Capped RNA Transcripts of Full-Length cDNA Clones
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病菌学杂志 2005年第3期
Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia
College of Veterinary Medicine, Iowa State University, Ames, Iowa
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.
INTRODUCTION
Hepatitis E virus (HEV), the causative agent of human hepatitis E, is a single positive-sense RNA virus in the new genus Hepevirus (8). HEV is transmitted by the fecal-oral route through contaminated drinking water. The mortality rate among hepatitis E patients is usually <1%, but it can reach up to 20% for infected pregnant women (12, 14). Hepatitis E is rarely diagnosed in industrialized countries, even though a significant proportion of healthy individuals in these countries are positive for antibodies to HEV (19, 31). Antibodies to HEV have also been reported for various animal species (1, 10, 15, 22), suggesting that hepatitis E may be a zoonotic disease (21).
In 1997, the first animal strain of HEV, swine HEV, was isolated and characterized from a pig in the United States (25). Experimental infections of specific-pathogen-free (SPF) pigs with swine HEV (23) and cross-species infections of HEV between swine and nonhuman primates (24) have been demonstrated. Swine HEV has since been identified in pigs in many other countries; in each case, it was found to be closely related to genotype 3 or 4 strains of human HEV (5, 16, 22). The prototype strain of swine HEV and two closely related U.S. strains of human HEV (US1 and US2) belong to genotype 3 (9). Although the US2 strain of human HEV infected pigs and the prototype swine HEV strain infected nonhuman primates, the infected animals did not develop clinical symptoms of hepatitis (24), even though both viruses replicated in various tissues and organs of infected pigs (34). Genotype 1 or 2 human HEV was unable to infect pigs under experimental conditions (23). For humans, it has been reported that pig handlers have an increased risk of HEV infection compared to healthy blood donors (4, 26), suggesting that hepatitis E may be a zoonosis. Recently, a cluster of hepatitis E cases was linked to the consumption of raw deer meats (30), and several cases of acute hepatitis E were also epidemiologically linked to the ingestion of undercooked pork livers in Japan (35), thus providing more convincing evidence of zoonotic HEV transmission.
The molecular biology of HEV is poorly understood. The HEV genome is 7.2 kb long and consists of a short 5' nontranslated region, three open reading frames (ORFs), and a short 3' nontranslated region followed by a poly(A) tract (5). ORF1 encodes a nonstructural protein containing putative functional domains characteristic of a methyltransferase, a Y domain, a papain-like protease, a helicase, and an RNA-dependent RNA polymerase (RdRp) (18). The ORF2 gene encodes the capsid protein, which contains a signal sequence at its N terminus. The N-terminal region of the capsid protein is postulated to interact with the negative charge-containing genomic RNA (36). The C-terminal region of the capsid protein contains several antigenic sites, including a neutralization epitope located at residues 452 to 617 (20). The ORF3 gene overlaps with ORFs 1 and 2 and encodes an immunogenic protein with an unknown function.
Due to the lack of an efficient cell culture system to propagate HEV, studies of the replication and transcription mechanisms of HEV are still very challenging. The reverse genetic system, which allows direct genetic manipulation of RNA viruses, is an extremely powerful tool for structural and functional studies of HEV genes. Although infectious cDNA clones of genotype 1 human HEV have been reported (7, 27), it was important to construct an infectious cDNA clone of a swine strain of HEV so that chimeric viruses between human and swine HEVs can be made in order to dissect the structural and functional relationships of HEV genes. More importantly, the availability of a swine HEV infectious cDNA clone will afford us an opportunity to use pigs as a model system to study the pathogenesis and replication of HEV and to understand the mechanism of cross-species infection.
We report here the construction and in vitro and in vivo characterizations of three infectious cDNA clones of the prototype genotype 3 swine HEV.
MATERIALS AND METHODS
Virus and cells. The virus used for this study was the prototype genotype 3 swine HEV isolated from a pig in the United States in 1997 (25). Huh7 cells were originally isolated in Japan and were cultured as previously described (6).
Construction of a full-length cDNA clone of swine HEV. The complete nucleotide sequence of the swine HEV genome was determined previously (13, 24). For construction of a full-length cDNA clone, swine HEV RNA was extracted from an infectious stock of swine HEV (24) by use of the Trizol reagent. Reverse transcription-PCR (RT-PCR) was used to amplify the swine HEV genome as previously described (23, 24). A total of eight overlapping fragments covering the entire swine HEV genome were amplified and subsequently ligated together at unique restriction enzyme sites present in each fragment (Fig. 1). Specific modifications were used to produce the 5' and 3' termini. A unique HindIII restriction site and a T7 RNA polymerase core promoter were introduced at the extreme 5' terminus. A short stretch of 15 adenosines (A) was engineered at the 3' end of the swine HEV genome, followed by a unique XbaI site for plasmid linearization (Fig. 1). The full-length genomic cDNA was ligated into the pGEM-9zf(–) vector (Promega) between the HindIII and XbaI sites of the polylinker to produce a full-length cDNA clone designated clone C.
Site-directed mutagenesis to produce three new full-length cDNA clones of swine HEV with various mutations at different genomic regions. After determining the complete sequence of clone C and comparing it to the consensus sequence of swine HEV, we identified a total of 14 mutations scattered across the entire genome of clone C (Table 1). Site-directed mutagenesis was used to create three new unique full-length cDNA clones. Three genomic fragments, BX, XAf, and AvN, were excised from clone C by the use of unique restriction enzymes (BamHI and XhoI for fragment BX, XhoI and AflII for fragment XAf, and AvrII and NdeI for fragment AvN) (Fig. 1), subcloned, and used as templates for site-directed mutagenesis according to the manufacturer's protocol (QuikChange Multi site-directed mutagenesis kit; Stratagene). Briefly, three mutations in fragment BX, four mutations in fragment XAf, and five mutations in fragment AvN were changed back to the consensus sequence (Fig. 1; Table 1). The corrected fragment BX replaced the corresponding region in clone C to produce clone pSHEV-1. The corrected fragment AvN was introduced into the corresponding region of clone pSHEV-1 to generate clone pSHEV-2. Clone pSHEV-3 contained all three corrected fragments (Fig. 1). Two silent mutations at nucleotides (nt) 38 and 1028 in all three clones were retained and used as genetic markers.
In vitro RNA transcription. The full-length cDNA clones of swine HEV were linearized with XbaI and purified by phenol-chloroform extraction and ethanol precipitation. For in vitro characterizations of the infectivity of the cDNA clones for Huh7 cells, 5 μg of linearized plasmid DNA was transcribed in a 50-μl reaction mixture containing 10 μl of 5x transcription buffer (Promega), 5 μl of 10 mM dithiothreitol (Promega), 2 μl of 40-U/ml RNasin (Promega), 5 μl of nucleoside triphosphates (a 5 mM concentration [each] of ATP, CTP, and UTP and 0.5 mM GTP), 5 μl of 5 mM 7-methyl-GTP (Ambion), and 2 μl of 20-U/ml T7 polymerase (Promega). The mixtures were incubated at 37°C for 2 h, with an additional 2 μl of polymerase added after the first hour. Each transcription mixture was cooled on ice and used "as is" for in vitro transfection of Huh7 cells (6) or of a subclone of Huh7 cells that were transfected more efficiently. Five microliters of transcript was treated with RQ1 DNase (Promega) and electrophoresed in a 1% agarose gel; single bands of equal intensities and sizes were observed for all samples used for each experiment.
