Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins

Deepshi Thakral, Baibaswata Nayak{dagger}, Shagufta Rehman, Hemlata Durgapal and Subrat Kumar Panda

Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, 110029 New Delhi, India

Correspondence
Subrat Kumar Panda
pandask{at}hotmail.com


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3–EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV–IRES–EGFP(+)] and antisense [HEV–IRES–EGFP(–)] orientations. HepG2 cells transfected with HEV–IRES–EGFP(+) and HEV–IRES–EGFP(–) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV–IRES–RLuc(+) showed approximately 10-fold higher luminescence than HEV–IRES–RLuc(–). There was complete loss of activity when the helicase–replicase domain in HEV–IRES–RLuc(–) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.

{dagger}Present address: Department of Pathology, Ward 6-080, Feinberg School of Medicine, North Western University, 215 East Chicago Avenue, IL-60611, USA.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis E virus (HEV) is a water-borne pathogen that is responsible for large epidemics and sporadic cases of acute viral hepatitis (Khuroo, 1980; Wong et al., 1980; Purcell & Ticehurst, 1988; Bradley, 1990). Serological surveys show a low HEV seroprevalence in most parts of the world, including industrialized countries (Skidmore et al., 1991; Dawson et al., 1992; Skidmore & Sherratt, 1996; Wu et al., 1998; Ooi et al., 1999; Tokita et al., 2003). Recent data suggest that hepatitis E may be a zoonosis (Clayson et al., 1995; Meng et al., 1997, 1998; Hsieh et al., 1999; Garkavenko et al., 2001; Nishizawa et al., 2003).

HEV is non-enveloped and carries a single-stranded, positive-sense RNA genome (approx. 7·2 kb in length) that is capped and polyadenylated. The genome is organized into three partially overlapping open reading frames (ORFs), designated 1, 2 and 3 (Tam et al., 1991; Purdy et al., 1993; Kabrane-Lazizi et al., 1999). The 27–35 nt long 5' non-coding region (NCR) is followed by the largest ORF (ORF1), which encodes the non-structural polyprotein of 1693 aa. Computer-assisted assignments for the putative functions of HEV ORF1 suggested the presence of methyltransferase-, putative cysteine protease-, putative RNA helicase- and RNA-dependent RNA polymerase (RdRp/replicase)-homology domains (Koonin et al., 1992). RdRp (Agrawal et al., 2001) and methyltransferase (Magden et al., 2001) activities have been verified experimentally. ORF2 encodes the major viral capsid protein, which contains a signal peptide and potential glycosylation sites (Jameel et al., 1996; Zafrullah et al., 1999). The non-glycosylated form of pORF2 has been demonstrated to be more stable than the glycosylated form (Torresi et al., 1999). Overlapping the two major reading frames is ORF3, which encodes a 123/122 aa cytoskeleton-associated phosphoprotein that may be associated with signal transduction (Zafrullah et al., 1997; Korkaya et al., 2001). No discrete function has been assigned to ORF3 so far. The 3' NCR is 65–74 nt long, ends in a poly(A) tail and has been demonstrated to bind specifically to the viral RdRp in vitro (Agrawal et al., 2001).

HEV replication is poorly understood, due to the absence of a reliable cell-culture system. In a rhesus macaque (Macaca mulatta) model, the presence of both positive- and negative-sense RNAs of HEV has been demonstrated in the liver (Nanda et al., 1994). HEV is related closely to alphaviruses, based on its genomic organization (Purdy et al., 1993). A sequence stretch in the negative strand of HEV is similar to the Sindbis alphavirus subgenomic promoter for transcription of the structural gene (Levis et al., 1990). No information is available regarding the subgenomic promoter function or the mechanism of translation of structural genes of HEV. The presence of one HEV genomic (~7·5 kb) and two subgenomic (~3·7 and ~2 kb) RNAs has been demonstrated in infected animal liver (Tam et al., 1991) and in cell culture (Xia et al., 2000). We have previously described the preparation of a full-length cDNA clone of an Indian strain (Hyderabad isolate) of HEV (Panda et al., 2000). Transfection of cultured HepG2 cells with in vitro-generated RNA transcripts from this clone was shown to produce negative-strand RNA and processed viral proteins, as well as infectious virions. Full-length cDNA clones of HEV have been described and in vitro-transcribed, capped RNA was reported to be infectious for chimpanzees (Emerson et al., 2001). However, in a follow-up study, Emerson et al. (2004) reported an HEV replicon expressing green fluorescent protein that demonstrated replication, albeit at a lower level, even in the absence of the 7mG cap structure. Neither of these studies described either negative-strand RNA synthesis or subgenomic RNA production, but rather depended on protein expression as an indicator of replication.

In order to extend our range of experimental tools to understand HEV replication, we modified the infectious cDNA clone (Panda et al., 2000) to generate: (i) HEV replicons carrying in-frame fusions of the ORF2 and ORF3 genes with enhanced green fluorescent protein (EGFP); (ii) dicistronic replicons carrying EGFP/Renilla luciferase (RLuc) reporter genes driven by the encephalomyocarditis virus internal ribosome entry site (EMCV-IRES); (iii) a short-term cell line carrying the full-length HEV genome that produces HEV RNA and viral proteins. These virus-based expression systems may be useful for understanding the factors that are involved in HEV replication.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
A human hepatoma cell line (HepG2) was sustained in maintenance medium [Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10 % heat-inactivated fetal calf serum (FCS; Life Technologies ), 100 U penicillin ml–1, 10 µg streptomycin ml–1 and 25 µg amphotericin B ml–1 in 0·85 % saline (Sigma)] at 37 °C in 5 % CO2.

Construction of plasmids.
The full-length HEV cDNA clone pSG1-HEV(I) (GenBank accession no. AF076239) was modified to introduce the EGFP reporter gene as an in-frame fusion with the ORF2 and ORF3 genes. The EGFP gene was amplified by PCR from pEGFP1 (Clontech) and inserted into the unique HindIII site at nt 5679 of pSG1-HEV(I) and pSG1-ORF2 (Jameel et al., 1996), to obtain an in-frame fusion of ORF2 with EGFP (Fig. 1). The HEV-ORF2–EGFP vector was digested with NruI, removing the fragment from nucleotide positions 4010 to 5141 and resulting in a replicase-deletion vector [HEV-ORF2{Delta}(4010–5141)].



