Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth inCell Culture Depends on an Inserted Human Gene Segment Acquiredby Recombination

P. Shukla, H. T. Nguyen, K. Faulk, K. Mather, U. Torian, R. E. Engle, and S. U. Emerson

Molecular Hepatitis and Hepatitis Viruses Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health,Bethesda, Maryland, USA

An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells wasconstructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertionthat encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomeswith this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advan-tage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin,the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cellculture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein,rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by�3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequenceresulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent proteinhad no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 se-quence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain,but this chimeric genome acquired a dramatic ability to replicate in hamster cells.

Hepatitis E virus (HEV) is a small RNA virus that belongs to thegenus Hepevirus in the family Hepeviridae (18). It causes hep-

atitis E, a disease clinically indistinguishable from hepatitis A (7).Initially, it was thought to occur only in underdeveloped regionsof the world, where it caused waterborne epidemics and sporadicdisease. However, in the past decade it has emerged as a sporadicdisease in industrialized countries, including Great Britain, theEuropean Union, and the United States (17, 23). This “emer-gence” is due to the recognition that there are not one, but fourgenotypes of HEV (genotypes 1 to 4) that infect humans and thatgenotypes 3 and 4 also routinely infect swine and occasionallyother species (16). Genotypes 1 and 2 are found mainly in under-developed countries, where they are spread via contaminated wa-ter: in contrast, genotypes 3 and 4 are zoonotic and are found inindustrialized countries, where they are spread mainly througheating undercooked pork or game products.

HEV infection was long thought to be an acute infection, last-ing 2 to 7 weeks, that never progressed to chronicity. Recently,however, chronic HEV infection has been identified in immuno-suppressed organ transplant patients or in AIDS patients (3, 10–12, 24, 25). Even more unexpectedly, some of these chronically illpatients have developed neurological symptoms (11, 12), andHEV has been isolated from cerebrospinal fluid (11). Thesechronic cases have been identified as genotype 3 infections.

The 7.2-kb genome of HEV (Fig. 1) is a single strand of posi-tive-sense RNA with three overlapping reading frames (ORFs)(18). Approximately the first 5 kb serve as mRNA for the ORF1polyprotein; it is not known whether the polyprotein is proteolyti-cally processed. ORF1 contains regions encoding methyl trans-ferase/guanylyltransferase, NTPase/helicase, RNA-dependentRNA polymerase and deubiquinating activities (13). In addition,ORF1 encodes a Y region and an X or macro region of unknown

function and a hypervariable region (HVR) of approximately 250nucleotides (nt) that is located near the middle of the ORF (1). TheHVR varies in length and sequence among strains and genotypes:it tolerates small deletions, but replication levels of deletion mu-tants are severely depressed in cell culture (27). ORF2 and ORF3are translated from a single bicistronic, subgenomic RNA to pro-duce a 660-amino-acid (aa) capsid protein and a 113- to 114-aamultifunctional protein, respectively (9). The ORF3 protein is re-quired for efficient release of virus particles from cultured cellsand is required for the infection of macaques (5, 8, 33).

HEV studies were long hindered by the absence of an efficientcell culture system for any of the genotypes. That deficit was par-tially overcome when Okamoto and coworkers (22) succeeded inadapting a genotype 3 (29) and a genotype 4 (30) strain, isolatedfrom acutely infected patients, to grow in cultured PLC/PRF/5human hepatoma cells. These researchers also constructed an in-fectious cDNA clone of the genotype 3 virus and implicated pointmutations as the basis for the adaptation (32). Shukla et al. (28)subsequently adapted the Kernow C-1 strain of genotype 3 virus,isolated from a chronically infected patient, to grow in culturedHepG2/C3A cells. This adapted virus differed strikingly from thetwo adapted acute strains by containing, in addition to point mu-tations, a 58-aa human S17 ribosomal protein fragment inserted

Received 18 January 2012 Accepted 29 February 2012

Published ahead of print 7 March 2012

Address correspondence to S. U. Emerson, semerson@niaid.nih.gov.

P.S. and H.T.N. contributed equally to this article.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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into the HVR. In the present study, the adapted Kernow strain wascloned as an infectious cDNA, and mutagenesis studies were per-formed to determine whether the inserted S17 sequence played arole in cell culture adaptation and/or host range.

MATERIALS AND METHODScDNA construction. Passage 1 Kernow-C1 virus RNA was extracted fromthe medium of infected HepG2/C3A cells with TRIzol LS reagent (LifeTechnologies). Reverse transcription-PCR (RT-PCR) was performedwith SuperScript II reverse transcriptase (Life Technologies), HerculaseHotStart Taq (Stratagene), and PrimeStar HS DNA polymerase (Ta-KaRa). A total of six overlapping fragments were joined by fusion PCRand ligation to generate the full-length genome. A unique XbaI restrictionsite and a T7 RNA polymerase core promoter were engineered into the 5=end and a stretch of 36 adenosines, followed by a unique MluI site and aHindII site engineered into the 3= end. The full-length genomic cDNA wasligated into pBlueScript SK(�) (Stratagene) between the XbaI and HindIIsites of the polylinker to generate p1. p6 was synthesized using standardmethods to replace restriction fragments of p1 with the correspondingfragments amplified by RT-PCR from the medium of passage 6 viruscultures. The GTPase sequence was amplified from a cDNA clone of theHVR region recovered from the feces of a U.S. patient (21). The greenfluorescent protein (GFP) sequences were amplified from a modified ge-notype 1 HEV cDNA clone described previously (4). The gaussia lucifer-ase gene was amplified from the pGLuc basic vector purchased from NewEngland Biolabs. Unique NruI and AatII restriction sites introduced at the5= and 3= termini of the S17 sequence were used to construct the S17truncations. RNA folding was predicted with Mfold (34).

