Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Price's Fork Road, Blacksburg, VA 24061-0342, USA
Correspondence
X. J. Meng
xjmeng{at}vt.edu
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ABSTRACT |
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INTRODUCTION |
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Although acute hepatitis E occurs only sporadically in the USA and other industrialized countries, an unexpected high seroprevalence rate has been reported in healthy individuals of these countries (Fukuda et al., 2004; Meng et al., 2002
; Thomas et al., 1997
). Anti-HEV antibodies have also been detected in various animal species including pigs, sheep, cats, cows, chickens and rodents, suggesting that HEV or a related agent infects these animals (Arankalle et al., 2001
; Favorov et al., 2000
; Kabrane-Lazizi et al., 1999
; Meng et al., 1999
; Okamoto et al., 2004
; Wang et al., 2002
). Increasing evidence indicates that HEV is a zoonotic agent and that animal reservoirs exist for HEV (Meng, 2000
, 2003
). The first animal strain of HEV, swine HEV, was identified from a pig in the USA in 1997 (Meng et al., 1997
). The prototype strain of swine HEV identified in the USA is genetically most closely related to US strains of human HEV (Schlauder et al., 1998
, 1999
). Similarly, numerous swine HEV isolates identified from pigs in other countries are also genetically closely related to strains of human HEV, especially genotype 3 or 4 human HEV strains, from the same geographical regions (Hsieh et al., 1999
; Pina et al., 2000
; Takahashi et al., 2003
; Wu et al., 2002
; Yazaki et al., 2003
). Cross-species infection of genotype 3 human and swine HEVs has been documented: swine HEV infected non-human primates and the US-2 strain of human HEV infected pigs (Halbur et al., 2001
; Meng et al., 1998b
). Seroepidemiological studies have revealed that swine veterinarians and other pig handlers have an increased risk of HEV infection compared with normal blood donors (Drobeniuc et al., 2001
; Meng et al., 2002
; Withers et al., 2002
). Recently, cases of acute hepatitis E in Japan were epidemiologically and genetically linked to consumption of raw pig and deer meat, thus providing more direct evidence of zoonotic HEV transmission (Matsuda et al., 2003
; Tei et al., 2003
; Yazaki et al., 2003
).
In 2001, another animal strain of HEV, designated avian HEV, was identified from chickens with hepatitissplenomegaly syndrome in the USA (Haqshenas et al., 2001). Avian HEV also shares about 80 % nucleotide sequence identity with the Australian big liver and spleen disease virus (BLSV) in the short stretch of sequence available for BLSV (Haqshenas et al., 2001
; Payne et al., 1999
). The complete genomic sequence of avian HEV has been determined (Huang et al., 2004
). Sequence analysis has revealed that avian HEV is genetically related to human HEV with approximately 5060 % nucleotide sequence identity (Huang et al., 2004
). Avian HEV also shares common antigenic epitopes on the capsid protein, similar genomic organization and conserved functional motifs in ORF1 with human and swine HEVs (Haqshenas et al., 2002
; Huang et al., 2004
). Avian HEV is enzootic in chicken flocks in the USA and has the ability to cross the species barrier and infect turkeys (Huang et al., 2002
; Sun et al., 2004a
, b
). Phylogenetic analysis has revealed that avian HEV forms a branch distinct from human and swine HEVs, and may represent a fifth genotype (Huang et al., 2004
).
Due to the lack of an efficient cell-culture system and a practical animal model for HEV, little is known about the mechanisms of HEV pathogenesis and replication. Infectious cDNA clones of genotype 1 and genotype 3 HEV have been reported (Emerson et al., 2001; Huang et al., 2005
; Panda et al., 2000
). Here, we report the construction and characterization of infectious cDNA clones of avian HEV, which is genetically very divergent from mammalian HEVs.
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METHODS |
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Construction of full-length cDNA clones of avian HEV.