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of HEV replicons expressing reporter genes. Organization of the full-length HEV genome (GenBank accession no. AF076329) where numbers represent nucleotide positions and open boxes represent the HEV coding region, including non-structural ORF1 [homology domains are designated methyltransferase (Met), protease (Pro), helicase (Hel), RNA-dependent RNA polymerase/replicase (RdRp) and the structural region (ORF3 and ORF2)]. For HEV-ORF2–EGFP, the 760 bp EGFP-coding region (solid box) was inserted in-frame with ORF2 at nucleotide position 5679 without removing any region of the full-length cDNA clone of HEV. The remaining ORF2 region after the EGFP stop codon is not translated (dashed box). HEV-ORF2–EGFP{Delta}(4010–5141) is identical to HEV-ORF2–EGFP except for the deletion (dashed box) from nucleotide positions 4010 to 5141, removing the replicase domain. HEV-ORF3–EGFP contains an in-frame fusion of ORF3 with EGFP in the full-length viral genome. pSG1–HEV{Delta}(5679–6818)MCS carries a major deletion in the structural region of ORF2 from nucleotide positions 5679 to 6818 and carries unique cloning sites, including EcoRV. Dicistronic HEV replicons with EMCV-IRES–EGFP/RLuc inserted in the partially deleted ORF2 in the forward [HEV–IRES–EGFP(+)] and reverse [HEV–IRES–EGFP(–)] orientations. The orientation of IRES–EGFP/RLuc defines the translation of EGFP/RLuc genes from either the plus (genomic sense) or minus (antisense) strands of dicistronic HEV replicon RNA, respectively. In HEV–IRES–RLuc(–){Delta}(2546–5679), a deletion of HEV–IRES–RLuc(–) from nucleotide positions 2546 to 5679 (dashed box) removed the helicase and replicase domains. pSG1–ORF2–EGFP and pSG1–ORF3–EGFP represent the EGFP fusion controls for pORF2 and pORF3, respectively.

 
The ORF3 termination codon was mutated and fused in-frame with EGFP as a carboxy-terminal fusion, resulting in HEV-ORF3–EGFP (Fig. 1). The ORF3–EGFP fusion cassette was amplified from the HEV-ORF3–EGFP template and cloned into vector pSG1 to serve as a subgenomic fusion control.

The full-length HEV cDNA clone pSG1-HEV(I) was further modified to introduce the IRES–EGFP/RLuc cassette in both the forward and reverse orientations to generate HEV–IRES–EGFP/RLuc(+) and HEV–IRES–EGFP/RLuc(–) expression vectors (Fig. 1), using conventional cloning. The region from nucleotide positions 5679 to 6818 of recombinant HEV was deleted to insert an 80 bp EcoRI–XhoI polylinker from vector pSG1 (Jameel et al., 1996) to produce pSG1-HEV{Delta}(5679–6818)MCS (Fig. 1). EGFP/RLuc was fused downstream of the IRES and cloned into the EcoRV site of pSG1-HEV{Delta}(5679–6818)MCS to produce HEV–IRES–EGFP/RLuc(+/–). Digestion of HEV–IRES–EGFP/RLuc(–) with NotI and deletion of the region encompassing the helicase and replicase domains (nt 2546–5679) resulted in the construct HEV–IRES–RLuc(–){Delta}(2546–5679) (Fig. 1). The resulting clones were characterized by restriction-enzyme digestion and confirmed by sequencing using a model ABI 310 automated sequencer (Applied Biosystems). Primer sequences and cloning details are available on request.

For the generation of a cell line transfected stably with the HEV genome, vector pSG1-HEV(I) was digested with EcoRI and XhoI to release full-length HEV cDNA, which was ligated into equivalent sites in vector pCDNA3 (Invitrogen), generating the construct pCDNA3-HEV(I).

RNA transcription.
Plasmids were digested with XhoI (Amersham Biosciences) to produce DNA templates for run-off transcription. Linearized, purified plasmids were resuspended in 10 µl RNase-free water (0·5 µg µl–1). mMESSAGE mMACHINE kits (Ambion) were used to in vitro-transcribe capped RNA in a 20 µl reaction with an additional 1·0 µl 30 mM GTP solution added. Following transcription, the DNA template was removed by DNase I treatment. The integrity of purified transcripts was determined by 1 % formaldehyde agarose-gel electrophoresis; the RNA was then quantified by spectrophotometry (Pye Unicam 8800 UV/visible spectrophotometer; Philips) and stored at –80 °C in aliquots for use within 24 h.

DNA and RNA transfection.
Cells at a confluence of 60–80 % were used for DNA transfection. Plasmid transfections were carried out by the liposome-induction method (Lipofectamine 2000; Invitrogen) in accordance with the manufacturer's guidelines. For each 30 mm petri dish or slide flask (Nunc), 3–5 µg purified plasmid mixed with 10 µl Lipofectamine 2000 in 1 ml Opti-Mem (Life Technologies) was used. Cells were incubated in 5 % CO2 at 37 °C, to be analysed at different time points.

For RNA transfection, approximately 1x107 HepG2 cells in DMEM without FCS were electroporated with 10 µg in vitro-transcribed capped RNA in 0·4 cm cuvettes using a Gene Pulser II apparatus (Bio-Rad) set at 200 V and 1000 µF. After electroporation, the cell suspension was kept for 5 min at room temperature and then diluted into DMEM supplemented with 10 % FCS and divided equally at subconfluent levels (2x106 cells) into 30 mm petri dishes. For fluorescence analysis of the expressed proteins, approximately 2x105 transfected cells were cultured onto 22 mm glass coverslips. Cells were fixed with 4 % paraformaldehyde in PBS (pH 7·5) at room temperature for 10 min, washed with PBS and used for analysis. RNA transfection was also carried out by liposome induction using 2–4 µg RNA for each transfection, as described above.

Immunofluorescence assay (IFA).
The fixed cells on coverslips were incubated at room temperature for 1 h in 1 : 500-diluted anti-ORF1 (anti-methyltransferase domain) or 1 : 1000-diluted anti-ORF3 or anti-ORF2 specific rabbit polyclonal sera in dilution buffer (0·5 % BSA and 0·1 % saponin in PBS). The cells were washed and further incubated with 1 : 200-diluted goat anti-rabbit IgG conjugated with fluorescein or Texas red (DakoPatts) at room temperature for 30 min. The monolayer was washed with PBS, mounted with mounting medium and observed under a fluorescence microscope (Eclipse E-600).