For the pig intrahepatic inoculation study, RNA transcripts were synthesized with an mMessage mMachine T7 transcription kit (Ambion). For each cDNA clone, in vitro transcription reactions were performed in a 500-μl reaction mixture containing 20 μg of linearized plasmid DNA, 50 μl of 10x reaction buffer, 250 μl of 2x nucleoside triphosphate-Cap, 50 μl of enzyme mix, and an additional 25 μl of a 30 mM GTP stock for capping. After the reaction mixture was incubated at 37°C for 1.5 h, 5 μl of the transcription mixture was electrophoresed in a 1% agarose gel to check the quality of the transcripts. The remaining reaction mixture for each clone was diluted with 3 volumes of cold RNase-, DNase-, and proteinase-free PBS buffer (1.5 ml), divided into aliquots in two vials, and immediately frozen on dry ice until used for the inoculation of pigs the next day.
In vitro transfection and immunofluorescence assay (IFA). Prior to in vitro transfection, a subclone of Huh7 cells in a 24-well plate were washed with Optimem (Gibco). Ten microliters of the transcription mixture was mixed with 200 μl of Optimem containing 4 μl of DMRIE-C (Invitrogen), and the mixture was immediately added to a drained well. After 5 h of incubation at 34.5°C, the mixture was aspirated, fresh medium (Dulbecco's modified Eagle's medium containing 5% fetal bovine serum) was added, and the cultures were incubated at 34.5°C.
On day 5 or 6 after transfection, the cells were trypsinized and replated in eight-well glass chamber slides. The slides were fixed and stained on day 6 or 7 as previously described (6). Briefly, cells on slides were rinsed in phosphate-buffered saline (PBS), fixed with acetone at room temperature, and air dried. The cells were then overlaid with a 1:1 mixture of 10% bovine serum albumin and PBS containing chimpanzee 1313 anti-HEV convalescent-phase serum or mouse anti-ORF3 hyperimmune serum. After 20 min at room temperature, the slides were rinsed in PBS and overlaid with PBS containing Alexa fluor 488-conjugated goat anti-human or goat anti-mouse IgG, respectively (Molecular Probes). After 20 min at room temperature, the slides were washed in PBS, Vectashield (Vector Laboratories) was added, and the slides were viewed by indirect immunofluorescence microscopy with a fluorescein isothiocyanate filter. The chimpanzee 1313 (infected with genotype 1 HEV) immune serum is specific for the ORF2 protein, as serum taken from chimpanzee 1313 prior to infection with HEV did not stain transfected cells (6). The mouse anti-ORF3 hyperimmune serum was generated by immunizing BALB/c mice with an affinity-purified recombinant ORF3 protein of swine HEV expressed in bacteria (Z. F. Sun and X. J. Meng, unpublished data).
Intrahepatic inoculation of SPF pigs with capped RNA transcripts from full-length swine HEV cDNA clones. To determine the infectivity of clone C, we intrahepatically inoculated two 6-week-old SPF pigs (pigs 3 and 4) with 1 ml of diluted full-length capped RNA transcripts from clone C. The RNA inocula were quickly thawed and immediately injected by an ultrasound-guided technique into five or six different sites in the liver, at about 200 μl per injection site. Another two SPF pigs (pigs 23 and 30), which were housed in a separate room, were not inoculated and served as negative controls. All pigs were monitored daily for clinical signs of disease for a total of 12 weeks, at which time they were necropsied.
A separate animal experiment was performed to determine the infectivities of the three corrected clones. Briefly, eight 6-week-old SPF pigs that were seronegative for HEV were randomly assigned into four groups of two each (groups A, B, C, and D). Each group of pigs was housed separately in a biosafety level 2 facility and maintained under conditions that met all requirements of the Institutional Committee on Animal Care and Use. The RNA inocula were similarly injected by the ultrasound-guided technique into the liver of each pig. The two pigs (286 and 293) in group A were each injected with 1 ml of the RNA inoculum from clone pSHEV-1, pigs 294 and 295 (group B) were each injected with 1 ml of the RNA inoculum from clone pSHEV-2, and pigs 297 and 298 (group C) were each inoculated with the RNA inoculum from clone pSHEV-3. The two pigs in group D were not inoculated and served as negative controls. Pigs were monitored daily for clinical signs of disease for a total of 10 weeks. All pigs were necropsied at 10 weeks postinoculation.
ELISA and RT-PCR. Serum and fecal samples were collected from all pigs for both animal experiments prior to inoculation and weekly postinoculation until the end of the studies. Serum samples were tested for anti-HEV IgG by an enzyme-linked immunosorbent assay (ELISA) as previously described (11, 23). Viremia and fecal virus shedding were tested by RT-PCR as described previously (11, 23, 24, 34). In addition, all positive serum samples were retested by a semiquantitative RT-PCR to determine the swine HEV genomic equivalent (GE) titers in sera as previously described (17). One GE is defined as the number of viral genomes present in the highest 10-fold dilution that is positive by RT-PCR (17).
Detection of genetic markers in viruses recovered from experimentally infected pigs. Selected regions of viruses recovered from serum and fecal samples from pigs in groups A, B, and C were amplified by RT-PCR and then sequenced. For the marker at nt 38, a reverse primer (5'-CTCGGATGGGCTCCAACTTCCA-3') was used for reverse transcription and cDNA synthesis. A PCR was performed to amplify this region by use of a sense primer (5'-CCACGTATGTGGTCGATGCC-3') and the reverse primer. For the marker at nt 1028, a reverse primer (5'-ATTTCTGGGCGTGCTCAACCTCC-3') and a sense primer (5'-ATGCCTTATGTCCCCTACCCTCG-3') were used for PCR amplification. For the markers at nt 4175 and 4307 in clones pSHEV-1 and pSHEV-2, a reverse primer (5'-CCGGCATACAGCCCAATAGG-3') and a sense primer (5'-CCTGTTCAGGCCACCACATGTGAGT-3') were used.
In addition, the entire ORF2 gene of viruses recovered from pigs that were inoculated with clones pSHEV-1 and pSHEV-3 was amplified with two pairs of primers (first pair, 5'-ATAACATGTCTTTTGCATCGCC-3' and 5'-GGGCTGAACCAAAATCCTGACATC-3'; second pair, 5'-TCAAACTACAACCACCCCTACTT-3' and 5'-GGGGCACAAAAGGAATTAATTA-3'). The PCR products were directly sequenced by use of the respective PCR primers.
Nucleotide sequence accession numbers. The complete sequences of the three cDNA clones have been deposited in the GenBank database (accession no. AY575857 to AY575859).