Total RNAs were extracted from the avian HEV infectious stock with TRIzol reagent and used for cDNA synthesis with SuperScript II reverse transcriptase (Invitrogen) and avian HEV-specific reverse primers (Table 1). Four cDNA fragments were amplified by PCR with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) using four sets of primers (Table 1
). Fragment I representing the 5' end of the viral genome was amplified with primers XbaIT7F1 and PstI3340R. The sense primer XbaIT7F1, 5'-gctctagataatacgactcactataGCATGACCCCATGCCAGGGT-3', contained an engineered XbaI restriction enzyme site (bold), a T7 core promoter sequence (underlined), followed by the extreme 5' end of avian HEV sequence (upper case). Fragment I' was the same as fragment I except for an extra G added between the T7 core promoter and the viral sequence using sense primer XbaIT7GF1 (Table 1
). Fragment III representing the 3' end of the viral genome was amplified with primers EcoRIF5019 and T18MluIXhoIR. The antisense primer, T18MluIXhoIR, introduced 18 adenosine nucleotides and MluI and XhoI restriction sites at the 3' end of the avian HEV genome. Fragment II covering the internal part of the avian HEV genome was amplified with primers KpnIF and EcoRIR5258 and partially overlapped fragments I (or I') and III. Using the convenient restriction sites BamHI and EcoRI naturally present in the overlapping regions (Fig. 1
), these PCR fragments were ligated in order into the pGEM-7zf(+) vector between the XhoI and XbaI sites in the polylinker. The full-length cDNA clone assembled with fragments I, II and III was designated pT7-aHEV and the clone assembled with fragment I', II and III was designated pT7G-aHEV.
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In vitro transfection and immunofluorescence assay.
Prior to transfection, LMH cells growing on a 12-well plate were washed with Waymouth's MB 752/1 medium. Different amounts of RNA transcripts (8, 14 and 18 µl) from each of the avian HEV cDNA clones were mixed with 4 µl Plus Reagent (Invitrogen) in 25 µl Waymouth's MB 752/1 medium. After a 15 min incubation, the mixtures were combined with 1 µl Lipofectamine (Invitrogen) diluted in 25 µl Waymouth's MB 752/1 medium and incubated for 15 min. The RNA transcripts, Plus Reagent and Lipofectamine mixtures were then added to washed LMH cells covered with 200 µl Waymouth's MB 752/1 medium. After incubation at 37 °C for 3 h, 600 µl fresh culture medium was added and the transfected cells were incubated at 37 °C.
On day 5 post-transfection, the transfected cells were trypsinized and replated on eight-well LabTek chamber slides. Slides were fixed and stained on day 6. Briefly, cells on slides were rinsed with PBS, fixed with a solution containing 80 % acetone, 20 % methanol and air dried. A 1 : 100-diluted anti-avian HEV convalescent serum from a specific-pathogen-free (SPF) chicken experimentally infected with avian HEV was added to the fixed cells and incubated for 25 min at 37 °C. After washing with cold PBS, 1 : 100-diluted fluorescein-labelled goat anti-chicken IgG (KPL) was added and incubated at 37 °C for 25 min. The slides were then washed with PBS, covered with fluoromount-G (Southern Biotechnology Associates) and viewed under a fluorescence microscope.
Intrahepatic inoculation of SPF chickens with RNA transcripts from three full-length cDNA clones of avian HEV.
The animal experiments in this study were approved by the Institutional Animal Care and Use Committee.
Experiment 1.