Immunofluorescence analysis by confocal microscopy.
Fixed cells were permeabilized with 100 % methanol at –20 °C for 3 min. Cells were then rehydrated with PBS for 30 min at room temperature. Blocking was done with 0·5 % BSA in PBS for 2 h followed by 5 % normal goat serum in PBS-T (PBS/0·5 % Tween 20) for 1 h. Cells were incubated with a 1 : 1000 dilution in PBS-T of rabbit anti pORF2/pORF3 primary antibody, washed three times with PBS-T and incubated with a 1 : 1000 dilution in PBS-T of goat anti-rabbit IgG Alexa 546 (red)-conjugated secondary antibody. The cells were mounted in 50 % glycerol in PBS on a glass slide and sealed. Fluorescence images were collected by using a 63x oil immersion objective at 1024x1024 resolution format on a Leica TCS-SP2 confocal microscope. For EGFP excitation, Ar/Kr 488 nm LASER lines were used, whereas for the excitation of Texas red/Alexa 546, Gre/Ne LASER lines were used. Cells were scanned in the sequential mode and images were processed by using Leica confocal software and Adobe Photoshop version 7.0.

Strand-specific anchored RT-PCR.
Cells were harvested and lysed in TRIzol reagent (Invitrogen Life Technologies) at various time points post-transfection. Total RNA was isolated by chloroform extraction and 2-propanol precipitation, followed by a wash in 70 % ethanol. The RNA pellet was air-dried, resuspended in diethyl pyrocarbonate-treated water and quantified spectrophotometrically. For strand-specific RT-PCR, reverse transcription was carried out by using 2 µg total cellular RNA with 200 U Superscript RT-II enzyme (Invitrogen Life Technologies) into cDNA and either a sense or an antisense primer.

For antisense-strand detection, RNA was reverse-transcribed by using a forward primer (5'-G11CCGCGCCCATACTTTTGATGA-3') and, for sense-strand detection, with the reverse primer (described below). Following cDNA synthesis, the RNA in the reaction mixture was degraded by digestion with 2 U RNase H (Amersham Biosciences) and 1 µg RNase A (Promega). The cDNA was used for anchored PCR; amplification was carried out by using poly(G17) as the forward primer and 5'-CAGGGAGCGCGGAACGGAACGCAG-3' as the reverse primer. Mock-transfected cells and RNA without reverse transcriptase were included as negative controls. The amplified products were analysed by electrophoresis on an agarose gel and photographed with a gel-documentation system (UVP). HEV-infected rhesus macaque (M. mulatta) bile RNA with a viral titre of >1x1012 particles ml–1 was used for standardization of the experiment (data not shown). The minus strand was undetectable with 100 ng RNA.

A comparative analysis of capped and uncapped full-length HEV transcripts was carried out by running parallel experiments. Briefly, 4 µg capped or uncapped RNA was transfected into HepG2 cells. RNA was extracted and reverse-transcribed into cDNA by using a strand-specific forward or reverse primer, as described. The cDNA was amplified by real-time PCR using SYBR green PCR mix (Applied Biosystems). The reaction was carried out with 2x SYBR green PCR mastermix in a 25 µl volume. The samples were aliquotted into a MicroAmp Optical 96-well reaction plate (Perkin Elmer Applied Biosystems) and sealed. Each reaction was done in triplicate in the Perkin Elmer ABI Prism 7700 Sequence Detection system (Applied Biosystems). A 1000-fold serial dilution of in vitro-transcribed full-length HEV RNA was used as the standard for quantification.

In vitro coupled transcription and translation.
In vitro synthesis of the desired polypeptide was carried out by using a coupled transcription and translation system (Promega) according to the manufacturer's guidelines. The protein was synthesized in the presence of [35S]-labelled methionine–cysteine [Promix; specific activity ~37 TBq (1000 Ci) mmol–1; BRIT, Mumbai, India] in a volume of 25 µl. The reaction was carried out at 30 °C for 90 min. The translated protein was analysed by SDS-PAGE followed by autoradiography.

Luciferase assay.
RLuc expression was detected in HepG2 cells co-transfected with the expression vectors/replicons carrying RLuc. A firefly luciferase reporter vector (Sriram et al., 2003) was used as an internal equalizer. Briefly, growth medium was removed from the transfected cells and the monolayer was rinsed with PBS. RLuc and firefly luciferase activity was determined by using the Dual Luciferase Assay system (Promega) according to the manufacturer's instructions. All assays were done in triplicate and the results reported were reproducible and expressed as means±SD in relative light units (r.l.u.) (mg protein)–1 or as percentage activities. A TD 20/20 Luminometer (Promega) was used to measure light emission.

Selection, passaging and storage of the cell line.
To select for geneticin-resistant HepG2 cells maintaining the pCDNA3-HEV(I) plasmid, we transfected ~2x106 cells at a confluence of 60–70 %. After 72 h transfection, cells were subcultured into 25 cm2 flasks and kept under G418 (Sigma-Aldrich) selection at a concentration of 500 µg ml–1. The selection medium was changed after every 4 days with fresh G418 until colonies were observed (after 3–4 weeks). The selected colonies were cloned by limiting dilution, expanded and stored frozen in 10 % DMSO and 20 % FCS in liquid nitrogen or propagated in maintenance medium containing 250 µg G418 ml–1.

Western blot analysis and immunoprecipitation.
Cellular proteins, isolated from geneticin-resistant clones by boiling in 1x SDS gel-loading buffer, were subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane (Hybond-C Extra, 0·45 µm; Amersham Biosciences) by using a semi-dry Western blot apparatus (Sigma-Aldrich) as recommended by the manufacturer. The nitrocellulose membrane was blocked (5 % skimmed-milk powder and 0·05 % Tween 20 in PBS) and incubated with the primary antibody at a dilution of 1 : 1000 in blocking solution (rabbit anti-pRdRp/anti-pORF2/anti-pORF3 antibody). After three washes with PBS containing 0·05 % Tween 20, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit antibody (DakoPatts) at a dilution of 1 : 500 for 1 h at room temperature. The membrane was washed three times with PBS and 6 mg diaminobenzidine substrate dissolved in 9 ml 0·01 M Tris/HCl (pH 7·6), 10 µl 30 % hydrogen peroxide was added. It was incubated at room temperature until the coloured bands developed.