RT-PCR. RNA was extracted with TRIzol LS (Invitrogen). The HVRwith flanking regions (nt 1986 to 2762) was amplified by nested RT-PCRwith a Qiagen Long-Range 2 Step RT-PCR kit. The visible product, in-cluding the neighboring regions above and below it, was eluted from anagarose gel and directly sequenced (consensus sequence) or cloned withthe Zero Blunt TOPO PCR cloning kit (Invitrogen). RNA genomes inculture medium were quantified by real-time RT-PCR (TaqMan). Primersequences and amplification conditions will be provided on request.

In vitro transcription and transfection of cultured cells. S10-3 cellsare a subclone of human Huh-7 cells isolated in-house. Human HepG2/C3A (CRL-10741), swine LLC-PK1 (CL-101), and hamster BHK-21(CCL-10) cells were purchased from the American Type Culture Collec-tion; HepG2/C3A cells were grown on rat tail collagen type 1 (Millipore).Cells were propagated in Dulbecco modified Eagle medium supple-

mented with L-glutamine, penicillin-streptomycin, gentamicin, and 10%fetal bovine serum (Ultra-Low immunoglobulin G from Invitrogen).

Plasmids were linearized at a 3= terminal MluI (Kernow-related) orBglII (Sar-related, GenBank accession no. AF444002) sites. Capped RNAtranscripts were generated with a T7 riboprobe in vitro transcription sys-tem (Promega) and Anti-Reverse Cap Analog (Ambion) as described pre-viously (6). For transfection of S10-3 and BHK-21 cells, 23 �l of RNAtranscription mixture, 1 ml of Opti-MEM (Gibco), and 20 �l of DMRIE-C (Invitrogen) were mixed and added to cells in a T25 flask. After incu-bation with transfection mixture for 5 h at 34.5°C in a CO2 incubator, thetransfection mixture was replaced with culture medium, and incubationwas continued at 34.5°C.

HepG2/C3A and LLC-PK1 cells were killed by DMRIE-C, so they weretransfected by electroporation using a Bio-Rad Gene Pulser II at settingsof 240 V and 950 capacitance using Bio-Rad cuvette 165-2086. RNA tran-scripts from a 100-�l transcription mixture were extracted with TRIzol LS(Invitrogen), precipitated with isopropanol, washed with 75% ethanol,and resuspended in 50 �l of water. Confluent cells in a 100-mm dish were,treated with trypsin, mixed with an equal volume of 1% crystalline bovineserum albumin in phosphate-buffered saline (PBS), and pelleted at 525 �g at 4°C for 5 min. The cells were resuspended in 400 �l of Opti-MEM(Gibco), mixed with the RNA, pulsed, added to culture medium contain-ing 20% fetal bovine serum, and incubated at 37°C (HepG2/C3A) or34.5°C (LLC-PK1) overnight; HepG2/C3A electroporated cells in a T25flask were supplemented with one-fourth of the untreated cells from a T25flask in order to provide a dense enough culture to promote growth. Thenext morning, medium was replaced with fresh medium containing 10%serum, and the incubation was continued.

Infection of cultured cells. Approximately 100,000 cells/well wereseeded onto eight-well Lab-Tek II CC2 slides (Nunc) a day before infec-tion. Virus samples were diluted in Opti-MEM or cell culture medium,and 100 �l of the diluted virus was added to each well, followed by incu-bation for 5 h at 34.5°C in a CO2 incubator. The virus mixture was re-moved, and cell culture medium was added, followed by incubation at34.5°C.

Immunofluorescence analysis and focus-forming assay. Transfectedor infected cells on chamber slides were washed with PBS and fixed andpermeabilized with acetone. ORF2 and ORF3 proteins were detected witha mixture of HEV ORF2-specific hyperimmune plasma from an HEV-infected chimpanzee (Ch1313) (6), and rabbit anti-ORF3 peptide anti-body (4): the chimpanzee plasma was preadsorbed on the respective cellsto minimize background staining. Secondary antibodies were a mixture ofAlexa Fluor 488-conjugated goat anti-human IgG (Molecular Probes) andAlexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes).Stained cells were overlaid with Vectashield mounting medium withDAPI (4=,6=-diamidino-2-phenylindole; Vector Laboratories) and visual-ized at 40� with a Zeiss Axioscope 2 Plus fluorescence microscope. Pos-itive cells or foci were manually counted.

Flow cytometric analysis for ORF2 protein. Trypsinized cells werepelleted at 525 � g, mixed with 1 ml of methanol for 15 min at 4°C, andstored at �80°C until all of the samples from one experiment were stainedin parallel. The cells were pelleted out of methanol, washed once with 5 mlof PBS, and resuspended in 100 �l of blocking solution (0.5% skim milk,0.5% crystalline bovine serum albumin, and 0.1% Triton X-100 in PBS) atroom temperature for 30 min before the addition of 100 �l of 2� chimp1313 preadsorbed serum; the cells were then washed with 10 ml of PBSand resuspended in 100 �l of anti-human Alexa Fluor 488-conjugatedantibody. After 30 min, the cells were washed with 10 ml of PBS, resus-pended in �0.5 ml of PBS, and analyzed using a FACScan flow cytometer(Becton Dickinson). A total of 20,000 to 40,000 events were acquired foreach sample, and the data were analyzed using BD CellQuest software.

Luciferase assay. Every 24 h posttransfection, all of the medium wasremoved from the culture, filtered through a 0.45-�m-pore-size MillexHV filter (Millipore), and stored at �80°C. Fresh medium was then addedto the culture, and incubation was continued. Media from the entire ex-

FIG 1 Diagram of the HEV genome summarizing the location and size, inamino acids, of nonviral sequences inserted into the HVR region. S17, humanS17 ribosomal protein fragment in the Kernow strain; GTPase, GTPase-acti-vating protein fragment in the Kernow strain; S19, S19 ribosomal proteinfragment in the LBPR strain (21).