Eight 6-week-old SPF chickens that were negative for avian HEV RNA and antibodies by testing serum and faecal samples with an RT-PCR and ELISA were randomly divided into four groups of two (groups AD). Two chickens in each group were housed in a single isolator. The RNA inocula were quickly thawed and immediately injected, through a percutaneous procedure, into two different sites on each side of the liver with approximately 100 µl per injection site. Two chickens in group A (#3765 and #3768) were each injected with 400 µl RNA transcripts from clone pT7-aHEV-5, chickens #2572 and #2568 in group B with 400 µl RNA transcripts from clone pT7G-aHEV-6, and chickens #3761 and #3767 in group C with 400 µl RNA transcripts from clone pT7G-aHEV-10. The two chickens in group D (#4494 and #2569) were not inoculated and served as negative controls. Faecal swabs were collected from each chicken every 3 days and serum from each chicken was collected every week. Faecal swabs and sera were tested by RT-PCR for avian HEV RNA and sera were tested by ELISA for seroconversion to avian HEV antibodies (Huang et al., 2002, 2004
). All inoculated chickens were killed and examined at 9 weeks post-inoculation (p.i.).
Experiment 2.
The percutaneous injection of RNA transcripts into chicken livers is a blind procedure. For confirmation purposes, we conducted a second chicken experiment using a surgical procedure. In this modified procedure, intrahepatic inoculation was performed using a right parasternal incision and the RNA transcripts were directly injected into the right lobe of the liver following visualization. Two chickens (#3762 and #4498) of 14 weeks of age in group A' were each injected with 400 µl RNA transcripts from clone pT7-aHEV-5, and chickens #4493 and #4500 in group C' with 400 µl RNA transcripts from clone pT7G-aHEV-10. Chickens #2569 and #4494 in group D were not inoculated to serve as negative controls. Faecal swabs and sera were collected as described in experiment 1. Faecal swabs and sera were tested by RT-PCR for avian HEV RNA and sera were tested by ELISA for seroconversion to avian HEV antibodies (Huang et al., 2002, 2004
). All chickens were killed and examined at 6 weeks p.i.
Detection of genetic markers in viruses recovered from experimentally infected chickens.
Viruses recovered from faecal material of inoculated chickens #3768, #2572 and #3767 at 14 days p.i. were used to amplify selected genomic regions by RT-PCR to determine the presence of genetic markers. A nested PCR with external primer pairs F2422 and R2931 and internal primer pairs F2495 and R2919 (Table 1) was used to amplify the helicase gene region containing genetic markers at nt 2586, 2592, 2622 and 2886. Another nested PCR with external primers F4914/R5301 and internal primers F4992/R5254 (Table 1
) was used to amplify the ORF2 gene region containing genetic markers at nt 5093, 5102, 5111 and 5210. Primers F5973, R6350 and R6407 (Table 1
) were used to detect the genetic marker at nt 6110 in ORF3. PCR products were purified with a GENECLEAN kit (Bio 101) and directly sequenced at the Virginia Tech DNA Sequencing Facility.
Experimental inoculation of SPF chickens with viruses recovered from chickens inoculated with capped RNA transcripts of avian HEV clones.
To confirm further the infectivity of the cDNA clones, viruses recovered from chickens inoculated with the RNA transcripts of avian HEV cDNA clones were used to infect naïve SPF chickens via the intravenous route of inoculation. Briefly, faeces collected from chickens #3767 and #3768 at 14 days p.i. were used to prepare a 10 % faecal suspension in PBS (Sun et al., 2004b), which tested positive for avian HEV RNA. Approximately 1 ml of each faecal suspension was used to intravenously inoculate two 7-week-old SPF chickens. Two chickens were not inoculated and housed in a different room as negative controls. Faecal swabs and sera were collected similarly as described above and tested by RT-PCR for avian HEV RNA and by ELISA for seroconversion to avian HEV antibodies (Huang et al., 2002
, 2004
). All chickens were killed and examined at 8 weeks p.i.
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RESULTS |
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The consensus genomic sequence of avian HEV, determined by direct sequencing of RT-PCR products amplified from the virus stock, differed at 24 positions from the sequence obtained from cloned cDNA fragments (Huang et al., 2004). Compared with the consensus sequence, the cDNA clone pT7-aHEV contained 21 mutations including nine non-silent mutations, and the cDNA clone pT7G-aHEV had 19 mutations including nine non-silent mutations. The non-silent mutations were changed back to the consensus sequence using site-directed mutagenesis to create three new clones, pT7-aHEV-5, pT7G-aHEV-6 and pT7G-aHEV-10 (Table 2
). These three new clones were subsequently used for in vitro and in vivo characterization.