RESULTS
Assembly of a full-length cDNA clone of swine HEV and determination of its infectivity. Eight overlapping DNA fragments covering the entire viral genome were amplified by RT-PCR and subsequently assembled into a full-length cDNA by ligation at appropriate restriction enzyme sites (Fig. 1). The nucleotide G, instead of the A nucleotide present in many other known HEV strains, was found at the extreme 5' end of the swine HEV genome. A HindIII site and a T7 core promoter were introduced immediately preceding the first G nucleotide to facilitate in vitro transcription of the viral genomic RNA. The assembled cDNA was cloned into plasmid pGEM-9zf(–) to produce a full-length cDNA clone of swine HEV, which we designated clone C.
The infectivity of clone C was determined by the intrahepatic inoculation of two pigs with full-length capped RNA transcripts from clone C. Swine HEV RNA was not detected by RT-PCR in the weekly sera or fecal swab materials of the two inoculated or two control pigs throughout the 12-week study. Seroconversion to HEV was not detected in either of the two inoculated or two control pigs after 12 weeks, suggesting that clone C was nonviable. In addition, Huh7 cells transfected with RNA transcripts from clone C did not contain detectable ORF2 or ORF3 protein, whereas those transfected in parallel with transcripts from a known infectious cDNA clone did (6; data not shown), suggesting that clone C was not viable in vitro as well as in vivo.
In order to determine the possible reasons for the nonviability of clone C, we redetermined the consensus sequence of the swine HEV genome by direct sequencing of RT-PCR products amplified from a swine HEV infectious stock (24). We found that the new consensus sequence of the swine HEV genome had a total of 17 nucleotide differences compared to the published sequence (accession no. AF082843 and AF011921), and 8 of these differences were nonsilent mutations. A sequence comparison revealed a total of 14 mismatches between clone C and the new consensus sequence (Table 1). Ten of these mismatches were silent mutations, and three mutations in the ORF2 capsid gene (T5338C, A5362G, and C6356T) resulted in amino acid changes. A deletion point mutation at nt 756 (A-756) which resulted in a downstream frameshift of the ORF1 gene was also found in clone C. These mutations in clone C may reflect a quasispecies in the virus stock or may be due to mistakes introduced by RT-PCR and/or cloning procedures, especially in the case of the deletion mutation A-756.
A full-length cDNA clone with a new consensus sequence and two mutant clones of swine HEV are replication competent when transfected into Huh7 cells. Three regions containing mutations in clone C were sequentially replaced by corrected fragments (BX, XAf, and AvN) to produce two mutant clones (pSHEV-1 and pSHEV-2) and a consensus clone (pSHEV-3). Capped full-length RNA transcripts from each clone and from clone C were transfected into a subclone of Huh7 cells to determine the replication competence of the clones. Viral antigens were detected by an IFA using a chimpanzee anti-HEV convalescent-phase serum or a mouse anti-swine HEV ORF3 hyperimmune serum to stain cells that were transfected individually with RNA transcripts from each of the four clones. A fluorescent signal was not detected with either antibody in cells transfected with clone C, but a signal was detected with each antibody in cells transfected with pSHEV-1, pSHEV-2, and pSHEV-3 (Fig. 2).
Capped RNA transcripts from the three swine HEV cDNA clones were infectious when injected intrahepatically into the livers of SPF pigs. Both pigs in groups A, B, and C, which were inoculated with RNA transcripts from one of the three clones, seroconverted to anti-HEV IgG, indicating that active swine HEV infections had occurred in the inoculated pigs (Table 2). Both inoculated and control pigs had no increase in rectal temperature or signs of illness or distress during the entire study, which was consistent with our earlier reports of swine HEV and human HEV infections in pigs (11, 23, 34). Pigs in groups B (pSHEV-2) and C (pSHEV-3) all seroconverted at about 4 to 5 weeks postinoculation, whereas the two pigs in group A (pSHEV-1) had a delayed seroconversion at 9 to 10 weeks postinoculation (Table 2; Fig. 3). The two uninoculated pigs in group D remained seronegative throughout the experiment. Swine HEV RNA was detected in the fecal samples of pigs inoculated with pSHEV-2 and pSHEV-3 starting at 14 days postinoculation (dpi) and lasting for 5 to 6 weeks. Similar to seroconversion, fecal virus shedding in pigs inoculated with pSHEV-1 also appeared late, at 35 (pig 286) and 42 (pig 293) dpi (Table 2).
Viremia was detected variably in inoculated pigs (Table 2). For group B (pSHEV-2), viremia was detected in pig 294 only at 28 dpi and in pig 295 at 21 to 35 dpi. The swine HEV genome titers in the sera of the two group B pigs were 102 GE/ml (Table 2). For group C (pSHEV-3), pig 298 was viremic for 5 weeks, starting at 21 dpi, and the antibody titers of this pig were the highest among all six inoculated pigs (Fig. 3). The swine HEV genome titer in this pig was 104 GE/ml at 21 dpi, 103 GE/ml at 28 to 42 dpi, and 102 GE/ml at 49 dpi. Interestingly, pig 297, which was also in group C, did not have detectable viremia, even though this pig shed virus in its feces for 6 weeks (Table 2). The two pigs in group A (pSHEV-1) also did not have detectable viremia. The lack of viremia, together with the delayed appearances of seroconversion and fecal virus shedding, suggested that the virus encoded by clone pSHEV-1 may be attenuated. Additional studies with larger number of animals are needed to draw a definitive conclusion regarding the attenuation of clone pSHEV-1. The bile sample collected from pig 293 of group A during necropsy at 70 dpi was positive by RT-PCR for swine HEV RNA, with a genome titer of 102 GE/ml, but the bile samples collected from all other pigs during necropsy were negative. Viremia and fecal virus shedding were not detected in either group D negative control pig. All eight pigs appeared clinically normal throughout the experiment.
Viruses recovered from experimentally infected pigs retained their respective genetic markers. To determine if the genetic markers of the three cDNA clones were retained in the respective rescued viruses, we amplified and sequenced genomic regions containing the markers from selected samples from inoculated pigs. For group C pigs, the two silent mutations at nt 38 and 1028 were still present in the viral genomes amplified from fecal samples at 21 dpi. For group B pigs, three genetic markers, nt 1028 in fragment BX and nt 4175 and 4307 in fragment XAf, were also retained in the sequence amplified from fecal samples at 21 dpi. The sequences recovered from all positive samples of the two group A pigs retained the silent mutation C at nt 5811 as well as the markers at nt 4175 and 4307. Taken together, these results confirmed that the viruses infecting the pigs originated from the respective cDNA clones pSHEV-1, pSHEV-2, and pSHEV-3.
The complete ORF2 capsid gene was also amplified and sequenced from the two pSHEV-1-inoculated pigs, from a bile sample from pig 293 at 70 dpi and a fecal sample from pig 286 at 42 dpi. Sequence analysis revealed that the three nonsilent mutations in the capsid gene of clone pSHEV-1 were retained in the genome and that new mutations had not arisen in the ORF2 gene. Additionally, the complete ORF2 gene of the virus infecting pig 298 (pSHEV-3) was amplified, and the sequence was found to be identical to that of clone pSHEV-3.