Immunoprecipitation of the [35S]methionine–cysteine-labelled proteins was carried out as described previously (Panda et al., 2000). Images of the autoradiographs were generated by using a gel-documentation system (UVP).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Detection of full-length HEV replication
In vitro-generated transcripts of full-length HEV were shown to produce negative-strand RNA as well as virally encoded proteins in transfected HepG2 cells in our previous report (Panda et al., 2000). Expression of processed non-structural pORF1 and pORF3 and structural pORF2 virally encoded proteins was shown previously by using immunofluorescence and immunoprecipitation. In the present study, a comparison between HEV replication with either uncapped or capped full-length HEV transcripts was carried out. A 10-fold greater amount of the minus strand was detected with capped transcripts (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Comparative analysis of capped and uncapped HEV transcripts using real-time RT-PCR

Levels of both plus- and minus-strand RNA were quantified by real-time RT-PCR in cells transfected with either capped or uncapped full-length HEV transcripts after 48–72 h. A standard curve was generated from a 1000-fold serial dilution of full-length HEV in vitro RNA (R constant, 0·99). Values (RNA copies ml–1) of replicates and means from parallel experiments are reported.

 
Therefore, capped RNA transcripts generated from full-length HEV were used in further experiments. IFA was performed with anti-ORF1/anti-ORF3/anti-ORF2 specific rabbit polyclonal sera and anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) as primary and secondary antibodies, respectively, to monitor viral protein synthesis (Fig. 2). The expression of virally encoded proteins increased from 24 to 72 h post-electroporation. Almost no cytopathic effect was observed in the IFA-positive cells at any time after transfection and the mock-transfected cells were negative for staining (Fig. 2). To confirm that the transfected RNA was replicating, synthesis of both positive and negative strands of HEV RNA was initially determined by strand-specific anchored RT-PCR of total cellular RNA, with HEV-specific primers (Fig. 3a). The strand specificity of the anchored RT-PCR assay was tested with HEV-infected rhesus macaque (M. mulatta) bile (data not shown). Minus-strand RNA synthesis increased from the time of transfection until 6 h, remained constant and decreased by 72 h (Fig. 3a). In addition, cells transfected with capped RNA from a deletion construct [pSG1-HEV{Delta}(4010–5141)] that had its replicase domain removed did not show any antisense RNA synthesis (Fig. 3b).



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 2. Indirect immunofluorescence microscopy of HepG2 cells 48 h after transfection with capped full-length HEV genome (a–c) or mock-transfected (d–f). Cells on coverslips were fixed with 4 % paraformaldehyde, stained with anti-ORF1 (a, d), anti-ORF3 (b, e) or anti-ORF2 (c, f) rabbit polyclonal sera and subsequently with FITC-conjugated goat anti-rabbit IgG.

 


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3. Detection of plus and minus strands of HEV RNA in transfected cells. Total cellular RNA (2 µg) isolated at 6, 24, 48 and 72 h after electroporation with capped HEV RNA was subjected to anchored RT-PCR analysis. (a) Strand-specific RT-PCR of the HEV plus strand at 6 (lane 1), 24 (2), 48 (3) and 72 (4) h post-electroporation. Plain cells (5), reactions without reverse transcriptase (6) and a no-template control (7) served as negative controls; M, DNA molecular size marker (Promega). Minus-strand HEV RNA was detected 6, 24, 48 and 72 h after electroporation with identical negative controls. (b) Similarly, cells transfected with a polymerase-defective HEV RNA, pSG1–HEV{Delta}(4010–5141), were tested for both plus- and minus-strand synthesis from total cellular RNA, isolated at 6 (lane 4), 24 (5) and 48 (6) h after electroporation with similar negative controls (1–3).

 
HEV replicons carrying reporter genes
Several versions of capped replicons were prepared from the replication-competent cDNA clones of HEV (Fig. 1). The RNA generated contained the authentic viral 3' non-coding sequence, followed by five A residues and an extra, non-viral G residue. More than 95 % of the in vitro transcripts from full-length HEV and of other replicons were of the expected length, as analysed by 1 % formaldehyde agarose-gel electrophoresis (data not shown).

Characterization of the HEV replicon expressing EGFP fused in-frame with ORF2
The HEV-ORF2–EGFP fusion resulted in a replicon of 7944 nt (Fig. 1). The fusion junction had eight additional nucleotides upstream of the EGFP start codon, inserting a 3 aa (Phe–Gly–Thr) spacer at the ORF2–EGFP junction. The 5' NCR, ORF1, ORF3 and the 3' NCR in this replicon were intact, with part of the amino terminus of ORF2 (176 aa) fused to EGFP. HepG2 cells transfected with capped HEV-ORF2–EGFP RNA were positive for fluorescence (Fig. 4a). Total cellular RNA from these cells was positive for the minus strand (Fig. 4b). Deletion of the replicase region of HEV-ORF2–EGFP (Fig. 4c) resulted in loss of the EGFP signal, as well as the minus strand (Fig. 3b). This confirmed that expression of the fusion protein was dependent on virus replication.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 4. Characterization of an HEV replicon carrying an ORF2–EGFP in-frame fusion. (a) HepG2 cells were transfected with in vitro-capped RNA generated from HEV-ORF2–EGFP and visualized 72 h after transfection under a confocal microscope. (b) Total cellular RNA (2 µg) isolated 6, 24, 48 and 72 h after transfection with capped HEV-ORF2–EGFP RNA was subjected to anchored RT-PCR analysis. Upper panel: strand-specific RT-PCR of the HEV plus strand at 6 (lane 4), 24 (5), 48 (6) and 72 (7) h post-electroporation. Plain cells (1), RNA without reverse transcriptase (2) and a no-template control (3) served as negative controls; 8, DNA molecular size marker (Promega). Lower panel: minus-strand HEV RNA was detected 6 (lane 5), 24 (6), 48 (7) and 72 (8) h after electroporation with identical negative controls (2–4). (c) HEV-ORF2–EGFP{Delta}(4010–5141) RNA-transfected HepG2 cells were visualized 72 h after transfection under a confocal microscope. EGFP expression was observed in pSG1-ORF2–EGFP fusion control-transfected cells (data not shown). (d) In vitro coupled transcription and translation of pSG1-ORF2–EGFP fusion-control vector yielded the expected 46 kDa protein (lane 1) that could be immunoprecipitated by using anti-ORF2 specific polyclonal sera (3); a pre-immune serum was used as a negative control (2). (e) HepG2 cells transfected with HEV-ORF2–EGFP RNA expressing EGFP were stained with anti-ORF2 polyclonal sera followed by Texas red-conjugated secondary antibody. Superimposition of EGFP-positive cells (green) and Texas red-stained cells (red) confirmed the presence of the fusion protein (merged).

 
In vitro transcription-coupled translation of pSG1-ORF2–EGFP resulted in the expected 46 kDa fusion protein (Fig. 4d), which could be immunoprecipitated by anti-ORF2 polyclonal sera (Fig. 4d). HepG2 cells transfected with pSG1-ORF2–EGFP fusion control showed positive EGFP expression (data not shown). The fusion protein could be detected in HEV-ORF2–EGFP RNA-transfected cells by IFA using a Texas red-conjugated secondary antibody. Superimposition of EGFP-positive cells and Texas red-stained cells confirmed the presence of the fusion protein (Fig. 4e).