Capped RNA transcripts from the avian HEV cDNA clones are replication competent when transfected into LMH chicken liver cells
RNA transcripts from each of the three clones were transcribed in vitro in the presence of a cap analogue. The addition of an extra G between the T7 core promoter and the 5' end of the viral sequence of clones pT7G-aHEV-6 and pT7G-aHEV-10 had no significant effect on the transcription efficiency of avian HEV cDNA clones, as similar amounts of RNA transcripts were produced from each of the three clones (data not shown). Capped RNA transcripts from each of the three full-length cDNA clones were subsequently transfected into LMH chicken liver cells to determinate infectivity. Viral antigens were detected in transfected LMH cells using an immunofluorescence assay with anti-avian HEV convalescent serum (Fig. 2). Like Huh7 cells transfected with RNA transcripts from a genotype 1 human HEV cDNA clone, the fluorescent signals were mainly located in the cytoplasm of LMH cells. About 1015 % of the cells were positive for avian HEV antigens (Fig. 2
); however, virus spread from cell-to-cell was not evident. Mock-transfected cells were negative for avian HEV antigens.
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Capped RNA transcripts from the avian HEV cDNA clones are infectious when inoculated into the livers of SPF chickens via a laparoscopic surgical procedure that can visualize the livers
Retrospective sequencing and sequence analysis revealed that clones pT7G-aHEV-6 and pT7G-aHEV-10 had an identical full-length genomic sequence. Therefore, only clones pT7-aHEV-5 and pT7G-aHEV-10 were included in this experiment. Compared with the percutaneous intrahepatic injection procedure in which the liver cannot be visualized, the right parasternal laparoscopic injection approach in this experiment allowed direct visualization of the liver prior to inoculation. Viral RNA was detected in faecal material from all four chickens inoculated with RNA transcripts from the two clones (Table 3). Two of the four inoculated chickens also had detectable viral RNA in their serum (Table 4
). All four chickens seroconverted to avian HEV antibodies, although chicken #4493 had low levels of antibodies and chicken #4498 had a delayed seroconversion (Fig. 3b
).
Infection of chickens with viruses recovered from chickens transfected in vivo with avian HEV RNA transcripts
For confirmation purposes, we attempted to transmit avian HEV infection to SPF chickens using the viruses recovered from the faecal material of the in vivo-transfected chickens. A 10 % suspension of PCR-positive faecal material from chickens #3767 and #3768 was prepared separately and each was used to inoculate two SPF chicken intravenously. The two inocula each had a titre of 10 genomic equivalents of avian HEV genome ml1 as determined by PCR. Faecal virus shedding and seroconversion were variably detected in all inoculated chickens (Table 3, Fig. 3c
) and viral RNA in serum was also detected in three of the four inoculated chickens (at days 35 and 42 p.i. for chicken #1589, at day 28 p.i. for chicken #1597 and at day 21 p.i. for chicken #1596), indicating that the recovered viruses originating from clones pT7G-aHEV-10 and pT7-aHEV-5 were infectious in chickens. Negative-control chickens remained negative for avian HEV infection throughout the study (Table 3
, Fig. 3c
).