DISCUSSION
Since there is a lack of an efficient in vitro cell culture system for HEV, the fundamental biology of HEV is poorly understood. Therefore, an infectious cDNA clone of HEV is critical for the study of the molecular biology of this important human pathogen by recombinant DNA technology. Two infectious cDNA clones of genotype 1 human HEV have been reported (7, 27). However, for an understanding of the tissue tropism and mechanisms of cross-species HEV infection, infectious cDNA clones of swine HEV and other genotypes of HEV are needed. For this study, we established a reverse genetic system for a prototype strain of swine HEV. Capped RNA transcripts from each of three full-length cDNA clones of swine HEV were viable both upon transfection into Huh7 cells and after intrahepatic inoculation into the livers of SPF pigs. The detection of genetic markers confirmed that the viruses recovered from the infected pigs originated from the respective cDNA clones.
The successful generation of infectious cDNA clones of RNA viruses is a laborious process which is often hampered by many obstacles, such as unexpected mutations introduced during the RT-PCR and cloning steps, the inherent quasispecies populations of the virus stock, the instability and toxicity of cDNA clones during propagation in Escherichia coli, the infidelity of the RNA polymerase used for in vitro transcription, the specific structures in the viral RNA genome [cap and poly(A) tail] required for efficient replication, and the potential harmful effects of nonviral nucleotides at the ends of RNA transcripts (2). It has been demonstrated that a cap structure is essential for the infectivity of human HEV RNA transcripts (7), and therefore in this study a cap analog was added during the in vitro transcription of swine HEV cDNA clones. Since it was previously shown that 15 adenosine nucleotides in the poly(A) tract are sufficient to rescue human HEV (7), we added 15 A's to the 3' end of the swine HEV genome. There are no nonviral nucleotides at the 5' ends of the RNA transcripts and only one nonviral nucleotide (T) at the 3' ends of the swine HEV cDNA clones. The nonviral nucleotide T at the 3' end of the genome did not abolish the infectivity of the cDNA clones. We did not encounter any problem regarding the stability of the clones during the construction and propagation of the three pSHEV plasmids in E. coli, even though the 7.2-kb genomic cDNA was inserted into the high-copy-number vector pGEM-9zf(–). After multiple-step mutagenesis and subcloning processes, the sequence of the final pSHEV-3 clone was identical to the newly determined consensus sequence of swine HEV, with the exception of the two genetic markers.
The clone C sequence differed from the consensus sequence due to the presence of a deletion mutation at nt 756 and 13 point mutations scattered across the genome. All three new cDNA clones derived from clone C had the corrected BX fragment which reversed the deletion at nt 756. In contrast to the nonviability of clone C, all three corrected clones replicated in vitro and were infectious in vivo. The viral genome titers in sera of inoculated pigs were relatively low (102 to 104 GE/ml) and peaked in the first week of the viremia period (Table 2). This low level of viremia was consistent with our previous studies with live swine HEV (23, 24). The GE titers of swine HEV in the sera of pigs inoculated with the consensus clone pSHEV-3 were at least 2 log higher than those in the two pigs inoculated with the mutant clone pSHEV-2, suggesting that the consensus clone pSHEV-3 replicated better in pigs than did the two mutant clones. The patterns of seroconversion, viremia, and fecal virus shedding in pigs inoculated with pSHEV-2 and pSHEV-3 RNA transcripts were similar, suggesting that the nucleotide differences between these two cDNA clones were not critical for virus replication. In contrast, pigs inoculated with RNA transcripts from clone pSHEV-1 had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia, suggesting that the virus encoded by clone pSHEV-1 may be attenuated in pigs. There are five mutations in the ORF2 capsid gene that are unique to clone pSHEV-1, including three nonsilent mutations at nt 5338, 5362, and 6356. The amino acid residues F-51, T-59, and S-390, corresponding to the three nonsilent mutations in the capsid protein of swine HEV, and the amino acid sequences in the proximity of them are completely conserved among all other known mammalian HEV sequences published to date (data not shown). Therefore, the substitutions at these three positions were unique to the swine HEV clone pSHEV-1. The three nonsilent mutations likely occurred as a result of natural strain variations, since the capsid protein was under immunological pressure. The changes from polar residues to nonpolar residues at amino acid positions 59 (Thr to Ala) and 390 (Ser to Leu) were likely important since such a change may drastically alter the structure of a viral protein and therefore influence its functions. It has been well documented that one or more amino acid changes in capsid or envelope proteins can contribute to the attenuation phenotype of viruses (3, 28, 29, 32, 33). Therefore, additional studies with a larger number of pigs and with infectious cDNA clones containing a single mutation and combination double mutations of the three nonsilent mutations are warranted to definitively determine the roles of these mutations in swine HEV infection.
An infectious human HEV cDNA clone, pSK-HEV-3, which contains a silent mutation at the C terminus of the ORF2 capsid gene, produces an attenuated virus in chimpanzees (7). This mutation slightly changed the secondary structure of a cis-reactive element, which was believed to be related to the attenuation. However, additional nearby mutations were generated in infected chimpanzees to restore the cis-reactive element (7). In the present study, the three nonsilent mutations in the capsid were stable in the virus, and other compensatory mutations were not identified in the capsid genes of recovered viruses. The stability of these mutations in clone pSHEV-1 should be very useful for further pathogenesis studies. The availability of these swine HEV infectious cDNA clones now affords us an opportunity to use pigs as a model to dissect the basic biology of HEV and to study the potential mechanisms of zoonotic transmission by constructing chimeric viruses between swine and human HEVs.
ACKNOWLEDGMENTS
This study was supported by grants from the National Institutes of Health (AI01653, AI50611, and AI46505) and in part by a grant from the U.S. Department of Agriculture (NRI 2002-35204-12531).
We thank Robert Purcell for his support, Denis Guenette for his technical assistance, and Z. F. Sun for anti-swine HEV ORF3 mouse hyperimmune serum preparation.
Y.W.H. and G.H. contributed equally to this study.
Present address: Virology Section, National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran.