Analysis of HEV replicon with the ORF3–EGFP in-frame fusion
EGFP was fused to ORF3 in full-length HEV by mutating its stop codon. Transfection of HEV-ORF3–EGFP capped RNA into HepG2 cells did not yield any EGFP fluorescence (Fig. 5a), although the minus strand could be detected in the transfected cells (Fig. 5b).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 5. Expression analysis of an HEV replicon carrying an ORF3–EGFP in-frame fusion. (a) HepG2 cells transfected with HEV-ORF3–EGFP capped RNA was visualized after 72 h under a confocal microscope. (b) Upper panel: strand-specific RT-PCR of total cellular RNA from HEV-ORF3–EGFP RNA-transfected cells for the plus strand at 24 (lane 4) and 48 (5) h post-transfection. Plain cells (1), RNA without reverse transcriptase (2) and a no-template control (3) served as negative controls; M, DNA molecular size marker (Promega). Lower panel: minus-strand HEV RNA was detected 24 (lane 4) and 48 (5) h after electroporation with identical negative controls (1–3). (c) pSG1-ORF3–EGFP fusion-control vector-transfected cells were visualized after 72 h under a confocal microscope. (d) In vitro coupled transcription and translation of the pSG1-ORF3–EGFP fusion control yielded the expected 40 kDa protein (lane 1) that was immunoprecipitated by using anti-ORF3 specific polyclonal sera (3); a pre-immune serum was used as a negative control (2). (e) HepG2 cells expressing EGFP were stained with anti-ORF3 antibody and Alexa 546-conjugated secondary antibody for colocalization of EGFP–ORF3 fusion protein. Superimposition of EGFP-positive cells (green) and Alexa 546-stained cells (red) confirmed the presence of the fusion protein (merged).

 
However, positive fluorescence was obtained upon transfection of the subgenomic fusion control pSG1-ORF3–EGFP (Fig. 5c), of which coupled transcription and translation resulted in a protein of ~40 kDa that could be immunoprecipitated by an ORF3-specific antibody (Fig. 5d). ORF3–EGFP fusion protein showed a typical distribution of EGFP at localized foci. Fusion protein was detected and confirmed by overlapping the EGFP-positive cells with immunofluorescence detection of pORF3 (Fig. 5e).

In the absence of information on the role of ORF3 in virus biology, we chose ORF2 as the target for deletion without affecting the replication competency of the recombinant clone of HEV.

HEV carrying a deletion in the structural region of its genome
For the construction of bicistronic replicons of HEV, a segment of the HEV genome encompassing nucleotide positions 5679 to 6818 was deleted from the full-length HEV [pSG1-HEV{Delta}(5679–6818)] (Fig. 1). This leaves the terminal part of ORF2 and the 3' NCR intact, as this region folds into stem–loop structures that bind specifically to RdRp (Agrawal et al., 2001). It has been shown that initiation of replication in sense-strand RNA viruses requires this replicase complex and interactions with host-cell proteins for initiating negative-strand RNA synthesis (Song & Simon, 1995). The replication potential of the recombinant vector with a partial deletion in ORF2 was determined by strand-specific RT-PCR analysis of total cellular RNA isolated from pSG1-HEV{Delta}(5679–6818)-transfected HepG2 cells with HEV-specific primers. Both positive and negative strands were detected, whereas no amplification was observed in negative controls (Fig. 6a). This indicates that pSG1-HEV{Delta}(5679–6818), produced by the targeted deletion in the HEV structural region, was capable of replication. Hence, the pSG1-HEV{Delta}(5679–6818) construct was evaluated for the possibility of being used as a reporter-gene vector to detect the production of the plus and minus strands of HEV RNA. The reporter genes EGFP and RLuc, driven by EMCV-IRES, were utilized for this purpose.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 6. (a) Both plus (lane 1, 24 h; lane 2, 48 h) and minus (lane 3, 24 h; lane 4, 48 h) strands of HEV were detected in total RNA isolated from HepG2 cells transfected with pSG1-HEV{Delta}(5679–6818)MCS; mock-transfected cells (5) and RNA without reverse transcriptase (6) were used as negative controls; M, DNA molecular size marker (100 bp DNA ladder; Promega). (b–d) Dicistronic constructs carrying the EGFP reporter gene driven by EMCV-IRES in the forward orientation [HEV–IRES–EGFP(+)] (b), reverse orientation [HEV–IRES–EGFP(–)] (c) and replicase-defective vector HEV–IRES–EGFP(–){Delta}(2546–5679) (d) were transfected into HepG2 cells and visualized after 72 h under a confocal microscope. (e) Strand-specific nested RT-PCR analysis of total RNA of the HEV minus strand 24 (lane 3) and 48 (lane 4) h after transfection. Negative controls included mock-transfected cells (lane 1) and RNA without reverse transcriptase (lane 2); lane 5, positive control; M, DNA molecular size marker. (f) Representative experiment showing RLuc activity after 72 h in HepG2 cell lysates transfected with pSG1-IRES–RLuc, HEV–IRES–RLuc(+), HEV–IRES–RLuc(–), HEV–IRES–RLuc(–){Delta}(2546–5679) and plain cells. Values are plotted as the mean relative luminescence units from three parallel transfections versus various constructs.

 
Bicistronic HEV replicon expressing EGFP
Insertion of the IRES–EGFP cassette resulted in a net increase in length of the viral genome of 606 nt. Insertion of the IRES–EGFP cassette in both orientations into pSG1-HEV{Delta}(5679–6818) was carried out in order to monitor the synthesis of plus and minus strands of the replicon during replication, via expression of EGFP driven by the IRES. Only about 5–10 % of the transfected cells were positive for fluorescence with both HEV–IRES–EGFP(+) (Fig. 6b) and HEV–IRES–EGFP(–) constructs (Fig. 6c). As a defective control, HEV–IRES–EGFP(–) vector with a deletion in the helicase and replicase domains used for transfection did not show any fluorescence (Fig. 6d). Minus-strand HEV RNA could be detected in test cells (Fig. 6e), implying that the sense-strand viral RNA was generated in the HEV–IRES–EGFP(–) vector-transfected HepG2 cells, followed by production of antisense RNA, hence supporting the occurrence of HEV replication. Due to the limited percentage of cells showing fluorescence for EGFP, it offered little help in serving as a quantitative assay for the detection of HEV replication. Therefore, the RLuc reporter was used to replace EGFP, making quantification possible.