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DISCUSSION |
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It has been demonstrated that a cap structure is required for the infectivity of a genotype 1 HEV cDNA clone (Emerson et al., 2001). The 5'-terminal sequence of the avian HEV genome was amplified by a pyrophosphatase-dependent elongation reaction, suggesting that the avian HEV genomic RNA is also capped (Huang et al., 2004
). Therefore, capped RNA transcripts were synthesized in this study to test their infectivity in vivo and in vitro. It is known that an extra G at the end of the T7 core promoter sequence is important for the in vitro transcription efficiency of RNA (Milligan et al., 1987
; Sasaki et al., 2001
). Therefore, we added an extra G at the 5' end of the avian HEV genome in clone pT7G-aHEV. In addition, non-viral sequences (MluI restriction site and a C nucleotide resulting from digestion by XhoI) were also introduced at the 3' end of the viral genome. Our results showed that these extra non-viral nucleotides at both 5' and 3' ends of the avian HEV genome did not affect the infectivity of these clones in chickens or in LMH chicken liver cells. Unexpected mutations, introduced during RT-PCR steps, cloning or due to infidelity of the RNA polymerase during in vitro transcription steps, are of concern for the construction of infectious cDNA clones. We found in this study that the silent mutations in the avian HEV clones had no effect on the infectivity of the clones and were still present in the viruses rescued from the infected chickens. A non-silent mutation, A5089, not present in the original cDNA clone, was detected in the virus recovered from a chicken intrahepatically inoculated with RNA transcripts from clone pT7-aHEV-5. This non-silent mutation is most likely due to the quasispecies nature of the virus, which is evidenced by the heterogeneity of avian HEV isolates from chickens in different regions of the USA (Huang et al., 2002
; Sun et al., 2004a
).
Faecal virus shedding, viraemia and seroconversion were detected variably in chickens intrahepatically injected with RNA transcripts from each of the cDNA clones, indicating that the clones are infectious. The infectivity of the clones was further confirmed by successfully passaging the viruses recovered from RNA-transfected chickens to naïve SPF chickens via the intravenous route of inoculation. Viraemia in inoculated chickens was transient or absent, and chickens without viraemia generally had delayed seroconversion or relatively low levels of anti-avian HEV antibodies. Lack of viraemia has also been reported in chickens and pigs experimentally infected with avian HEV and swine HEV, respectively (Billam et al.; 2005; Meng et al., 1998a
). Ultrasound-guided intrahepatic injection of viral RNA transcripts has been used for in vivo testing of HEV infectious clones in non-human primates and pigs (Emerson et al., 2001
; Huang et al., 2005
). However, this procedure is not applicable to chickens due to their small size. Therefore, we attempted to use percutaneous and the laparoscopic surgical procedures for intrahepatic inoculation of avian HEV RNA transcripts. Our results showed that both procedures are equally efficient in inducing avian HEV infection.
Capped RNA transcripts from a human HEV strain Sar-55 infectious clone have been shown to be replication competent in two human liver cell lines, PLC/PRF/5 and Huh-7, as well as in an intestinal cell line, Caco-2, although the virus progeny produced from transfected cells did not spread (Emerson et al., 2004b). Cell lines derived from non-human primates, pigs and rodents transfected with RNA transcripts from the Sar-55 human HEV clone did not support virus replication, indicating that species-specific factors are involved in virus replication. It has been shown that avian HEV can cross species barriers and infect turkeys, although it failed to infect two rhesus monkeys (Huang et al., 2004
; Sun et al., 2004b
). Therefore, to test the infectivity of the avian HEV cDNA clones in vitro, we used a liver cell line of chicken origin, LMH. As expected, RNA transcripts from the avian HEV clones were replication competent in LMH cells. Like the human HEV Sar-55 infectious clone (Emerson et al., 2004b
), the fluorescent signals in LMH cells transfected with avian HEV RNA were also detected in the cytoplasm of dividing cells, but the virus progeny did not spread from cell-to-cell. Nevertheless, the LMH cell culture system identified in this study is still useful for future studies of HEV biology, as we can now test the viability of mutant clones of avian HEV in LMH cells and study the mechanism of HEV replication. Most importantly, we can now construct chimeric viruses from avian, swine and human HEVs to study the mechanism of cross-species infection.
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ACKNOWLEDGEMENTS |
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Received 30 March 2005;
accepted 16 May 2005.
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