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periment were thawed together, and 20 �l was assayed for luciferase ac-tivity using the Renilla luciferase assay system (Promega) according to themanufacturer’s protocol. Briefly, 20 �l of culture medium was added perwell of a 96-well black, flat-bottom microplate (Corning), followed by theaddition of Renilla luciferase assay substrate and the detection of lumines-cence using a Synergy 2 Multi-Mode microplate reader (Bio-Tek, Win-ooski, VT). The microplate reader was set to dispense 50 �l of substrate,followed by shaking for 2 s and reading for 5 s. Samples were assayed intriplicate and read sequentially.

GenBank accession numbers. Sequences have been deposited inGenBank under accession numbers JQ679013 to JQ679025.

RESULTS

The fact that the Kernow virus adapted by six serial passages inHepG2/C3A cells was a recombinant virus containing part of ahuman S17 gene was not discovered until the passage 6 virus wassequenced (28). Although viral genomes containing the 171-ntS17 sequence could be detected in the feces by nested RT-PCRwith virus-human primer pairs (25), they constituted such a mi-nor quasispecies that they were not represented in 120 cDNAclones of the HVR region of viruses in the fecal inoculum (data notshown). In order to determine when in the passage series the viruscontaining this insert first emerged and when it became the dom-inant species, the HVR region of viruses in the medium at each ofthe six cell culture passage levels was amplified by RT-PCR,cloned, and sequenced. Two of eleven clones from the first passagealready contained the S17 sequence and from passage 2 onward; itwas present in the majority of clones (Table 1). Amazingly, a dif-

ferent mammalian gene insert, 114 nt long, was present in 5 otherof the 11 clones from the first cell culture passage and, in this case,an almost identical sequence was found in 2 of the 120 clones fromthe feces (Fig. 1). This 114-nt sequence lacked 10 nt in the middleof the GTPase-activating protein gene sequence (GenBank acces-sion no. AB384614.1) and consisted of a rearranged gene segmentin which GTPase nt 3009 to 3105 were followed by GTPase nt 2981 to3008, and the reading frame was changed so that the sequence, asinserted, encoded an unrelated amino acid sequence that did notmatch anything when BLAST analyzed against all known nonredun-dant protein databases. However, this insert was not detected in anyof the clones from subsequent passages 2 through 6.

Infectious cDNA virus clones. The medium of cultured cellsshould contain the members of a virus quasispecies that are bestable to infect and complete a replication cycle in these cells. There-fore, the first full-length cDNA clone of the Kernow virus wasconstructed from uncloned cDNA fragments amplified from themedium (passage 1 virus) of HepG2/C3A cells that had been in-oculated 111 days previously with a stool suspension containingthe original Kernow strain. This Kernow passage 1 cDNA clone,p1 (GenBank accession no. JQ679014), lacked the S17 insert anddiffered from the consensus sequence of virus in the feces (Gen-Bank HQ389543) by 16 aa, including an extra proline (Table 2). Itwas transfected into S10-3 hepatoma cells, which were monitored5 to 6 days later by immunofluorescence microscopy for cellsstained for ORF2 protein. Less than 2% of the S10-3 cells trans-fected with in vitro transcripts of the passage 1 clone produceddetectable ORF2 protein, suggesting that this virus genome, al-though infectious, lacked elements essential for robust replica-tion. Incorporation of the S17 insert into the cDNA clone to yieldp1/S17 increased the number of cells successfully transfected, butthe levels remained below �10%. In an attempt to derive a morerobust virus and to identify regions that contributed to cell cultureadaptation, convenient restriction fragments of p1/S17 cDNAswere sequentially replaced with the quasispecies of uncloned PCRproduct amplified from passage 6, cell-culture-adapted virus (Ta-ble 2). Transcripts from multiple clones of these new full-lengthgenomes were transfected into S10-3 cells and examined for ORF2production by immunofluorescence microscopy. The clone pro-ducing the highest percentage of transfected cells was used as thebackbone for the next substitution, and this process was repeatedfour more times. Finally, all clones were compared by flow cyto-metry in the same experiment (Fig. 2). The first three sequential

TABLE 1 Comparison of HVR clones from each passage level

Passage (totalno. of clones)

No. of clones

S17a GTPaseb

Deletion(s)(no. of nt)c

1 (11) 2 5 1*, 1 (612), 2 (501)2 (10) 9 0 1 (738)3 (8) 8 0 04 (10) 8 0 2 (435)d

5 (8) 8 0 06 (11) 10 0 1 (381)a That is, the number of clones with an S17 insert encoding 58 aa.b That is, the number of clones containing 114 nt of the GTPase gene and lacking S17.c Containing a large deletion compared to passage 6 but with the S17 insert intact(except as noted). *, S17 insert absent.d These were two identical clones with deletions removing the 3= 45 nt of S17.

TABLE 2 Stepwise modification of passage 1 virus clone by swapping in fragments from passage 6 virus

Name p6 sequence added Mutation(s) addeda

p1 None aa838[extra P]p1/S17 S17 insert aa750[58 additional amino acids]3=ORF2,nc SnaB1-MluI[6812-poly(A)] aa593-594[TL/AS] 652-653[MK/TE]

nt7355-6[tt/cc] 7381[c/t] 7405[c/t] 7407[t/c] [A36/A85]b

ORF2/3 NsiI-SnaB1[4608-6812] aa483[T/A];ORF3:aa1[M/T] 69[M/I]5=ORF1 AsiS1-NotI[671-2182] aa220[A/T] 598[R/C]650[G/A]ORF1/CCA NotI-BsiW1[2182-3063] aa838[P/-] 882[L/P] 904[S/P] 965[R/Q]

nt2520-2534[ccc/cca 4X]p6/ORF3-null BsiW1-NsiI[3063-4608] No amino acid changesp6 NsiI-Pml1[4608-5743] ORF3:aa1[T/M]a Amino acid numbering is based on the passage 6 consensus sequence. Brackets denote mutations as “[passage 1/passage 6]”. Underlining indicates the amino acid mutations inORF2.b Length of polyadenosine tract.