Tenfold-higher luciferase activity was observed in cells receiving the positive-orientation construct HEV–IRES–RLuc(+), in comparison to the negative-orientation construct HEV–IRES–RLuc(–) (Fig. 6f). These results indicated the relative abundance of plus-sense HEV RNA in comparison to the minus-sense viral RNA in the transfected cells and is in agreement with our earlier report (Panda et al., 2000). A negative control similar to that used for EGFP replicons yielded no RLuc expression. Therefore, this transient-transfection reporter assay can be used for relative quantitative analysis of HEV replication.

pcDNA3-HEV(I) allows short-term selection of cells persistently expressing viral proteins
HepG2 cells were transfected with a plasmid carrying the neomycin-resistance gene (neo) and full-length HEV under the control of the cytomegalovirus promoter. Culturing the cells in the presence of the antibiotic geneticin (G418) eliminated cells that did not harbour the plasmid and allowed the selected clones to propagate. The selected clones were tested for viral RNA by RT-PCR with HEV-specific primers (Fig. 7a). The cells, as well as the culture supernatant, were positive for HEV RNA even after the seventh passage (Fig. 7b). Viral proteins were detected by Western blot analysis for pORF2 (~72 kDa), pORF3 (~13·5 kDa) and replicase (~37 kDa) (Fig. 7c). The band was diffuse in case of pORF3. Similarly, immunoprecipitation analysis carried out for the structural proteins revealed the presence of both pORF2 (~72 kDa) and pORF3 (~13·5 kDa) (Fig. 7d).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7. Selection and characterization of cells maintaining pCDNA3-HEV(I) vector. (a) HepG2 cells transfected with pCDNA3-HEV(I) vector were subjected to selection with G418 at 500 µg ml–1. Cells selected under G418 for 35 days (lane 4), normal HepG2 cells (3), an RNA-extraction control (2), a no-template control (1) and a positive control (5) were tested by RT-PCR for total cellular RNA. M, DNA molecular size marker. (b) RT-PCR of total RNA from selected cells (passage 5, lane 6), cell-culture supernatant (passage 7, lane 5), cell-culture supernatant (passage 5, lane 4) and negative controls (lanes 1–3). (c) Western blot analysis of virally encoded proteins with anti-ORF2 (i), anti-ORF3 (ii) and anti-RdRp (iii). In all three panels (i–iii), lane 1 represents bacterially induced protein as a positive control, lane 2 represents the negative control with a pre-immune serum and lane 3 represents bands detected with either anti-ORF2 (i), anti-ORF3 (ii) or anti-RdRp (iii) specific antibodies, respectively. (d) Immunoprecipitation of virally encoded proteins isolated from selected cells with anti-ORF2 (i; lane 2) and anti-ORF3 (ii; lane 2) with both positive (lane 3) and negative (lane 1) controls.

 
These results indicate that the G418-resistant HepG2 cells continuously produced HEV RNA and proteins without affecting the apparent morphology of the cells. These cells can be used for analysis of the transcriptional- and translational-control systems of the virus.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In vitro-transcribed uncapped RNAs generated from a full-length cDNA clone of HEV have been shown to produce negative-strand RNA (Panda et al., 2000), viral proteins and infectious virions (Emerson et al., 2001; Panda et al., 2000). As it has been demonstrated that the HEV genome is capped (Kabrane-Lazizi et al., 1999), we generated full-length HEV transcripts with a 5' cap and tested them by transfection into HepG2 cells. A comparative analysis with 4 µg transcript demonstrated a 10-fold advantage in the case of capped RNA (Table 1). Immunofluorescence analysis (Fig. 2) and strand-specific RT-PCR (Fig. 3) demonstrated the replication of wild-type viral RNA in the transfected HepG2 cells. HEV-specific negative strand was detected in the transfected cells by an anchored RT-PCR developed in our laboratory. This also agrees with recent observations by IFA in a cell-culture system (Emerson et al., 2004). In this study, the methodology was partially modified and optimized with 2–4 µg capped transcripts instead of 20 µg uncapped RNA, which was used previously (Panda et al., 2000).

Lower transfection efficiency limits the use of Northern hybridization in these studies, although we have detected both the positive and negative strands of HEV RNA by slot-blot hybridization in our previous study (Panda et al., 2000). Early protein synthesis in HEV occurs within first few hours post-infection/-transfection, as has been seen by the presence of replicase up to 36 h (Panda et al., 2000) and subgenomic RNAs at 6·5–7·5 h post-infection (Xia et al., 2000).

In the absence of information on the subgenomic promoter sequences or the mechanism of translation of the internal ORFs of HEV, our initial approach was to determine the region of the HEV genome that could be manipulated without affecting the replication competency of the virus. Therefore, we attempted to insert the reporter gene EGFP as an in-frame fusion with both ORF2 and ORF3. EGFP was expressed successfully when fused to amino-terminal ORF2, but no signal was observed when a deletion was introduced upstream of the ORF2–EGFP sequence, removing a major region spanning the replicase as well as the non-coding spacer between ORF1 and ORF2 (Fig. 4). Loss of EGFP expression from the deleted ORF2–EGFP replicon could be attributed to the absence of the replicase domain and possible lack of the cis-acting upstream sequences. Primer-extension analysis data that had mapped the 5' end of the large subgenomic RNA to the initiative region within RNA polymerase region, and the small subgenomic RNA to the intergenic region between ORF1 and ORF2 (Xia et al., 2000), indicated that the expression of ORF2 is probably mediated by subgenomic RNA.

EGFP fluorescence was absent in case of the HEV–ORF3–EGFP construct. ORF3 encodes a phosphoprotein and has been suggested to be a regulatory molecule in signal transduction (Zafrullah et al., 1997; Korkaya et al., 2001). The ORF3 reading frame was intact with its termination codon mutated, such that EGFP could be translated as a carboxy-terminal fusion protein. This was confirmed by sequencing, as well as by expression of EGFP from the subgenomic ORF3–EGFP fusion control (Fig. 5c). The ORF3–EGFP protein showed a typical distribution pattern, unlike the uniform cytoplasmic expression of the ORF2–EGFP fusion product. This kind of distribution could be attributed to the cytoskeletal association of ORF3–EGFP, as has been described previously (Zafrullah et al., 1997). In a recent report, EGFP has been expressed from an HEV replicon with fused ORF2–EGFP. The amino-terminal methionine of ORF2 was fused to EGFP, thereby removing most of the ORF2 and ORF3 proteins (Emerson et al., 2004). The reason that the ORF3–EGFP fusion in the context of full-length HEV replication did not express the reporter is unknown, although the minus strand was detected, indicating the occurrence of replication, and the control expression with the subgenomic construct rules out misfolding. Any abnormality in generation of the RNA producing this protein needs further in-depth analysis, as does other transcriptional regulation of HEV.