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College of Veterinary Medicine, Iowa State University, Ames, Iowa
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
ABSTRACT
Swine hepatitis E virus (swine HEV), the first animal strain of HEV to be isolated, is a zoonotic agent. We report here the construction and in vitro and in vivo characterizations of infectious cDNA clones of swine HEV. Eight overlapping fragments spanning the entire genome were amplified by reverse transcription-PCR and assembled into a full-length cDNA clone, clone C, which contained 14 mutations compared to the consensus sequence of swine HEV. RNA transcripts from clone C were not infectious, as determined by intrahepatic inoculation into pigs and by in vitro transfection of Huh7 cells. Multiple site-based site-directed mutagenesis was performed to generate three new cDNA clones (pSHEV-1, pSHEV-2, and pSHEV-3) which differed from each other. The transfection of capped RNA transcripts into human liver Huh7 cells resulted in the synthesis of both ORF2 capsid and ORF3 proteins, indicating that the cDNA clones were replication competent. Each of the three clones resulted in active swine HEV infections after the intrahepatic inoculation of pigs with capped RNA transcripts. The patterns of seroconversion, viremia, and fecal virus shedding for pigs inoculated with RNA transcripts from clones pSHEV-2 and pSHEV-3 were similar to each other and to those for pigs inoculated with wild-type swine HEV, suggesting that the nucleotide differences between these two cDNA clones were not critical for replication. Pigs inoculated with RNA transcripts from clone pSHEV-1, which contained three nonsilent mutations in the ORF2 capsid gene, had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia. The availability of these infectious cDNA clones affords us an opportunity to understand the mechanisms of cross-species infection by constructing chimeric human and swine HEVs.
INTRODUCTION
Hepatitis E virus (HEV), the causative agent of human hepatitis E, is a single positive-sense RNA virus in the new genus Hepevirus (8). HEV is transmitted by the fecal-oral route through contaminated drinking water. The mortality rate among hepatitis E patients is usually <1%, but it can reach up to 20% for infected pregnant women (12, 14). Hepatitis E is rarely diagnosed in industrialized countries, even though a significant proportion of healthy individuals in these countries are positive for antibodies to HEV (19, 31). Antibodies to HEV have also been reported for various animal species (1, 10, 15, 22), suggesting that hepatitis E may be a zoonotic disease (21).
In 1997, the first animal strain of HEV, swine HEV, was isolated and characterized from a pig in the United States (25). Experimental infections of specific-pathogen-free (SPF) pigs with swine HEV (23) and cross-species infections of HEV between swine and nonhuman primates (24) have been demonstrated. Swine HEV has since been identified in pigs in many other countries; in each case, it was found to be closely related to genotype 3 or 4 strains of human HEV (5, 16, 22). The prototype strain of swine HEV and two closely related U.S. strains of human HEV (US1 and US2) belong to genotype 3 (9). Although the US2 strain of human HEV infected pigs and the prototype swine HEV strain infected nonhuman primates, the infected animals did not develop clinical symptoms of hepatitis (24), even though both viruses replicated in various tissues and organs of infected pigs (34). Genotype 1 or 2 human HEV was unable to infect pigs under experimental conditions (23). For humans, it has been reported that pig handlers have an increased risk of HEV infection compared to healthy blood donors (4, 26), suggesting that hepatitis E may be a zoonosis. Recently, a cluster of hepatitis E cases was linked to the consumption of raw deer meats (30), and several cases of acute hepatitis E were also epidemiologically linked to the ingestion of undercooked pork livers in Japan (35), thus providing more convincing evidence of zoonotic HEV transmission.
The molecular biology of HEV is poorly understood. The HEV genome is 7.2 kb long and consists of a short 5' nontranslated region, three open reading frames (ORFs), and a short 3' nontranslated region followed by a poly(A) tract (5). ORF1 encodes a nonstructural protein containing putative functional domains characteristic of a methyltransferase, a Y domain, a papain-like protease, a helicase, and an RNA-dependent RNA polymerase (RdRp) (18). The ORF2 gene encodes the capsid protein, which contains a signal sequence at its N terminus. The N-terminal region of the capsid protein is postulated to interact with the negative charge-containing genomic RNA (36). The C-terminal region of the capsid protein contains several antigenic sites, including a neutralization epitope located at residues 452 to 617 (20). The ORF3 gene overlaps with ORFs 1 and 2 and encodes an immunogenic protein with an unknown function.
Due to the lack of an efficient cell culture system to propagate HEV, studies of the replication and transcription mechanisms of HEV are still very challenging. The reverse genetic system, which allows direct genetic manipulation of RNA viruses, is an extremely powerful tool for structural and functional studies of HEV genes. Although infectious cDNA clones of genotype 1 human HEV have been reported (7, 27), it was important to construct an infectious cDNA clone of a swine strain of HEV so that chimeric viruses between human and swine HEVs can be made in order to dissect the structural and functional relationships of HEV genes. More importantly, the availability of a swine HEV infectious cDNA clone will afford us an opportunity to use pigs as a model system to study the pathogenesis and replication of HEV and to understand the mechanism of cross-species infection.
We report here the construction and in vitro and in vivo characterizations of three infectious cDNA clones of the prototype genotype 3 swine HEV.
MATERIALS AND METHODS
Virus and cells. The virus used for this study was the prototype genotype 3 swine HEV isolated from a pig in the United States in 1997 (25). Huh7 cells were originally isolated in Japan and were cultured as previously described (6).
Construction of a full-length cDNA clone of swine HEV. The complete nucleotide sequence of the swine HEV genome was determined previously (13, 24). For construction of a full-length cDNA clone, swine HEV RNA was extracted from an infectious stock of swine HEV (24) by use of the Trizol reagent. Reverse transcription-PCR (RT-PCR) was used to amplify the swine HEV genome as previously described (23, 24). A total of eight overlapping fragments covering the entire swine HEV genome were amplified and subsequently ligated together at unique restriction enzyme sites present in each fragment (Fig. 1). Specific modifications were used to produce the 5' and 3' termini. A unique HindIII restriction site and a T7 RNA polymerase core promoter were introduced at the extreme 5' terminus. A short stretch of 15 adenosines (A) was engineered at the 3' end of the swine HEV genome, followed by a unique XbaI site for plasmid linearization (Fig. 1). The full-length genomic cDNA was ligated into the pGEM-9zf(–) vector (Promega) between the HindIII and XbaI sites of the polylinker to produce a full-length cDNA clone designated clone C.
Site-directed mutagenesis to produce three new full-length cDNA clones of swine HEV with various mutations at different genomic regions. After determining the complete sequence of clone C and comparing it to the consensus sequence of swine HEV, we identified a total of 14 mutations scattered across the entire genome of clone C (Table 1). Site-directed mutagenesis was used to create three new unique full-length cDNA clones. Three genomic fragments, BX, XAf, and AvN, were excised from clone C by the use of unique restriction enzymes (BamHI and XhoI for fragment BX, XhoI and AflII for fragment XAf, and AvrII and NdeI for fragment AvN) (Fig. 1), subcloned, and used as templates for site-directed mutagenesis according to the manufacturer's protocol (QuikChange Multi site-directed mutagenesis kit; Stratagene). Briefly, three mutations in fragment BX, four mutations in fragment XAf, and five mutations in fragment AvN were changed back to the consensus sequence (Fig. 1; Table 1). The corrected fragment BX replaced the corresponding region in clone C to produce clone pSHEV-1. The corrected fragment AvN was introduced into the corresponding region of clone pSHEV-1 to generate clone pSHEV-2. Clone pSHEV-3 contained all three corrected fragments (Fig. 1). Two silent mutations at nucleotides (nt) 38 and 1028 in all three clones were retained and used as genetic markers.
