or biophysical characterizations of HEV X-domain associated Appr-1″-pase activity would only confirm its biological significance in virus or host-pathogenesis.

The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.

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.

Among the known human HEV strains (genotypes 1, 2, 3 and 4), the genotype 2 Mexican isolate has two 'double-base' substitutions (5'U5100G5101→CU/3'C5117U5118→GG) flanking the conserved cis-reactive element (CRE) in the intergenic-junction sequences. While the 'C5100U5101' natural mutations in the upstream ORF1 coding region replace 'alanine' for the conserved 'valine', the 'G5117G5118' doublet resides in the downstream non-coding/promoter region of ORF3 gene. Though a stable 'stem-loop' structure containing CRE, critical for virus replication had been reported, the phenotypic effect of genotype 2 'CU/GG' variations were neither mentioned nor explored. In this study, the evolutionary significance of such tolerable mutations in the conserved regulatory-sequences was investigated. Multiple sequence alignment of intergenic-junction of human HEV strains showed further base conservations flanking the CRE sequences. In silico analysis of the conserved sequences (nts. 5099-5121) of the representative genotypes revealed a stable RNA 'stem-loop' structure (CREX). Of the four genotype-specific CREX, the Mexican mutant bases 'CU/GG' very interestingly, compensated and complemented themselves (5'C5100:3'G5118 and 5'U5101:3'G5117) in the 'lower-stem'. The substitution of 'GG' bases in the ORF3 promoter-region did not affect its 'optimal-context' and therefore, negated its regulatory role at 'nucleotide' level. Virtual mutations introduced to break the two base-pairings in the CREX 'lower-stem', completely destabilized the secondary structure. Further molecular characterization of the CREX mutants in HEV-SAR55 replicon background showed a drastic downregulation of viral RNA replication in S10-3 cells. Though the CREX-mutant RNA were encapsidated into trans-complemented viral capsids (ORF2), and produced virions, they were poorly infectious to naïve HepG2/C3A cells. In conclusion, the compensatory mutations in the intergenic-junction of Mexican isolate suggest strict conservation of the CREX 'stem-loop' structure, essential for HEV genome replication. This could have a greater regulatory role in viral life cycle, including RNA packaging.

We assessed hepatitis E virus (HEV) seroprevalence in patients with hepatic disorders as well as in human immunodeficiency virus (HIV)-infected patients and emphasised the issue of possible non-specific anti-HEV seroresponse and need for combining diagnostic methods for hepatitis E diagnosis. Over a two-year period, from March 2011 to February 2013, we determined anti-HEV immunoglobulin M (IgM) and IgG by enzyme immunoassays (EIA; Mikrogen, Germany) in 504 hepatitis patients negative for acute viral hepatitis A-C. Furthermore, 88 samples from randomly selected consecutive HIV-infected patients were also analysed. All EIA reactive samples were additionally tested by line immunoblot assays (LIA; Mikrogen, Germany). HEV nested reverse transcription polymerase chain reaction (RT-PCR) was carried out in 14 anti-HEV IgM LIA-positive patients. Anti-HEV IgM or IgG were detected in 16.9 % of patients by EIA and confirmed by LIA in 10.7 % [95 % confidence interval (CI) 8.3-13.7 %] of hepatitis patients. HEV RNA was detected in five patients. The agreement between EIA and LIA assessed by Cohen's kappa was 0.47 (95 % CI 0.55-0.75) for IgM and 0.83 (95 % CI 0.78-0.93) for IgG. Anti-HEV IgM and IgG seroprevalence in HIV-infected patients was 1.1 %, respectively. Our findings show a rather high HEV seroprevalence in patients with elevated liver enzymes in comparison to HIV-infected patients. Discordant findings by different methods stress the need to combine complementary methods and use a two-tier approach with prudent interpretation of reactive serological results for hepatitis E diagnosis.

Hepatitis E Virus (HEV) ORF1 encodes the nonstructural polyprotein wherein a role of PCP-domain in ORF1 proteolysis and/or RNA replication still remains contested. A series of ORF1 mutants of HEV-SAR55 replicon were constructed and tested for viability in S10-3 cells. Six of PCP-'cysteine' (C457A, C459A, C471A, C472A, C481A and C483A) and three 'histidine' (H443L, H497L and H590L) mutants were lethal. Further, a highly conserved 'glycine-triad' (G815-G816-G817) in downstream X-domain, homologous to rubella virus protease-substrate (G1299-G1300-G1301) was identified where two of X-mutants (G816V and G817V) turned lethal. However, all ORF1 sequential nucleotide-mutants conserving the amino acids were viable, which clearly showed post-translational regulation of HEV replication by PCP- and X-domains. Moreover, while vector-expressed ORF1-fusion polyprotein yielded a ~191 kDa band in vitro, it produced ~78 and ~35 kDa fragments ex vivo. Collectively, the indispensability and functional effects of 'PCP-catalytic' and 'X-substrate' residues on HEV replication strongly supported a viral protease.

Hepatitis E virus is a single, positive-sense, capped and poly A tailed RNA virus classified under the family Hepeviridae. Enteric transmission, acute self-limiting hepatitis, frequent epidemic and sporadic occurrence, high mortality in affected pregnants are hallmarks of hepatitis E infection. Lack of an efficient culture system and resulting reductionist approaches for the study of replication and pathogenesis of HEV made it to be a less understood agent. Early studies on animal models, sub-genomic expression of open reading frames (ORF) and infectious cDNA clones have helped in elucidating the genome organization, important stages in HEV replication and pathogenesis. The genome contains three ORF's and three untranslated regions (UTR). The 5' distal ORF, ORF1 is translated by host ribosomes in a cap dependent manner to form the non-structural polyprotein including the viral replicase. HEV replicates via a negative-sense RNA intermediate which helps in the formation of the positive-sense genomic RNA and a single bi-cistronic sub-genomic RNA. The 3' distal ORF's including the major structural protein pORF2 and the multifunctional host interacting protein pORF3 are translated from the sub-genomic RNA. Pathogenesis in HEV infections is not well articulated, and remains a concern due to the many aspects like host dependent and genotype specific variations. Animal HEV, zoonosis, chronicity in immunosuppressed patients, and rapid decompensation in affected chronic liver diseased patients warrants detailed investigation of the underlying pathogenesis. Recent advances about structure, entry, egress and functional characterization of ORF1 domains has furthered our understanding about HEV. This article is an effort to review our present understanding about molecular biology and pathogenesis of HEV.