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fragment substitutions had introduced mutations into the 3=ORF2 and noncoding regions [nt 6812-poly(A)], into the 3= ORF1and ORF2/ORF3 overlap (nt 4608 to 6812), and into the 5= third ofORF1 (nt 671 to 2182): of the three fragments, only the 6812-An

substitution significantly increased the level of successful transfec-tions (Fig. 2). Unexpectedly, of the passage 6 PCR ampliconsspanning nt 4608 to 6812, the sequence that boosted successfultransfection levels the most contained mutations that eliminatedthe only two methionine codons (aa 1 and 69) in ORF3 (Table 2);immunofluorescence microscopy confirmed that viruses fromthis cDNA clone and the three subsequent cDNA clones did notproduce ORF3 protein (data not shown). The passage 6 fragmentwith the greatest enhancing effect spanned nt 2182 to 3063 andcontained three naturally occurring amino acid mutations in theX domain and a single proline deletion in the HVR: additionally,four proline codons in this fragment were changed by site-di-rected mutagenesis to CCA codons in order to preserve the aminoacid sequence while disrupting a cluster of C residues in the HVRthat greatly hindered PCR and sequence analyses. The fifth frag-ment substitution (nt 3063 to 4608) contained a highly conserved

region of the helicase and polymerase genes, did not introduce anyamino acid changes, and had no obvious effect (P � 0.067). Fi-nally, the methionine initiation codon of ORF3 was restored sothat ORF3 protein could be produced by the p6 virus. The pres-ence or absence of this methionine codon had no apparent effecton levels of successful transfection of S10-3 cells (compare thep6/ORF3-null and p6 transfection levels in Fig. 1 [P � 1.0]). West-ern blots of cell lysates detected ORF3 protein in p6 transfectedcells but not in p6/ORF3 null transfected cells (data not shown).The p6 clone (GenBank accession no. JQ679013), excluding theinsert, differed from the stool consensus sequence (GenBank ac-cession no. HQ389543) by 16 aa and from p1 by 25 aa but from thepassage 6 consensus sequence (GenBank accession no.HQ709170) by only 2 aa (aa 598 � R to C in ORF1 and aa 593 �T to A in ORF2). Transcripts of the final p6 clone routinely trans-fected 10 to 45% of S10-3 cells.

Because the function of the X domain is unknown and theC-to-A changes in proline codons of the HVR were engineeredrather than natural, it was important to determine whether thethree mutations in the X domain or the C-to-A synonymous mu-tations in the HVR in fragment from nt 2182 to 3063 were themost important for enhancing transfection. Because back-muta-tion of the proline codons would recreate the sequencing prob-lems, the amino acid codons in the X domain were chosen for backmutation. All three mutations in the X domain were simultane-ously back-mutated to the original codons present in the p1 cDNAclone, and the level of successful transfections was quantified byflow cytometry at day 6 posttransfection (Fig. 3). Transcripts fromthe clone containing the three reverted X domain mutations weresignificantly (P � 0.0006) less efficient than those from the p6

FIG 2 Transfection of S10-3 cells with sequential plasmid constructs. Repre-sentative flow cytometry profiles of nontransfected and transfected cells areshown on the left. Numbers beneath each clone indicate the fragment positionon the genome that was replaced with the corresponding fragment amplifiedfrom the passage 6 virus quasispecies; the parentheses indicate gene regionsincluded in the fragment. The new construct served as the background plasmidfor the next replacement, and the procedure was repeated until all of the p1 hadbeen replaced with p6 sequences. Finally, all plasmids were transcribed, trans-fected and immunostained for ORF2 protein in the same experiment: tripli-cate samples were harvested and tested by flow cytometry at 3 days posttrans-fection. Student t test P values are given for adjacent samples. P � 0.05 wasconsidered significant. Error bars indicate the standard deviations.

FIG 3 Reversion of aa 882, 904, and 965 in the X region reduces the level ofsuccessful transfection. S10-3 cells were transfected with the 5=ORF1 plasmidlacking the CCA and X region mutations (fragment 671-2182), the ORF1/CCAplasmid containing both the CCA and X region mutations (fragment 2182-3063), p6, and a revertant plasmid containing the CCA but not the X regionmutations (p6/882; 904; 965 revert). Cells in triplicate samples were immuno-stained and analyzed by flow cytometry at 6 days posttransfection. P values aregiven, and error bars denote the standard deviations.

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cDNA clone in successfully transfecting S10-3 cells and not signif-icantly different (P � 0.12) from the 671-2182 clone, which lackedboth the HVR proline mutations and the X domain mutations,suggesting that the engineered changes that interrupted thepoly(C) tract had a minimal effect on successful transfections,whereas one or more of the three mutations in the X region playeda critical role.

Removal of the S17 sequence decreases successful transfec-tions. In order to determine whether the effect of the S17 sequencewas limited to the modest increase in transfection levels observedfollowing its insertion into the p1 cDNA clone, the S17 sequencewas selectively removed from the p6 cDNA clone containing all ofthe point mutations to yield p6delS17. Flow cytometry confirmedthat addition of S17 sequence to p1 virus genomes significantlyincreased successful transfections by these genomes, although thelevels did not approach those attained by the recombinant p6genomes (Fig. 4). Surprisingly, removal of the S17 sequence fromthe p6 cell culture-adapted cDNA clone dramatically decreasedsuccessful transfections by the genome transcripts to levels only3-fold better than those of the p1delS17 cDNA clone (Fig. 4). Thisresult suggested that the point mutations responsible for the in-cremental improvement in successful transfections by the serialclones were mostly ineffective in the absence of S17 sequence.