In vitro RNA transcription. The full-length cDNA clones of swine HEV were linearized with XbaI and purified by phenol-chloroform extraction and ethanol precipitation. For in vitro characterizations of the infectivity of the cDNA clones for Huh7 cells, 5 μg of linearized plasmid DNA was transcribed in a 50-μl reaction mixture containing 10 μl of 5x transcription buffer (Promega), 5 μl of 10 mM dithiothreitol (Promega), 2 μl of 40-U/ml RNasin (Promega), 5 μl of nucleoside triphosphates (a 5 mM concentration [each] of ATP, CTP, and UTP and 0.5 mM GTP), 5 μl of 5 mM 7-methyl-GTP (Ambion), and 2 μl of 20-U/ml T7 polymerase (Promega). The mixtures were incubated at 37°C for 2 h, with an additional 2 μl of polymerase added after the first hour. Each transcription mixture was cooled on ice and used "as is" for in vitro transfection of Huh7 cells (6) or of a subclone of Huh7 cells that were transfected more efficiently. Five microliters of transcript was treated with RQ1 DNase (Promega) and electrophoresed in a 1% agarose gel; single bands of equal intensities and sizes were observed for all samples used for each experiment.
For the pig intrahepatic inoculation study, RNA transcripts were synthesized with an mMessage mMachine T7 transcription kit (Ambion). For each cDNA clone, in vitro transcription reactions were performed in a 500-μl reaction mixture containing 20 μg of linearized plasmid DNA, 50 μl of 10x reaction buffer, 250 μl of 2x nucleoside triphosphate-Cap, 50 μl of enzyme mix, and an additional 25 μl of a 30 mM GTP stock for capping. After the reaction mixture was incubated at 37°C for 1.5 h, 5 μl of the transcription mixture was electrophoresed in a 1% agarose gel to check the quality of the transcripts. The remaining reaction mixture for each clone was diluted with 3 volumes of cold RNase-, DNase-, and proteinase-free PBS buffer (1.5 ml), divided into aliquots in two vials, and immediately frozen on dry ice until used for the inoculation of pigs the next day.
In vitro transfection and immunofluorescence assay (IFA). Prior to in vitro transfection, a subclone of Huh7 cells in a 24-well plate were washed with Optimem (Gibco). Ten microliters of the transcription mixture was mixed with 200 μl of Optimem containing 4 μl of DMRIE-C (Invitrogen), and the mixture was immediately added to a drained well. After 5 h of incubation at 34.5°C, the mixture was aspirated, fresh medium (Dulbecco's modified Eagle's medium containing 5% fetal bovine serum) was added, and the cultures were incubated at 34.5°C.
On day 5 or 6 after transfection, the cells were trypsinized and replated in eight-well glass chamber slides. The slides were fixed and stained on day 6 or 7 as previously described (6). Briefly, cells on slides were rinsed in phosphate-buffered saline (PBS), fixed with acetone at room temperature, and air dried. The cells were then overlaid with a 1:1 mixture of 10% bovine serum albumin and PBS containing chimpanzee 1313 anti-HEV convalescent-phase serum or mouse anti-ORF3 hyperimmune serum. After 20 min at room temperature, the slides were rinsed in PBS and overlaid with PBS containing Alexa fluor 488-conjugated goat anti-human or goat anti-mouse IgG, respectively (Molecular Probes). After 20 min at room temperature, the slides were washed in PBS, Vectashield (Vector Laboratories) was added, and the slides were viewed by indirect immunofluorescence microscopy with a fluorescein isothiocyanate filter. The chimpanzee 1313 (infected with genotype 1 HEV) immune serum is specific for the ORF2 protein, as serum taken from chimpanzee 1313 prior to infection with HEV did not stain transfected cells (6). The mouse anti-ORF3 hyperimmune serum was generated by immunizing BALB/c mice with an affinity-purified recombinant ORF3 protein of swine HEV expressed in bacteria (Z. F. Sun and X. J. Meng, unpublished data).
Intrahepatic inoculation of SPF pigs with capped RNA transcripts from full-length swine HEV cDNA clones. To determine the infectivity of clone C, we intrahepatically inoculated two 6-week-old SPF pigs (pigs 3 and 4) with 1 ml of diluted full-length capped RNA transcripts from clone C. The RNA inocula were quickly thawed and immediately injected by an ultrasound-guided technique into five or six different sites in the liver, at about 200 μl per injection site. Another two SPF pigs (pigs 23 and 30), which were housed in a separate room, were not inoculated and served as negative controls. All pigs were monitored daily for clinical signs of disease for a total of 12 weeks, at which time they were necropsied.
A separate animal experiment was performed to determine the infectivities of the three corrected clones. Briefly, eight 6-week-old SPF pigs that were seronegative for HEV were randomly assigned into four groups of two each (groups A, B, C, and D). Each group of pigs was housed separately in a biosafety level 2 facility and maintained under conditions that met all requirements of the Institutional Committee on Animal Care and Use. The RNA inocula were similarly injected by the ultrasound-guided technique into the liver of each pig. The two pigs (286 and 293) in group A were each injected with 1 ml of the RNA inoculum from clone pSHEV-1, pigs 294 and 295 (group B) were each injected with 1 ml of the RNA inoculum from clone pSHEV-2, and pigs 297 and 298 (group C) were each inoculated with the RNA inoculum from clone pSHEV-3. The two pigs in group D were not inoculated and served as negative controls. Pigs were monitored daily for clinical signs of disease for a total of 10 weeks. All pigs were necropsied at 10 weeks postinoculation.
ELISA and RT-PCR. Serum and fecal samples were collected from all pigs for both animal experiments prior to inoculation and weekly postinoculation until the end of the studies. Serum samples were tested for anti-HEV IgG by an enzyme-linked immunosorbent assay (ELISA) as previously described (11, 23). Viremia and fecal virus shedding were tested by RT-PCR as described previously (11, 23, 24, 34). In addition, all positive serum samples were retested by a semiquantitative RT-PCR to determine the swine HEV genomic equivalent (GE) titers in sera as previously described (17). One GE is defined as the number of viral genomes present in the highest 10-fold dilution that is positive by RT-PCR (17).
Detection of genetic markers in viruses recovered from experimentally infected pigs. Selected regions of viruses recovered from serum and fecal samples from pigs in groups A, B, and C were amplified by RT-PCR and then sequenced. For the marker at nt 38, a reverse primer (5'-CTCGGATGGGCTCCAACTTCCA-3') was used for reverse transcription and cDNA synthesis. A PCR was performed to amplify this region by use of a sense primer (5'-CCACGTATGTGGTCGATGCC-3') and the reverse primer. For the marker at nt 1028, a reverse primer (5'-ATTTCTGGGCGTGCTCAACCTCC-3') and a sense primer (5'-ATGCCTTATGTCCCCTACCCTCG-3') were used for PCR amplification. For the markers at nt 4175 and 4307 in clones pSHEV-1 and pSHEV-2, a reverse primer (5'-CCGGCATACAGCCCAATAGG-3') and a sense primer (5'-CCTGTTCAGGCCACCACATGTGAGT-3') were used.