Encouraged by EGFP expression from the ORF2–EGFP replicon, a major deletion was introduced into the structural ORF2 of the HEV cDNA clone with the 5' NCR, ORF1, ORF3 and 3' NCR intact and unique cloning sites were added by inserting multiple cloning sites. Both HEV–IRES–EGFP(+) and HEV–IRES–EGFP(–) constructs, with the reporter in either orientation, were positive for fluorescence, indicating the generation of plus and minus strands of HEV RNA, respectively (Fig. 6). The presence of sense and antisense RNA has been confirmed in the vector-transfected cells. Due to very low levels of expression, the relative expression levels produced from the forward- and reverse-orientation IRES–EGFP constructs could not be compared. Therefore, EGFP was replaced by the RLuc reporter gene in the same constructs. Tenfold-higher luminescence with HEV–IRES–RLuc(+)-transfected cells than those tranfected with HEV–IRES–RLuc(–) (Fig. 6f) is well in agreement with the higher detectable levels of the positive strand than the negative strand (Panda et al., 2000). Expression from the antisense orientation-expressing cassette HEV–IRES–EGFP/RLuc(–) is in contradiction to the earlier reported cases of Kunjin virus (Khromykh & Westaway, 1997) and West Nile virus (Shi et al., 2002), where no expression was observed when IRES–reporter gene was inserted in the reverse orientation. We believe that this expression may be due to the presence of a polyadenylation signal (SV40pA) in our insertion cassette after the reporter gene, which does not base pair and might result in the unwinding of the negative strand, hence facilitating IRES-mediated reporter-gene expression. In addition, there might be a difference in the helicases of HEV and Kunjin virus.

Similar reporter replicons have been described for members of the genus Alphavirus (Liljestrom & Garoff, 1991; Xiong et al., 1989) and flaviviruses, such as West Nile virus (Shi et al., 2002) and Kunjin virus (Khromykh & Westaway, 1997). These replicons can be utilized for studying adaptive mutations, thereby providing replicons with better replication efficiency. The advantage of these dicistronic constructs is the separation of RNA replication from virion assembly and maturation. Hence, they may permit accurate mapping of the protein and RNA motifs that are involved directly in HEV replication.

These RNA replicons do not encode structural proteins; thus, they are incapable of generating infectious particles and the level of heterologous product synthesized in transfected cells is related directly to the transfection efficiency of the recombinant RNA. Under the described conditions for RNA transfection using cationic liposomes or electroporation for these vectors, their usefulness is limited for high-level production or experiments where expression in every cell is required. Either packaging cell lines or vectors that can deliver at 100 % efficiency must be utilized (Polo et al., 1999).

With only a small percentage of cells being transfected with HEV–reporter replicons, we attempted to establish a cell line containing the full-length virus genome. Transfection of cells with an HEV-carrying plasmid (expressing neomycin phosphotransferase) allowed us to select G418-resistant cells maintaining the pCDNA3-HEV vector. The G418-resistant cells showed viral RNA and proteins (replicase, ORF2 and ORF3). Viral RNA could be detected in the culture supernatant until the seventh passage (50 days). Virally encoded proteins of the expected sizes were obtained for both ORF2 and ORF3 (Fig. 7), but analysis of RdRp revealed a smaller protein of approximately 37 kDa. Similar patterns of protein expression were obtained in transient-transfection studies with uncapped transcripts of full-length HEV (Panda et al., 2000). The selected G418-resistant cells should serve as a useful tool to define the components of the replication complex.


   ACKNOWLEDGEMENTS
 
This work was supported by a Grant-in-Aid programme from the Department of Biotechnology, Government of India, to S. K. P. D. T. and S. R. are Senior Research Fellows of the Council of Scientific and Industrial Research (CSIR) at the Department of Pathology, AIIMS, India.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Agrawal, S., Gupta, D. & Panda, S. K. (2001). The 3' end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp). Virology 282, 87–101.[CrossRef][Medline]

Bradley, D. W. (1990). Enterically transmitted non-A, non-B hepatitis. Br Med Bull 46, 442–461.[Abstract]

Clayson, E. T., Innis, B. L., Myint, K. S., Narupiti, S., Vaughn, D. W., Giri, S., Ranabhat, P. & Shrestha, M. P. (1995). Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. Am J Trop Med Hyg 53, 228–232.[Medline]

Dawson, G. J., Mushahwar, I. K., Chau, K. H. & Gitnick, G. L. (1992). Detection of long-lasting antibody to hepatitis E virus in a US traveller to Pakistan. Lancet 340, 426–427.[Medline]

Emerson, S. U., Zhang, M., Meng, X.-J., Nguyen, H., St Claire, M., Govindarajan, S., Huang, Y. K. & Purcell, R. H. (2001). Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element. Proc Natl Acad Sci U S A 98, 15270–15275.[Abstract/Free Full Text]

Emerson, S. U., Nguyen, H., Graff, J., Stephany, D. A., Brockington, A. & Purcell, R. H. (2004). In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. J Virol 78, 4838–4846.[Abstract/Free Full Text]

Garkavenko, O., Obriadina, A., Meng, J., Anderson, D. A., Benard, H. J., Schroeder, B. A., Khudyakov, Y. E., Fields, H. A. & Croxson, M. C. (2001). Detection and characterisation of swine hepatitis E virus in New Zealand. J Med Virol 65, 525–529.[CrossRef][Medline]

Hsieh, S.-Y., Meng, X.-J., Wu, Y.-H., Liu, S.-T., Tam, A. W., Lin, D.-Y. & Liaw, Y.-F. (1999). Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. J Clin Microbiol 37, 3828–3834.[Abstract/Free Full Text]

Jameel, S., Zafrullah, M., Ozdener, M. H. & Panda, S. K. (1996). Expression in animal cells and characterization of the hepatitis E virus structural proteins. J Virol 70, 207–216.[Abstract]

Kabrane-Lazizi, Y., Meng, X.-J., Purcell, R. H. & Emerson, S. U. (1999). Evidence that the genomic RNA of hepatitis E virus is capped. J Virol 73, 8848–8850.[Abstract/Free Full Text]