Luciferase expression from ORF2. The flow cytometry analy-ses based on ORF2 protein immunostaining revealed the percent-age of cells that produced detectable ORF2 protein, but they didnot provide a quantitative comparison of the amount of ORF2protein produced or of the duration of ORF2 synthesis. In order toconfirm and extend the flow cytometry data, the 5= portion ofORF2 was replaced with the in-frame gaussia luciferase reportergene to yield p6/luc: this luciferase has a signal sequence that re-sults in its secretion and accumulation in the cell culture medium.Therefore, sequential time points can be obtained from the sameculture.

The luciferase system was validated by measuring the amountof luciferase secreted into the medium by p6/luc virus containingeither a functional polymerase or a mutated, nonfunctional poly-merase that could not synthesize viral RNA. Whereas the lucifer-ase signal in medium from S10-3 cells transfected with the p6/lucdefective polymerase mutant or from untransfected S10-3 cellswas less than 111 U/24 h at its peak on day 2, that in the mediumof cells transfected with p6/luc rose from 2,163 U/24 h on day 1 toover 36 million units/24 h on days 4 through 6 (data not shown).

Therefore, luciferase production requires viral RNA synthesis,as predicted based on the ORF2 location of the luciferase gene inthe subgenomic mRNA. Luciferase production by p6/luc viruswas then compared to that by the p6/luc virus mutated to eitherdelete the S17 insert or to eliminate the three X gene mutations.The production of luciferase by p6/luc virus, either with or with-out the S17 insert, peaked on day 6 posttransfection, but the ratioof p6/luc units to p6/luc(del S17) units steadily increased andreached 52-fold at day 7, thus confirming that the S17 insert con-ferred a significant growth advantage and served as a cell culture-adaptive mutation (Fig. 5A). In addition, the luciferase data forthe three X gene back-mutations was consistent with that from theflow cytometry analyses (Fig. 5B); cultures transfected with thep6/luc X gene revertant produced less luciferase than those trans-fected with the p6/luc virus. It is interesting that, as shown in bothFig. 5A and B, the luciferase values on day 9 decreased substan-tially for the p6/luc virus but remained near plateau levels for themutants.

Synonymous mutations did not decrease transfection. Theenhancing effect of the S17 insert could be due to either the RNAor the protein sequence. In an attempt to distinguish betweenthese possibilities, the third base in 93 and 70% of the 58 codons inS17 was changed (purine to purine and pyrimidine to pyrimidine)in two clones without altering the encoded AA except for twoM-to-I changes. The number of cells successfully transfected byeach of these two clones did not differ significantly from that of p6(Fig. 6), even though the predicted RNA structures and �G (Gibbsfree energy) differed from those of p6 (�G � �120.38) and eachother (�G � �102.99 and 98.97 for clones 1 and 2, respectively).Therefore, it appeared that the enhancing effect probably oc-curred at the protein level.

Effect of insert size. The previous studies demonstrated theimportance of the S17 insert for growth of the Kernow virus in cellculture but did not provide any insights into how it functioned.Since the first passage of the stool inoculum had provided evi-dence for a possible enhancing effect of the GTPase insert ongrowth of the Kernow strain in cell culture, this insert was substi-tuted for that of the S17 insert in the p6 clone to evaluate its effect.Although the 114-nt GTPase insert increased the number of suc-cessfully transfected cells, it was only about half as effective as the171-nt S17 insert (Fig. 7A). In order to determine whether lengthof the insert per se was a factor, sequence encoding the N-terminalor C-terminal 58 aa (174 nt) of GFP was substituted for the S17sequence. GFP was chosen because it has been shown to be rela-tively benign when expressed as a fusion protein with many part-ners in many cell types. Indeed, fluorescence microscopy indi-cated that GFP was produced when the entire coding region wasfused in-frame to the 3= terminus of the S17 insert (data notshown); however, neither of the 174 nt encoding 58 aa of theN-terminal or C-terminal amino acids of GFP had a detectableeffect on the levels of transfection of S10-3 cells (Fig. 7A and B).

FIG 4 Removal of S17 sequence from p6 eliminates the adaptive effect of mostpoint mutations. S10-3 cells were transfected with p1 and p6 plasmids with orwithout S17 sequence. Triplicate samples were analyzed by flow cytometry atday 4 posttransfection. P values were all �0.0001 except for p1/S17 versusp6delS17. Error bars denote the standard deviations.

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Therefore, the number of nucleotides and/or amino acids in itselfis not a determining factor. The effect of size was tested also byremoving approximately half of the nucleotides from the S17 in-serted sequence in p6 to yield 87 to 90 nt of sequence encoding theN-terminal half, the C-terminal half, or the middle portion of theS17 insert. All three constructs successfully transfected cells to asimilar extent that averaged 2- to 6-fold less than if the entire insertwas present and 2.5-fold more than if it was absent (Fig. 7C).Finally, 117 nt of another mammalian gene sequence (S19 ribo-

somal protein) which we discovered inserted into the HVR ofanother genotype 3 strain from a different chronically infectedhepatitis E patient (21), was substituted for the S17 sequence in thep6 clone. Although genomes carrying the S19 sequence in thisdifferent genotype 3 strain had been selected during culture inHepG2/C3A cells, much as had the S17-containing Kernow ge-nomes (21), transfer of this sequence from that genotype 3 strainto the Kernow strain resulted only in a modest enhancement(Fig. 7D).

p6 encodes a virus that can infect both swine and humancells. Both the S10-3 cells used for transfection and the HepG2/C3A cells to which the passage 6 virus was adapted are humanhepatoma cells, so it was important to determine whether the p6virus retained the ability of the original fecal inoculum to growalso in swine cells (28). Transcripts of p6 and p6del S17 wereelectroporated into LLC-PK1 swine kidney cells, which were as-sayed by flow cytometry 5 days later. ORF2 protein was producedin swine cells by both constructs, demonstrating that both thenegative strand genomes and the subgenomic mRNAs had beensynthesized by each. More than 31% of the swine cells were trans-fected by the p6 clone compared to 12% by the p6 clone missingthe S17 insert, thus demonstrating that the S17 sequence en-hanced transfection of swine cells as it did human cells (Fig. 8A).Next, the p6 virus itself was tested for the ability to infect swinecells. Two different lots of p6 virus grown in HepG2/C3A cellswere titered in parallel on HepG2/C3A cells and LLC-PK cells (Fig.8B). In both cases, the infectivity titer was higher on the swine cellsthan on the human cells; although the difference varied for the twopreparations and reached significance in only one case, the p6cDNA clone clearly did encode a virus which could infect culturedcells originating from each of the two major host species for geno-type 3 HEV.

FIG 5 Expression of luciferase from ORF2 is substantial and prolonged in thepresence of the S17 insert. The ORF2 viral capsid protein was replaced with thegaussia luciferase gene in p6 genomes lacking the S17 insert or the X generegion mutations. After transfection of S10-3 cells, culture medium was com-pletely replaced every 24 h. (A) The ratio of luciferase units produced by p6/lucgenomes with (solid bars) or without (hatched bars) the S17 insert is shown inparentheses above each time point. Error bars are standard deviation. (B) Theaverage luciferase production from genomes encoded by two independentcDNA clones (stippled and crosshatched bars) lacking the three X gene muta-tions was decreased 2.3- to 5.1-fold compared to that from p6/luc genomes(ratios are shown in parentheses above each time point).

FIG 6 Synonymous mutations in the S17 insert have little effect on the effi-ciency of successful transfections. Mutations that preserved the amino se-quence were introduced into the third base of 54/58 codons (mutant 1) or41/58 codons (mutant 2) in theS17 insert, and RNA transcripts were trans-fected into S10-3 cells. The efficiency of successful transfection was determinedby flow cytometry of triplicate samples at 6 days posttransfection.

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Effect of S17 sequence on a genotype 1 strain. Transcriptsfrom a genotype 1 cDNA clone, Sar55, transfect S10-3 cells readily,but extensive attempts to adapt the virus to grow in cell culturehave thus far failed (S. Emerson, unpublished data). A previousexperiment had demonstrated that recombinant Sar55 genomescontaining the S17 sequence from p6 virus in their HVR (Sar55/S17) were able to transfect S10-3 cells, but a quantitative compar-ison with Sar55 genomes lacking the insert was not performed(28). Therefore, Sar55 and Sar55/S17 transcripts were transfectedinto S10-3 cells, which were subjected to flow cytometry 5 dayslater. Both sets of transcripts produced a similar number of ORF2-positive cells, suggesting that the S17 sequence neither enhancednor diminished successful transfection Sar55 genomes in this sys-tem (Fig. 9A).

Since the Kernow virus had displayed such a diverse host rangepreviously (28), p6 transcripts were tested for the ability to trans-fect hamster BHK-21 cells and were found to produce ORF2-positive cells, although with low efficiency (3.8% for BHK-21compared to 30.1% for S10-3). Therefore, the Sar55 and Sar55/S17 transcripts also were tested by flow cytometry for the ability to

FIG 7 Comparison of efficiency of successful transfection by p6 genomic tran-scripts encoding different HVRs. Triplicate samples were subjected to flow cyto-metry at day 5 posttransfection. Error bars represent the standard deviation, andbrackets denote Student t test P values. (A) The 174 nt encoding the 58-aa S17insert were deleted or replaced with the 114-nt GTPase insert from passage 1 orwith the 3=-terminal 174 nt of green fluorescent protein (GFP). #1 and #2 are twoindependent clones. P values for p6 versus any other genome were �0.0003. (B) 5=174 nt encoding the first 58 aa of GFP. # 1 to #3 are independent clones. P values forp6 versus any other genome were �0.001. (C) The 5=half, middle, or 3=half (87 nt)of the S17 insert replaced full-length S17. P values for p6 versus any other genomewere �0.0002. (D) 117 nt S19 ribosomal protein gene insert. #1 and 2 are inde-pendent clones. The P values for p6 versus any other genome were �0.001, and theP values among the three GFP clones were �0.27.

FIG 8 Genomes or viruses encoded by p6 can replicate in, and infect swineLLC-PK1 cells. (A) Swine cells transfected with transcripts from p6delS17 orp6 containing the S17 insert were assayed by flow cytometry at day 5 posttrans-fection. (B) Triplicate samples of p6 virus harvested from the medium oftransfected HepG2/C3A cells were titered in parallel on HepG2/C3A cells(open bars) and LLP-CK1 cells (stippled bars) under code.

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transfect BHK-21 cells, even though these cells were an unlikelyhost given the restricted host range of genotype 1 viruses. Amaz-ingly, not only were the hamster cells transfected by the Sar55genomes, the number of transfected cells was boosted almost7-fold by inclusion of the S17 insert (Fig. 8B, P � 0.0001). Themarked enhancement of transfection by the S17 insert was con-firmed by immunofluorescence microscopy in an independentexperiment (Fig. 9C).

p6 encodes a virus that grows in and spreads among HepG2/C3A cells. Since the p6 cDNA genome was derived from virusadapted to grow in HepG2/C3A cells, the virus encoded by thiscDNA clone was predicted to replicate and spread efficiently incultures of these cells: in contrast, previous studies implicatingORF3 protein in virus egress (5, 33) suggested that a p6/ORF3-null virus genome incapable of producing ORF3 might transfectas many cells as did p6 genomes but that virus would not spread to

other cells. p6 virus genomes and p6/ORF3-null genomes wereelectroporated into HepG2/C3A cells, and virus production andspread were monitored by flow cytometry. The p6 virus and theORF3-null mutant displayed surprisingly similar patterns, andboth appeared to replicate and spread efficiently throughout theculture: in both cases, the percentage of ORF2 protein-positivecells increased from ca. 15% on day 5 to more than 70% on day 14(Fig. 10). An independent experiment produced similar resultswith the percentage of positive cells increasing from 12.4% 1.96% to 59.7% 0.87% for p6 virus and from 13.3% 0.31% to67.8% 5.57% for the ORF3-null mutant between days 5 to 15.Although these results demonstrated that the p6 clone did indeedencode a cell-culture-adapted virus, the similar levels of cell-to-cell spread for the two viruses was puzzling because both our lab-oratory (5) and that of Okamoto (33) had published data, dem-onstrating that efficient viral egress required functional ORF3protein; in these reports, virus release in the absence of ORF3protein was only ca. 10% as much as that in its presence. Sequenceanalysis of the ORF3 region of the null mutant genomes amplifiedby RT-PCR from the day 9 medium confirmed that no methio-nine codons were present, and ORF3 protein was not detected byimmunofluorescence microscopy of the cells (data not shown).However, an infectious focus assay performed with the mediumfrom the two cultures identified an average of 11630 FFU of p6

FIG 9 Effect of S17 insert on Sar55 successful transfection of S10-3 andBHK-21 cells. The efficiency of transfection of S10-3 (A) and BHK-21 (B) cellswas monitored by flow cytometry. (C) Immunofluorescence microscopy oftransfected BHK-21 cells stained for ORF2 protein at day 5 posttransfection.

FIG 10 Lack of the ORF3 protein does not inhibit cell-to-cell spread inHepG2/C3A cultures. HepG2/C3A cells were electroporated with transcriptsfrom p6 or p6/ORF3-null plasmids, mixed with naive HepG2/C3A cells andcultured at 37°C. Triplicate samples were harvested on each of indicated days,fixed with methanol, and stored at �80°C until assayed by flow cytometry. Theresults of representative flow cytometry scans are shown. Error bars indicatethe standard deviation. P � 0.74 for day 5 values of the two viruses, indicatingthat a similar number of cells had been successfully transfected with eachconstruct.

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virus/ml and twice as many, 23,200 FFU/ml, of the ORF3-nullmutant (Table 3). Determination by real-time RT-PCR of thenumber of viral genomes in the medium was most revealing: therewere indeed �10-fold fewer viral genomes released into the me-dium for the ORF3-null mutant compared to the p6 virus (Table3). Calculations of the number of viral genomes per FFU indicatedthat the mean specific infectivity of the ORF3-null mutant viruswas �25-fold higher than that of p6 virus itself. Therefore, thedecrease in egress from cells due to a lack of ORF3 was more thanoffset by the increase in infectivity, thus enabling the null mutantto spread through the culture as efficiently as the parent p6 virus.

DISCUSSION

A nonhomologous recombination event involving the HEV ge-nome and a human RNA molecule was ultimately responsible forthe ability of the Kernow strain of HEV to flourish in cell culture.Such a dramatic change in virus phenotype following virus-hostRNA recombination is rare, but a few cases have been reportedpreviously. Thus, a poliovirus lethal mutation was pseudorevertedby introduction of 15 host nucleotides into a mutated cleavage siteduring replication in cell culture (2). Similarly, introduction, byrecombination, of 54 host nucleotides into the hemagglutinincleavage site of an apathogenic influenza virus produced a virusvariant with increased pathogenicity (14). Even more strikingly, arare recombination event which inserted 228 or more nucleotidesof host ubiquitin gene sequence into a noncytopathogenic variantof bovine viral diarrhea virus rendered the virus cytopathogenic(19).

The infectious genotype 3 cDNA clone we constructed pro-vides an additional tool for HEV research. Since the liver is thetarget organ for this virus, the ability to transfect or infect humanliver (HepG2/C3A) cells and to produce large quantities of viablevirus may provide a more authentic model system in which torevisit numerous, well-executed studies that produced intriguingdata but were limited by their reliance on overexpression of singleviral proteins out of context. In addition, the ability of p6 virus toinfect both human and swine cells may prove useful for identify-ing parameters that restrict the host range of genotype 1 and 2strains to humans and nonhuman primates. The luciferase repli-con we developed should be especially useful for some studiessince it permits convenient sequential sampling and is exquisitelysensitive: since the luciferase gene is located on the subgenomicmRNA, luciferase production can act as an indirect indicator of

subgenomic RNA synthesis and stability. This new model systemhas already provided the first evidence that the previously unchar-acterized X gene region has a function in viral replication sincethree mutations in it contributed substantially to establishment ofthe infected state following transfection (Fig. 3 and 5).

HEV is not noted for recombination and intergenotypic re-combination has been reported only rarely (31). In retrospect, thismight reflect the different transmission pathways and localizedgeographic distribution of the four human genotypes resulting ina low number of coinfections with two or more readily distin-guishable genomes; intragenotypic recombination might not benoticed unless specifically searched for. However, our discovery ofthree different human sequences embedded in HEV genomesfrom the only two patients examined suggests that HEV may un-dergo recombination more frequently than realized. For instance,a genotype 3 virus from France was reported to have an insert ofunidentified origin of �90 nt in the HVR (15). Additional studiesare required to determine whether insertion of ribosomal proteingenes occurred by chance or reflected some unknown aspect ofHEV replication.

The discovery of the human S17 gene sequence embedded inthe HEV genome (28) was especially surprising since it indicated(i) that the virus genome had recombined with host RNA and (ii)that this event had apparently imparted properties that resulted inselection of this extremely minor quasispecies virus in cell culture.This scenario was subsequently repeated with a genotype 3 strainfrom another chronically infected patient (21), suggesting thatillegitimate recombination by HEV is not necessarily a rare event.In the present study, we demonstrated that this recombinant virusemerged as soon as the first passage in cell culture (Table 1): itsdominance in all passages thereafter strongly suggested that theinsert played a critical role in cell culture adaptation. Mutagenesisstudies of the infectious cDNA clone demonstrated unequivocallythat the insert was a major factor in enabling efficient virus prop-agation in cell culture and therefore was an adaptive mutation.However, since the stepwise cloning strategy demonstrated thatmutations other than the S17 insert also contributed to adaptation(Fig. 2), it was surprising to find an almost total elimination ofenhancement of transfection by point mutations upon removal ofthe S17 insert from the final construct (Fig. 4). One possible ex-planation is that the inserted S17 sequence enhanced the stability/translatability of the RNA or aided the folding/processing/stabilityof ORF1 protein. The question of proteolytic processing has notyet been resolved for HEV: however, since introduction of synon-ymous mutations into 24 to 32% of the nucleotide positions in theS17 insert did not appreciably affect the level of transfection (Fig.6), it seems unlikely that the viral RNA is the critical factor, butrather suggests that the effect is at the protein level. Deletion ex-periments have shown that decreasing the size of the standardHVR could decrease the virulence of HEV or reduce its replicationin cell culture (27). Our database analysis of the 50% truncationsof the S17 insert demonstrated that the size of the insert, and henceof the HVR, matters, but the experiments substituting GFP,GTPase, or S19 gene fragments (7A, B, and D) suggested that theamino acid composition of both the insert and the genomic back-ground must also contribute to enhancement. This conclusion isin agreement with data showing decreased replication in vitrowhen the HVR of a genotype 1 and a genotype 3 strain wereswapped (26).

Very few HEV strains have been successfully propagated in cell

TABLE 3 Specific infectivity of virions released from HepG2/C3A cells

Virusa Culture% Positivecellsb

No. ofgenomes(SD)c FFU (SD)d

Specificinfectivitye

p6 1 73.92 33.3 (5.1) 1,270 (90) 26,2002 75.53 19.9 (1.4) 1,360 (384) 14,6003 74.92 35.4 (2.8) 860 (112) 41,200

p6-null 1 81.34 4.6 (1.8) 2,790 (329) 1,6002 79.49 2.7 (2.8) 2,430 (385) 1,1003 76.89 1.1 (0.4) 1,740 (413) 600

a Three cultures each of p6 and p6/ORF3-null were assayed.b ORF2-positive cells were quantified by flow cytometry at 12 days posttransfection.c That is, the mean number of viral genomes � 10�6 /100 �l of medium (triplicates).d That is, the number of focus-forming units (FFU)/100 �l of medium (triplicates).e Calculated by dividing mean number of genomes by the mean number of FFU.

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culture, and the selection of the S17 recombinant in cell cultureraised the hope that insertion of this sequence into other geno-types and strains might increase the success rate. Certainly, theability of Sar55-S17 to transfect human or hamster cells waspromising. Unfortunately, in toto, the data suggested that inser-tion of foreign sequences into the HVR of an HEV genome will nothave a predictable impact and that subjection to long-term selec-tive pressure in culture may be the only way to obtain culturablestrains. On the other hand, the fact that the S17 sequence in-creased the ability of Sar55 genomes to replicate in cells from suchan unlikely species as the hamster leads one to speculate that newsyndromes, such as neurological disorders recently associatedwith HEV infections, may reflect the ability to infect new cell typesbecause of changes in the HVR. Indeed, Kamar et al. have reportedthat genomes in the cerebrospinal fluid of a chronically infectedpatient had sequence differences from those that circulated in theserum (11). Certainly, this possibility merits further exploration.

Transfection and infection experiments with human HepG2/C3A and swine LLC-PK1 cells demonstrated that the p6 virusclone retained the ability of the fecal virus quasispecies to crossspecies boundaries and displayed a slight preference for swinecells. In contrast, the titer of the fecal inoculum was previouslyreported to be up to 13-fold higher on swine cells compared tohuman cells (28), which suggests that there might be other mem-bers of the fecal quasispecies that either had mutations favorablefor infection of swine cells or detrimental for infection of humancells (Fig. 8). It is not known whether receptors or other factorsdetermine host range. Between the p6 cloned virus and the con-sensus sequence of viruses in the feces, there are 4 aa differences inthe capsid protein which might affect receptor interactions. Twoof the four mutations were also present in the p1 virus clone whichrepresented the first selection step for HepG2/C3A cells, so it willbe interesting to determine whether reversion of any of these mu-tations to the consensus sequence in the feces will increase therelative titer on swine cells.

Although both p6 virus and ORF3-null virus eventually spreadand infected the majority of HepG2/C3A cells in a culture, theydid so relatively slowly, and the percentage of infected cells did notbegin to increase until after day 7 (Fig. 10). In contrast, luciferaseexpression was detected in the culture medium as soon as day 1posttransfection (2,163 U) and had jumped 38-fold by day 2 (Fig.5A). Since the luciferase is translated from the subgenomicmRNA, viral negative-strand and subgenomic RNA synthesismust have been greatest between days 0 and 2 in this experiment,suggesting that synthesis of viral RNA and/or proteins is probablynot rate limiting but rather that assembly, maturation, and/orexcretion are responsible for the relatively slow production of in-fectious HEV virions. It is worth noting that since the luciferaseconstruct lacks a capsid gene, it cannot spread, so the data in Fig.5B suggested that translation of p6 subgenomic mRNA continuedat peak rates through day 7 or 8 before declining.

Perhaps the most confounding result was the discovery that avirus unable to make ORF3 protein spread throughout the cultureas efficiently as one synthesizing ORF3 protein. This result posesmore questions than answers. The observed difference in specificinfectivities provides an explanation of why it happened, but thequestion of why the specific infectivities differed remains. Oka-moto has proposed that HEV egress from PLC/PRF/5 cells de-pends on ORF3 protein interaction with cellular protein Tsg101(20). So, is the null mutant exiting the HepG2/C3A cells by a

different pathway than p6 uses? Is a different pathway used indifferent cell lines? Are any of the available culture systems trulyreliable models for infection in vivo? It now may be possible toaddress some of these questions.

ACKNOWLEDGMENTS

This research was supported by the Intramural Research Program of theNational Institutes of Health, National Institute of Allergy and InfectiousDiseases.

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