In addition, the entire ORF2 gene of viruses recovered from pigs that were inoculated with clones pSHEV-1 and pSHEV-3 was amplified with two pairs of primers (first pair, 5'-ATAACATGTCTTTTGCATCGCC-3' and 5'-GGGCTGAACCAAAATCCTGACATC-3'; second pair, 5'-TCAAACTACAACCACCCCTACTT-3' and 5'-GGGGCACAAAAGGAATTAATTA-3'). The PCR products were directly sequenced by use of the respective PCR primers.
Nucleotide sequence accession numbers. The complete sequences of the three cDNA clones have been deposited in the GenBank database (accession no. AY575857 to AY575859).
RESULTS
Assembly of a full-length cDNA clone of swine HEV and determination of its infectivity. Eight overlapping DNA fragments covering the entire viral genome were amplified by RT-PCR and subsequently assembled into a full-length cDNA by ligation at appropriate restriction enzyme sites (Fig. 1). The nucleotide G, instead of the A nucleotide present in many other known HEV strains, was found at the extreme 5' end of the swine HEV genome. A HindIII site and a T7 core promoter were introduced immediately preceding the first G nucleotide to facilitate in vitro transcription of the viral genomic RNA. The assembled cDNA was cloned into plasmid pGEM-9zf(–) to produce a full-length cDNA clone of swine HEV, which we designated clone C.
The infectivity of clone C was determined by the intrahepatic inoculation of two pigs with full-length capped RNA transcripts from clone C. Swine HEV RNA was not detected by RT-PCR in the weekly sera or fecal swab materials of the two inoculated or two control pigs throughout the 12-week study. Seroconversion to HEV was not detected in either of the two inoculated or two control pigs after 12 weeks, suggesting that clone C was nonviable. In addition, Huh7 cells transfected with RNA transcripts from clone C did not contain detectable ORF2 or ORF3 protein, whereas those transfected in parallel with transcripts from a known infectious cDNA clone did (6; data not shown), suggesting that clone C was not viable in vitro as well as in vivo.
In order to determine the possible reasons for the nonviability of clone C, we redetermined the consensus sequence of the swine HEV genome by direct sequencing of RT-PCR products amplified from a swine HEV infectious stock (24). We found that the new consensus sequence of the swine HEV genome had a total of 17 nucleotide differences compared to the published sequence (accession no. AF082843 and AF011921), and 8 of these differences were nonsilent mutations. A sequence comparison revealed a total of 14 mismatches between clone C and the new consensus sequence (Table 1). Ten of these mismatches were silent mutations, and three mutations in the ORF2 capsid gene (T5338C, A5362G, and C6356T) resulted in amino acid changes. A deletion point mutation at nt 756 (A-756) which resulted in a downstream frameshift of the ORF1 gene was also found in clone C. These mutations in clone C may reflect a quasispecies in the virus stock or may be due to mistakes introduced by RT-PCR and/or cloning procedures, especially in the case of the deletion mutation A-756.
A full-length cDNA clone with a new consensus sequence and two mutant clones of swine HEV are replication competent when transfected into Huh7 cells. Three regions containing mutations in clone C were sequentially replaced by corrected fragments (BX, XAf, and AvN) to produce two mutant clones (pSHEV-1 and pSHEV-2) and a consensus clone (pSHEV-3). Capped full-length RNA transcripts from each clone and from clone C were transfected into a subclone of Huh7 cells to determine the replication competence of the clones. Viral antigens were detected by an IFA using a chimpanzee anti-HEV convalescent-phase serum or a mouse anti-swine HEV ORF3 hyperimmune serum to stain cells that were transfected individually with RNA transcripts from each of the four clones. A fluorescent signal was not detected with either antibody in cells transfected with clone C, but a signal was detected with each antibody in cells transfected with pSHEV-1, pSHEV-2, and pSHEV-3 (Fig. 2).
Capped RNA transcripts from the three swine HEV cDNA clones were infectious when injected intrahepatically into the livers of SPF pigs. Both pigs in groups A, B, and C, which were inoculated with RNA transcripts from one of the three clones, seroconverted to anti-HEV IgG, indicating that active swine HEV infections had occurred in the inoculated pigs (Table 2). Both inoculated and control pigs had no increase in rectal temperature or signs of illness or distress during the entire study, which was consistent with our earlier reports of swine HEV and human HEV infections in pigs (11, 23, 34). Pigs in groups B (pSHEV-2) and C (pSHEV-3) all seroconverted at about 4 to 5 weeks postinoculation, whereas the two pigs in group A (pSHEV-1) had a delayed seroconversion at 9 to 10 weeks postinoculation (Table 2; Fig. 3). The two uninoculated pigs in group D remained seronegative throughout the experiment. Swine HEV RNA was detected in the fecal samples of pigs inoculated with pSHEV-2 and pSHEV-3 starting at 14 days postinoculation (dpi) and lasting for 5 to 6 weeks. Similar to seroconversion, fecal virus shedding in pigs inoculated with pSHEV-1 also appeared late, at 35 (pig 286) and 42 (pig 293) dpi (Table 2).
Viremia was detected variably in inoculated pigs (Table 2). For group B (pSHEV-2), viremia was detected in pig 294 only at 28 dpi and in pig 295 at 21 to 35 dpi. The swine HEV genome titers in the sera of the two group B pigs were 102 GE/ml (Table 2). For group C (pSHEV-3), pig 298 was viremic for 5 weeks, starting at 21 dpi, and the antibody titers of this pig were the highest among all six inoculated pigs (Fig. 3). The swine HEV genome titer in this pig was 104 GE/ml at 21 dpi, 103 GE/ml at 28 to 42 dpi, and 102 GE/ml at 49 dpi. Interestingly, pig 297, which was also in group C, did not have detectable viremia, even though this pig shed virus in its feces for 6 weeks (Table 2). The two pigs in group A (pSHEV-1) also did not have detectable viremia. The lack of viremia, together with the delayed appearances of seroconversion and fecal virus shedding, suggested that the virus encoded by clone pSHEV-1 may be attenuated. Additional studies with larger number of animals are needed to draw a definitive conclusion regarding the attenuation of clone pSHEV-1. The bile sample collected from pig 293 of group A during necropsy at 70 dpi was positive by RT-PCR for swine HEV RNA, with a genome titer of 102 GE/ml, but the bile samples collected from all other pigs during necropsy were negative. Viremia and fecal virus shedding were not detected in either group D negative control pig. All eight pigs appeared clinically normal throughout the experiment.
Viruses recovered from experimentally infected pigs retained their respective genetic markers. To determine if the genetic markers of the three cDNA clones were retained in the respective rescued viruses, we amplified and sequenced genomic regions containing the markers from selected samples from inoculated pigs. For group C pigs, the two silent mutations at nt 38 and 1028 were still present in the viral genomes amplified from fecal samples at 21 dpi. For group B pigs, three genetic markers, nt 1028 in fragment BX and nt 4175 and 4307 in fragment XAf, were also retained in the sequence amplified from fecal samples at 21 dpi. The sequences recovered from all positive samples of the two group A pigs retained the silent mutation C at nt 5811 as well as the markers at nt 4175 and 4307. Taken together, these results confirmed that the viruses infecting the pigs originated from the respective cDNA clones pSHEV-1, pSHEV-2, and pSHEV-3.
The complete ORF2 capsid gene was also amplified and sequenced from the two pSHEV-1-inoculated pigs, from a bile sample from pig 293 at 70 dpi and a fecal sample from pig 286 at 42 dpi. Sequence analysis revealed that the three nonsilent mutations in the capsid gene of clone pSHEV-1 were retained in the genome and that new mutations had not arisen in the ORF2 gene. Additionally, the complete ORF2 gene of the virus infecting pig 298 (pSHEV-3) was amplified, and the sequence was found to be identical to that of clone pSHEV-3.
DISCUSSION
Since there is a lack of an efficient in vitro cell culture system for HEV, the fundamental biology of HEV is poorly understood. Therefore, an infectious cDNA clone of HEV is critical for the study of the molecular biology of this important human pathogen by recombinant DNA technology. Two infectious cDNA clones of genotype 1 human HEV have been reported (7, 27). However, for an understanding of the tissue tropism and mechanisms of cross-species HEV infection, infectious cDNA clones of swine HEV and other genotypes of HEV are needed. For this study, we established a reverse genetic system for a prototype strain of swine HEV. Capped RNA transcripts from each of three full-length cDNA clones of swine HEV were viable both upon transfection into Huh7 cells and after intrahepatic inoculation into the livers of SPF pigs. The detection of genetic markers confirmed that the viruses recovered from the infected pigs originated from the respective cDNA clones.
The successful generation of infectious cDNA clones of RNA viruses is a laborious process which is often hampered by many obstacles, such as unexpected mutations introduced during the RT-PCR and cloning steps, the inherent quasispecies populations of the virus stock, the instability and toxicity of cDNA clones during propagation in Escherichia coli, the infidelity of the RNA polymerase used for in vitro transcription, the specific structures in the viral RNA genome [cap and poly(A) tail] required for efficient replication, and the potential harmful effects of nonviral nucleotides at the ends of RNA transcripts (2). It has been demonstrated that a cap structure is essential for the infectivity of human HEV RNA transcripts (7), and therefore in this study a cap analog was added during the in vitro transcription of swine HEV cDNA clones. Since it was previously shown that 15 adenosine nucleotides in the poly(A) tract are sufficient to rescue human HEV (7), we added 15 A's to the 3' end of the swine HEV genome. There are no nonviral nucleotides at the 5' ends of the RNA transcripts and only one nonviral nucleotide (T) at the 3' ends of the swine HEV cDNA clones. The nonviral nucleotide T at the 3' end of the genome did not abolish the infectivity of the cDNA clones. We did not encounter any problem regarding the stability of the clones during the construction and propagation of the three pSHEV plasmids in E. coli, even though the 7.2-kb genomic cDNA was inserted into the high-copy-number vector pGEM-9zf(–). After multiple-step mutagenesis and subcloning processes, the sequence of the final pSHEV-3 clone was identical to the newly determined consensus sequence of swine HEV, with the exception of the two genetic markers.
The clone C sequence differed from the consensus sequence due to the presence of a deletion mutation at nt 756 and 13 point mutations scattered across the genome. All three new cDNA clones derived from clone C had the corrected BX fragment which reversed the deletion at nt 756. In contrast to the nonviability of clone C, all three corrected clones replicated in vitro and were infectious in vivo. The viral genome titers in sera of inoculated pigs were relatively low (102 to 104 GE/ml) and peaked in the first week of the viremia period (Table 2). This low level of viremia was consistent with our previous studies with live swine HEV (23, 24). The GE titers of swine HEV in the sera of pigs inoculated with the consensus clone pSHEV-3 were at least 2 log higher than those in the two pigs inoculated with the mutant clone pSHEV-2, suggesting that the consensus clone pSHEV-3 replicated better in pigs than did the two mutant clones. The patterns of seroconversion, viremia, and fecal virus shedding in pigs inoculated with pSHEV-2 and pSHEV-3 RNA transcripts were similar, suggesting that the nucleotide differences between these two cDNA clones were not critical for virus replication. In contrast, pigs inoculated with RNA transcripts from clone pSHEV-1 had a delayed appearance of seroconversion and fecal virus shedding and had undetectable viremia, suggesting that the virus encoded by clone pSHEV-1 may be attenuated in pigs. There are five mutations in the ORF2 capsid gene that are unique to clone pSHEV-1, including three nonsilent mutations at nt 5338, 5362, and 6356. The amino acid residues F-51, T-59, and S-390, corresponding to the three nonsilent mutations in the capsid protein of swine HEV, and the amino acid sequences in the proximity of them are completely conserved among all other known mammalian HEV sequences published to date (data not shown). Therefore, the substitutions at these three positions were unique to the swine HEV clone pSHEV-1. The three nonsilent mutations likely occurred as a result of natural strain variations, since the capsid protein was under immunological pressure. The changes from polar residues to nonpolar residues at amino acid positions 59 (Thr to Ala) and 390 (Ser to Leu) were likely important since such a change may drastically alter the structure of a viral protein and therefore influence its functions. It has been well documented that one or more amino acid changes in capsid or envelope proteins can contribute to the attenuation phenotype of viruses (3, 28, 29, 32, 33). Therefore, additional studies with a larger number of pigs and with infectious cDNA clones containing a single mutation and combination double mutations of the three nonsilent mutations are warranted to definitively determine the roles of these mutations in swine HEV infection.
An infectious human HEV cDNA clone, pSK-HEV-3, which contains a silent mutation at the C terminus of the ORF2 capsid gene, produces an attenuated virus in chimpanzees (7). This mutation slightly changed the secondary structure of a cis-reactive element, which was believed to be related to the attenuation. However, additional nearby mutations were generated in infected chimpanzees to restore the cis-reactive element (7). In the present study, the three nonsilent mutations in the capsid were stable in the virus, and other compensatory mutations were not identified in the capsid genes of recovered viruses. The stability of these mutations in clone pSHEV-1 should be very useful for further pathogenesis studies. The availability of these swine HEV infectious cDNA clones now affords us an opportunity to use pigs as a model to dissect the basic biology of HEV and to study the potential mechanisms of zoonotic transmission by constructing chimeric viruses between swine and human HEVs.
ACKNOWLEDGMENTS
This study was supported by grants from the National Institutes of Health (AI01653, AI50611, and AI46505) and in part by a grant from the U.S. Department of Agriculture (NRI 2002-35204-12531).
We thank Robert Purcell for his support, Denis Guenette for his technical assistance, and Z. F. Sun for anti-swine HEV ORF3 mouse hyperimmune serum preparation.
Y.W.H. and G.H. contributed equally to this study.
Present address: Virology Section, National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran.
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