Khromykh, A. A. & Westaway, E. G. (1997). Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol 71, 1497–1505.[Abstract]

Khuroo, M. S. (1980). Study of an epidemic of non-A, non-B hepatitis: possibility of another human hepatitis virus distinct from post-transfusion non-A, non-B type. Am J Med 68, 818–824.[Medline]

Koonin, E. V., Gorbalenya, A. E., Purdy, M. A., Rozanov, M. N., Reyes, G. R. & Bradley, D. W. (1992). Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses. Proc Natl Acad Sci U S A 89, 8259–8263.[Abstract/Free Full Text]

Korkaya, H., Jameel, S., Gupta, D. & 9 other authors (2001). The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. J Biol Chem 276, 42389–42400.[Abstract/Free Full Text]

Levis, R., Schlesinger, S. & Huang, H. V. (1990). Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription. J Virol 64, 1726–1733.[Medline]

Liljestrom, P. & Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 9, 1356–1361.[CrossRef][Medline]

Magden, J., Takeda, N., Li, T., Auvinen, P., Ahola, T., Miyamura, T., Merits, A. & Kääriäinen, L. (2001). Virus-specific mRNA capping enzyme encoded by hepatitis E virus. J Virol 75, 6249–6255.[Abstract/Free Full Text]

Meng, X.-J., Purcell, R. H., Halbur, P. G., Lehman, J. R., Webb, D. M., Tsareva, T. S., Haynes, J. S., Thacker, B. J. & Emerson, S. U. (1997). A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci U S A 94, 9860–9865.[Abstract/Free Full Text]

Meng, X. J., Halbur, P. G., Shapiro, M. S., Govindarajan, S., Bruna, J. D., Mushahwar, I. K., Purcell, R. H. & Emerson, S. U. (1998). Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. J Virol 72, 9714–9721.[Abstract/Free Full Text]

Nanda, S. K., Panda, S. K., Durgapal, H. & Jameel, S. (1994). Detection of the negative strand of hepatitis E virus RNA in the livers of experimentally infected rhesus monkeys: evidence for viral replication. J Med Virol 42, 237–240.[Medline]

Nishizawa, T., Takahashi, M., Mizuo, H., Miyajima, H., Gotanda, Y. & Okamoto, H. (2003). Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99 % identity over the entire genome. J Gen Virol 84, 1245–1251.[Abstract/Free Full Text]

Ooi, W. W., Gawoski, J. M., Yarbough, P. O. & Pankey, G. A. (1999). Hepatitis E seroconversion in United States travelers abroad. Am J Trop Med Hyg 61, 822–824.[Abstract/Free Full Text]

Panda, S. K., Ansari, I. H., Durgapal, H., Agrawal, S. & Jameel, S. (2000). The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. J Virol 74, 2430–2437.[Abstract/Free Full Text]

Polo, J. M., Belli, B. A., Driver, D. A. & 10 other authors (1999). Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors. Proc Natl Acad Sci U S A 96, 4598–4603.[Abstract/Free Full Text]

Purcell, R. H. & Ticehurst, J. R. (1988). Enterically transmitted non-A, non-B hepatitis: epidemiology and clinical characteristics. In Viral Hepatitis and Liver Diseases, pp. 131–137. Edited by A. J. Zuckerman. New York: Wiley-Liss.

Purdy, M. A., Tam, A. W., Huang, C. C., Yarbough, P. O. & Reyes, G. R. (1993). Hepatitis E virus: a non-enveloped member of the ‘alpha-like’ RNA virus supergroup? Semin Virol 4, 319–326.[CrossRef]

Shi, P.-Y., Tilgner, M. & Lo, M. K. (2002). Construction and characterization of subgenomic replicons of New York strain of West Nile virus. Virology 296, 219–233.[CrossRef][Medline]

Skidmore, S. J. & Sherratt, L. M. (1996). Hepatitis E infection in the UK. J Viral Hepat 3, 103–105.[Medline]

Skidmore, S. J., Yarbough, P. O., Gabor, K. A., Tam, A. W., Reyes, G. R. & Flower, A. J. E. (1991). Imported hepatitis E in UK. Lancet 337, 1541.[CrossRef][Medline]

Song, C. & Simon, A. E. (1995). Requirement of a 3'-terminal stem-loop in in vitro transcription by an RNA-dependent RNA polymerase. J Mol Biol 254, 6–14.[CrossRef][Medline]

Sriram, B., Thakral, D. & Panda, S. K. (2003). Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication. Virology 312, 350–358.[CrossRef][Medline]

Tam, A. W., Smith, M. M., Guerra, M. E., Huang, C.-C., Bradley, D. W., Fry, K. E. & Reyes, G. R. (1991). Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 185, 120–131.[CrossRef][Medline]

Tokita, H., Harada, H., Gotanda, Y., Takahashi, M., Nishizawa, T. & Okamoto, H. (2003). Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993. J Gen Virol 84, 421–427.[Abstract/Free Full Text]

Torresi, J., Li, F., Locarnini, S. A. & Anderson, D. A. (1999). Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells. J Gen Virol 80, 1185–1188.[Abstract]

Wong, D. C., Purcell, R. H., Sreenivasan, M. A., Prasad, S. R. & Pavri, K. M. (1980). Epidemic and endemic hepatitis in India: evidence for a non-A, non-B hepatitis virus aetiology. Lancet ii, 876–879.

Wu, J.-C., Sheen, I.-J., Chiang, T.-Y., Sheng, W.-Y., Wang, Y.-J., Chan, C.-Y. & Lee, S.-D. (1998). The impact of traveling to endemic areas on the spread of hepatitis E virus infection: epidemiological and molecular analyses. Hepatology 27, 1415–1420.[CrossRef][Medline]

Xia, X., Huang, R. & Li, D. (2000). Studies on the subgenomic RNAs of hepatitis E virus. Wei Sheng Wu Xue Bao 40, 622–627 (in Chinese).[Medline]

Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C. M. & Huang, H. V. (1989). Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science 243, 1188–1191.[Medline]

Zafrullah, M., Ozdener, M. H., Panda, S. K. & Jameel, S. (1997). The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. J Virol 71, 9045–9053.[Abstract]

Zafrullah, M., Ozdener, M. H., Kumar, R., Panda, S. K. & Jameel, S. (1999). Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein. J Virol 73, 4074–4082.[Abstract/Free Full Text]

Received 19 October 2004; accepted 23 December 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Thakral, D.
Articles by Panda, S. K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Thakral, D.
Articles by Panda, S. K.
Agricola
Articles by Thakral, D.
Articles by Panda, S. K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS