Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV

F. F. Huang1, Z. F. Sun1, S. U. Emerson2, R. H. Purcell2, H. L. Shivaprasad3, F. W. Pierson1, T. E. Toth1 and X. J. Meng1

1 Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Price's Fork Road, Blacksburg, VA 24060-0342, USA
2 Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3 California Animal Health and Food Safety Laboratory System, University of California-Davis, Fresno, CA 93725, USA

Correspondence
X. J. Meng
xjmeng{at}vt.edu


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Avian hepatitis E virus (avian HEV), recently identified from a chicken with hepatitis–splenomegaly syndrome in the United States, is genetically and antigenically related to human and swine HEVs. In this study, sequencing of the genome was completed and an attempt was made to infect rhesus monkeys with avian HEV. The full-length genome of avian HEV, excluding the poly(A) tail, is 6654 bp in length, which is about 600 bp shorter than that of human and swine HEVs. Similar to human and swine HEV genomes, the avian HEV genome consists of a short 5' non-coding region (NCR) followed by three partially overlapping open reading frames (ORFs) and a 3'NCR. Avian HEV shares about 50 % nucleotide sequence identity over the complete genome, 48–51 % identity in ORF1, 46–48 % identity in ORF2 and only 29–34 % identity in ORF3 with human and swine HEV strains. Significant genetic variations such as deletions and insertions, particularly in ORF1 of avian HEV, were observed. However, motifs in the putative functional domains of ORF1, such as the helicase and methyltransferase, were relatively conserved between avian HEV and mammalian HEVs, supporting the conclusion that avian HEV is a member of the genus Hepevirus. Phylogenetic analysis revealed that avian HEV represents a branch distinct from human and swine HEVs. Swine HEV infects non-human primates and possibly humans and thus may be zoonotic. An attempt was made to determine whether avian HEV also infects across species by experimentally inoculating two rhesus monkeys with avian HEV. Evidence of virus infection was not observed in the inoculated monkeys as there was no seroconversion, viraemia, faecal virus shedding or serum liver enzyme elevation. The results from this study confirmed that avian HEV is related to, but distinct from, human and swine HEVs; however, unlike swine HEV, avian HEV is probably not transmissible to non-human primates.

The complete genomic sequence of avian HEV has been deposited in the NCBI database under GenBank accession no. AY535004.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis E is an important public health disease in many developing countries of Asia and Africa and also occurs sporadically in some industrialized countries (Emerson & Purcell, 2003; Kabrane-Lazizi et al., 2001; Schlauder et al., 1999; Takahashi et al., 2002). The disease mainly affects young adults and has a relatively high mortality of up to 25 % in infected pregnant women (Emerson & Purcell, 2003). The causative agent, hepatitis E virus (HEV), is a small, non-enveloped RNA virus that is transmitted via the faecal–oral route, primarily through contaminated water supplies (Emerson & Purcell, 2003). It is the sole member of the genus Hepevirus. The genome of HEV is a single-stranded, positive-sense RNA molecule of approximately 7·2 kb and encodes three open reading frames (ORFs) (Tam et al., 1991). ORF1, located at the 5' end of the genome, encodes motifs for non-structural proteins, including the methyltransferase, protease, helicase and RNA-dependent RNA polymerase (RdRp) (Koonin et al., 1992). ORF2, located at the 3' end of the genome, encodes the capsid protein (Tam et al., 1991). The small ORF3, which partially overlaps ORF2, encodes a cytoskeleton-associated phosphoprotein (Tyagi et al., 2002; Zafrullah et al., 1997).

The first animal strain of HEV to be sequenced and characterized was isolated from a pig in the USA in 1997 and designated swine HEV (Meng et al., 1997). Since then, numerous strains of HEV have been isolated from pigs in both developing and industrialized countries (Arankalle et al., 2002; Choi et al., 2003; Garkavenko et al., 2001; Hsieh et al., 1999; Huang et al., 2002a; Pei & Yoo, 2002; Takahashi et al., 2003a; van der Poel et al., 2001). Swine HEV is enzootic in swine herds worldwide (Meng, 2003). Seroepidemiological studies have shown that pig handlers are at higher risk of HEV infection than normal blood donors (Drobeniuc et al., 2001; Meng et al., 2002). Experimental interspecies transmission of HEV has been demonstrated: swine HEV was found to infect non-human primates, and a US strain of human HEV infected pigs (Halbur et al., 2001; Meng et al., 1998a, b). Increasing evidence indicates that pigs are animal reservoirs for HEV and that hepatitis E may be zoonotically transmitted to humans (Meng, 2000a, b; Tei et al., 2003).

Big liver and spleen disease (BLS) in chickens was first recognized in Australia in 1980 and considered to be the most economically significant disease affecting commercial broiler breeder flocks in that country (Payne et al., 1999). The causative agent, BLS virus (BLSV), was isolated from diseased chickens in Australia (Payne et al., 1999). Based on the sequence of a very short genomic region, BLSV was found to be genetically related to human HEV (Payne et al., 1999). More recently, Haqshenas et al. (2001) identified a strain of HEV, avian HEV, from a chicken with hepatitis–splenomegaly syndrome (HS syndrome) in the USA using PCR primers primarily based on the sequence of BLSV (Haqshenas et al., 2001). Like BLSV, avian HEV is also distantly related to human HEV (Haqshenas et al., 2001). Avian HEV shared about 80 % nucleotide sequence identity with BLSV in a short stretch of sequence available for BLSV but shared only about 57–61 % sequence identity with mammalian HEVs in this region (Haqshenas et al., 2001).

The approximately 4 kb 3' sequence of avian HEV genome has been determined previously (Haqshenas et al., 2001). It is known that mammalian HEVs encode a methyltransferase at the 5' end of the genome (Koonin et al., 1992) and that the genome is capped (Kabrane-Lazizi et al., 1999b). Therefore, it was important to identify the remaining 5'-end sequence of avian HEV to compare it with known HEVs. In this study, we completed the sequencing of avian HEV genome and compared the sequence with those of known human and swine HEVs. We also attempted to infect rhesus monkeys with avian HEV.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus.
Avian HEV used in this study was originally recovered from a bile sample of a 56-week-old White Plymouth Rock chicken with HS syndrome (F93-5077) in California (Haqshenas et al., 2001). A standard infectious stock of avian HEV was prepared as a mixture of bile and 10 % suspension of faeces collected from experimentally infected specific-pathogen-free (SPF) chickens. This avian HEV stock had an infectivity titre of 5x105·0 50 % chicken infectious doses (CID50) ml –1 (Sun et al., unpublished data) and was used for the rhesus monkey transmission study. Bile containing avian HEV collected from an experimentally infected SPF chicken was used for construction of cDNA libraries.

Construction of cDNA libraries of avian HEV genomic RNA.
The 3'-terminal sequence of avian HEV has been previously reported (Haqshenas et al., 2001). To determine the complete genomic sequence, we constructed two cDNA libraries of avian HEV genomic RNA based on the known avian HEV sequences. Synthesis and cloning of cDNA were accomplished using the SuperScript Plasmid System with Gateway Technology for cDNA Synthesis and Cloning (Invitrogen). Briefly, total RNA was extracted with Trizol reagent from a bile sample containing avian HEV. Reverse transcription was performed at 42 °C for 1 h with SuperScript II reverse transcriptase (RT; Invitrogen) and the NotI RT primer-adapter (Table 1) in the first cDNA library or at 65 °C for 1 h with ThermoScript RT (Invitrogen) and the NotI RT-1 primer-adapter (Table 1) in the second cDNA library. Primer-adapter NotI RT was based on the known terminal sequence of avian HEV with a NotI restriction site engineered at the 5' end. Primer-adapter NotI RT-1, used in the second cDNA library construction, was based on the 5' sequence of cDNA clones identified in the first cDNA library. The second-strand cDNA was synthesized using DNA polymerase I, RNase H and DNA ligase at 16 °C for 2 h. After addition of a SalI adapter, the resulting double-stranded cDNA was digested by NotI restriction endonuclease. cDNA fractionated by column chromatography was ligated to the vector pSPORT 1 previously digested with NotI and SalI. The ligation mixture was transformed into One Shot TOP 10F' competent E. coli cells (Invitrogen) and 50 µl was spread on LB agar plates containing ampicillin and incubated at 37 °C overnight.


View this table:
[in this window]
[in a new window]
 
Table 1. Synthetic oligonucleotide primers used for cDNA amplification, library constructions and screening and DNA sequencing of the avian HEV genome

 
Synthesis of DIG-labelled avian HEV-specific cDNA probes for cDNA library screening.
Total RNA extracted from bile containing avian HEV was used to synthesize two avian HEV-specific cDNA probes by RT-PCR with a DIG labelling mix kit (Roche Molecular Biochemicals). Briefly, primers Labelling F1 and Labelling R1 were designed on the basis of the known 3'-terminal sequence of avian HEV upstream of the NotI RT primer-adapter used for the first cDNA library construction. A DIG-labelled 190 bp cDNA fragment was amplified as the probe with this primer set. The DIG-labelled cDNA probe used for colony screening in the second cDNA library was synthesized as a 100 bp cDNA fragment by the same method with primers Labelling F2 and Labelling R2, which were based on the 5'-terminal sequences of cDNA clones identified in the first cDNA library (Table 1).

Identification of authentic cDNA clones containing avian HEV genome by colony hybridization.
LB plates containing recombinant bacterial colonies growing at 37 °C were chilled at 4 °C for 2 h. A marked nylon membrane was then placed on each LB agar plate for 5 min to allow the transfer of the colonies to the membrane. The membranes were removed and dried for 5 min, followed by denaturation with 1·5 M NaCl and 0·5 M NaOH for 10 min and then neutralization with 1·5 M NaCl and 1 M Tris/HCl (pH 8·0) for 10 min. The plasmid DNA from the recombinant colonies was cross-linked to the membranes by baking at 80 °C for 30 min. Prehybridization of each membrane was performed at 55 °C for 2 h in a Reichert–Jung water bath shaker. The prehybridization solution was then replaced with 30 ml hybridization solution containing denatured DIG-labelled avian HEV-specific probes (15 ng ml–1) and incubated at 55 °C overnight. The hybridized membranes were washed twice for 10 min each with 2x wash solution (2x SSC, 0·1 % SDS) and then twice for 15 min each with 0·5x wash solution.

After colony hybridization, the positive colonies that hybridized to the DIG-labelled avian HEV-specific cDNA probes were detected using a DIG Nucleic Acid Detection Kit (Roche Molecular Biochemicals). Briefly, the hybridized membranes were incubated in a blocking solution, followed by an anti-DIG antibody solution, each for 30 min. After equilibration of the membranes for 5 min in a detection buffer, the membranes were incubated in a freshly prepared substrate solution in the dark. When positive signals appeared, the reaction was stopped by washing the membranes with sterile water. Colonies corresponding to the positive signals on the membranes were matched to the original plates, selected and cultured in LB media containing ampicillin overnight at 37 °C in an incubator shaker (New Brunswick Scientific, model I2400). Plasmid DNA was extracted with a GenElute Plasmid Miniprep Kit (Sigma) and verified by restriction enzyme digestion with NotI and SalI. Three independent cDNA clones of 508 bp, 540 bp and 551 bp from the first cDNA library and another three independent cDNA clones of 2440 bp, 2380 bp and 2305 bp from the second cDNA library were selected for sequencing.

5' RACE for the identification of the extreme 5' sequence of avian HEV genome.
It has been shown that the genomic RNA of HEV is capped at its 5' end (Kabrane-Lazizi et al., 1999b; Zhang et al., 2001). Therefore, to identify the extreme 5' genomic sequence of avian HEV, an RNA ligase-mediated RACE (RLM-RACE) was used (FirstChoice RLM-RACE Kit; Ambion). Briefly, total RNA extracted from bile containing avian HEV was treated with calf intestinal phosphatase to remove the 5' phosphate from degraded mRNAs but not from viral RNA with a 5' cap structure. The reaction was then treated with tobacco acid pyrophosphatase to remove the cap structure from the viral RNA, followed by ligation of an RNA adapter (5' RACE Adapter) (Table 1) to the viral RNA. The ligated RNA was used as the template for cDNA synthesis with an avian HEV-specific antisense primer (5' AHEV R1) (Table 1) and SuperScript II RT. The resulting cDNA was amplified by a nested PCR with two sense adapter primers supplied in the kit (5' RACE Outer primer and 5' RACE Inner primer) and two antisense primers specific to avian HEV (5' AHEV RACE Outer and 5' AHEV RACE Inner) (Table 1).

Nucleotide sequencing and sequence analysis.
Three independent cDNA clones from each of the two cDNA libraries were selected for sequencing using the primer-walking strategy. The PCR products of 5' RLM-RACE were purified with a GENECLEAN kit (Bio 101 Inc.) and directly sequenced at the Virginia Tech DNA Sequencing Facility. Sequences of the PCR products and cDNA clones were determined for both DNA strands.

The full-length genomic sequence was assembled and analysed using the MacVector computer program (Oxford Molecular Inc.). Multiple nucleotide and amino acid sequence alignments of avian HEV and strains of human and swine HEVs with known full-length or near full-length genomic sequences were analysed using the MacVector program. GenBank database searches of the avian HEV sequence were carried out with BlastN, BlastP and BlastX with default settings.

Phylogenetic analysis was conducted with the aid of the PAUP program (David Swofford, Smithsonian Institute, Washington, DC, distributed by Sinauer Associate Inc.). An heuristic search with 1000 replicates was used to produce a phylogenetic tree.

Verification of deletions and insertions in avian HEV genome.
After the full-length genomic sequence of avian HEV was assembled, numerous deletions and insertions were observed in ORF1 when aligned with genomic sequences of human and swine HEVs. To confirm these deletions and insertions, approximately 3 kb of ORF1 sequence at the 5' end of the avian HEV genome was amplified directly by RT-PCR as six overlapping fragments from a bile sample containing avian HEV. The RT-PCR reactions were performed with the addition of 5 % DMSO, using primer pairs ConF1/ConR1, ConF2/ConR2, ConF3/ConR3, ConF4/ConR4, ConF5/ConR5 and ConF6/ConR6 (Table 1).

Experimental inoculation of rhesus monkeys with avian HEV.
We have previously infected a chimpanzee and rhesus monkeys with swine HEV (Meng et al., 1998b). To assess whether avian HEV can also infect across species, two rhesus monkeys were each inoculated intravenously with 5x105·0 CID50 avian HEV. Pre-inoculation and weekly serum and faecal samples were collected from both monkeys. Sera were tested for avian HEV RNA using a nested RT-PCR, for IgG antibody to avian HEV using an ELISA and for serum liver enzymes by standard methods. Faecal samples were tested for viral RNA using a nested RT-PCR. The animals were monitored for evidence of viral infection and hepatitis for 22 weeks.

RT-PCR for detection of avian HEV RNA from experimentally inoculated monkeys.
To determine whether avian HEV RNA was present in faecal and serum samples from the experimentally inoculated monkeys, we performed a nested RT-PCR with avian HEV-specific primers (FAHEVEp/RAHEVEp and FAHEVEpF/RAHEVEpR) (Table 1). The PCR primers were based on the ORF2 sequence of the avian HEV used in the inoculum. The parameters for each round of the nested PCR included denaturation at 95 °C for 6 min, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 50 s at 58 °C and extension for 1 min at 72 °C, with a final incubation at 72 °C for 7 min. Positive and negative controls were included in each set of RT-PCR amplifications. RNA extraction and other pre-PCR amplification steps were performed in a separate clean room to minimize cross-contamination.

ELISA.
A purified truncated recombinant avian HEV ORF2 protein expressed in E. coli was used as the antigen for an ELISA to detect antibody to avian HEV in experimentally inoculated monkeys (Haqshenas et al., 2002; Huang et al., 2002b). All sera from the monkeys before or after inoculation were tested in duplicate at a dilution of 1 : 100 using peroxidase-labelled goat anti-human IgG (Sigma) as the secondary antibody.

GenBank accession numbers.
The accession numbers of the strains of human and swine HEVs with full-length or near full-length genomic sequences used in the study are: Arkell swine HEV (AY115488), JRA1 (AP003430), JKN-Sap (AB074918), swJ570 (AB073912), prototype swine HEV (AF082843), US1 (AF060668), US2 (AF060669), JMY-Haw (AB074920), HE-JA1 (AB097812), HEVNE8L (D10330), HeBei (M94177), Hyderabad (AF076239), HEVJapan (E17109), Madras (X99441), Haryana (AF459438), Burma (M73218), Xinjiang (D11092), KS2-87 (L25547), hev037 (X98292), Sar-55 (AF444003), T1 (AJ272108), Morocco (AY230202), Mexico (M74506), JSN (AB091395), JJT-Kan (AB091394), HE-JI4 (AB080575), JAK-Sai (AB074915), JKK-Sap (AB074917) and swJ13-1 (AB097811).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Complete sequence and organization of the avian HEV genome
Based on the available 3'-terminal sequence, avian HEV shares only about 50 % nucleotide sequence identity with mammalian HEVs (Haqshenas et al., 2001). Therefore, it would have been difficult to amplify the avian HEV genome by RT-PCR with degenerate primers based on the sequences of human and swine HEVs. For this reason, we constructed two cDNA libraries of avian HEV genomic RNA. In the first cDNA library, positive colonies containing avian HEV cDNA were detected with a DIG-labelled avian HEV-specific probe and the cDNA inserts were verified by sequencing. However, the positive cDNA inserts in the first cDNA library were all less than 600 bp. Therefore, based on the sequence of the three cDNA clones identified in the first cDNA library, a second cDNA library was constructed by using ThermoScript RT, which can melt secondary structures and produce longer cDNA fragments. Clones containing avian HEV cDNA inserts of up to 2·5 kb were detected in the second cDNA library using a DIG-labelled avian HEV-specific probe and the cDNA inserts were confirmed by sequencing. With RLM-RACE, the region at the extreme 5' end of the avian HEV genome was successfully amplified using a nested RT-PCR and sequenced.

The consensus sequence of the avian HEV genome was determined from at least three independent cDNA clones from each of the two cDNA libraries. The resulting consensus sequence in ORF1 was further confirmed by direct sequencing of six overlapping RT-PCR products spanning the ORF1 region. An additional 2723 nt were identified at the 5' end of the avian HEV genome in this study. Combined with the known 3' sequence, the complete genome of avian HEV, excluding the 3' poly(A) tail, was 6654 nt in length, which is about 600 bp shorter than that of human and swine HEVs. Similar to human and swine HEVs, the genome of avian HEV consisted of a short 5' non-coding region (NCR) followed by three partially overlapping ORFs and a 3'NCR. The 5'NCR of avian HEV consisted of 24 bp, which is 2–4 bp shorter than that of most human and swine HEV strains (Fig. 1). The ORF1 began at nt 25 and ended at nt 4620 and potentially encoded a polyprotein of 1531 aa. ORF2, from nt 4707 to 6527, comprised 1821 nt and encoded a putative capsid protein of 606 aa (Haqshenas et al., 2001). ORF3, from nt 4654 to 4917, consists of 264 nt and encoded a small protein of 87 aa (Haqshenas et al., 2001) (Fig. 2). As in human and swine HEVs, the ORF2 of avian HEV also overlapped ORF3. Complete sequence analysis revealed that the predicted polyprotein encoded by ORF1 of avian HEV contained several putative functional domains including the methyltransferase, papain-like cysteine protease, helicase and RdRp, which are also present in human and swine HEVs (Fig. 3). Amino acid sequence comparisons revealed that motifs typical of the helicase superfamily I and of the putative viral methyltransferase found throughout the alpha-like virus supergroup were conserved between avian HEV and mammalian HEVs (Koonin et al., 1992) (Fig. 3). For example, 6 of the 10 aa in motif I, 5 of the 7 aa in motif II and 7 of the 15 aa in motif III of the methyltransferase were identical between avian HEV and mammalian HEVs. Similarly, in the helicase, 9 of the 12 aa in motif I, 11 of the 15 aa in motif V and 9 of the 11 aa in motif VI were identical between avian HEV and mammalian HEVs (Fig. 3).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Nucleotide sequence alignment of the 5'NCR of avian HEV and selected strains of human and swine HEVs. The 5'NCR sequence of Sar-55 human HEV strain is shown at the top and only differences are indicated in other strains. The sequence of avian HEV is shown at the bottom. Deletions are indicated by dashes. Identical nucleotide sequences are indicated by dots.

 


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 2. Schematic diagram of the genomic organization of avian HEV, which contains a short 5'NCR, a 3'NCR and three partially overlapping ORFs: ORF1 encodes non-structural proteins including putative functional domains of the methyltransferase (Methyl), helicase (Heli) and RNA-dependent RNA polymerase (RdRp); ORF2 encodes putative capsid protein (CP) and ORF3 encodes a small protein with unknown function. The first and last nucleotide positions of the NCRs and ORFs are indicated in parentheses.

 


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 3. Alignment of the deduced amino acid sequence of avian HEV ORF1 (aHEV) with those of selected HEV strains, one from each of the four genotypes: Sar-55 (genotype 1), Mexico (genotype 2), sHEV (swine HEV, genotype 3) and T1 (genotype 4). The ORF1 sequence of Sar-55 human HEV is shown on the top and only differences in other strains are indicated. Deletions are indicated by dashes. Identical amino acids are indicated by dots. The first and last nucleotide positions of the putative functional domains in ORF1 of human and swine HEVs are indicated with arrows: methyltransferase (Methyl, aa 56–241), papain-like cysteine protease (PLP, aa 433–593), hypervariable region (HVR, aa 673–802), helicase (aa 984–1216) and RNA-dependent RNA polymerase (RdRp, aa 1231–1720). The conserved motifs I–VI typical of the helicase superfamily I and the conserved motifs I–III in viral methyltransferase found throughout the alpha-like virus supergroup are indicated. Asterisks indicate identical amino acid residues.

 
Avian HEV is genetically and phylogenetically related to, but distinct from, human and swine HEVs
The complete genomic sequence of avian HEV was compared with the corresponding sequences of human and swine HEV strains. It was found that avian HEV shared about 50 % nucleotide sequence identity over the entire genome, 48–51 % identity in ORF1 (Table 2), 46–48 % identity in ORF2 and only 29–34 % identity in ORF3 with human and swine HEV strains (Haqshenas et al., 2001). At the amino acid sequence level, the putative polyprotein encoded by ORF1 of avian HEV shared 41–42 % identity with strains of human and swine HEVs. Sequences of putative functional domains in ORF1 of avian HEV were also compared with those of human and swine HEV strains. The helicase gene was the most conserved between avian HEV and other HEV strains, displaying 56–59 % nucleotide sequence identity (Table 2). The ORF1 of avian HEV was 4596 nt in length, which is 480–521 nt shorter than that of human and swine HEVs. Significant genetic variation, characterized by multiple deletions and insertions, was observed in the ORF1 of avian HEV (Fig. 3). Most of the deletions were located in and between the proposed papain-like cysteine protease domain and the hypervariable region (HVR) of human and swine HEVs. However, the deletions and insertions did not alter the reading frame of avian HEV ORF1.


View this table:
[in this window]
[in a new window]
 
Table 2. Nucleotide sequence comparison of avian HEV with selected human and swine HEV strains

 
Phylogenetic analysis based on the complete genomic sequence of HEV confirmed that avian HEV was distantly related to swine and human HEVs. Avian HEV was segregated into a distinct branch separate from human and swine HEVs of the four known genotypes (Fig. 4).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. A phylogenetic tree based on the complete genomic sequences of avian HEV and 29 human and swine HEV strains. The tree was constructed with the aid of the PAUP program by using an heuristic search with 1000 replicates. The scale bar, indicating the number of character state changes, is proportional to the genetic distance.

 
Failure to infect rhesus monkeys experimentally with avian HEV
Swine HEV has been shown to infect rhesus monkeys and a chimpanzee (Meng et al., 1998b). Thus, we attempted to infect two rhesus monkeys with avian HEV. The two inoculated monkeys were not infected by avian HEV as evidenced by absence of seroconversion, viraemia, faecal virus shedding or elevation of serum liver enzymes.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HEV was originally misclassified as a calicivirus because of its genomic organization, which superficially resembles that of the Caliciviridae. To determine whether the avian HEV shared all of the important characteristics of the Hepeviridae, we wished to analyse all the motifs that now define this new family. The sequence data that we obtained in this study permitted us to make a more thorough comparison of avian HEV with other HEVs: the results suggested that avian HEV shares significant features with other HEVs and is related to them, in spite of the overall low sequence homologies. The genome organization of avian HEV is very similar to that of human and swine HEVs. The polyprotein encoded by ORF1 of avian HEV consists of functional domains and motifs similar to those found in human and swine HEVs. The viral methyltransferase found in some single-stranded RNA viruses is involved in mRNA capping, and a cap structure at the 5' terminus of mammalian HEVs has been demonstrated (Kabrane-Lazizi et al., 1999b; Okamoto et al., 2001; Zhang et al., 2001). In this study, we used a pyrophosphatase-dependent elongation reaction with RLM-RACE to amplify the extreme 5'-genomic sequence of avian HEV, suggesting that avian HEV genomic RNA is also capped. The identification of a methyltransferase gene in the avian HEV genome and the conserved motifs I, II and III in the methyltransferase gene between avian HEV and mammalian HEVs provided important support for classifying avian HEV with other HEVs. The 5'NCRs of HEVs are highly conserved among human and swine HEV strains except for 10 additional nucleotides at the 5' terminus of the US2 strain (Zhang et al., 2001). However, the 5'NCR of avian HEV was highly divergent from that of human and swine HEVs. The biological significance of the observed variation in the 5'NCR between avian HEV and other HEV strains is unclear but may be related to differences in the translation apparatus of mammals and birds.

The complete avian HEV genome was approximately 600 bp shorter than that of other HEV strains. Sequence comparison revealed multiple deletions in avian HEV across the genome, but especially in and around the domains of the putative papain-like cysteine protease and the HVR, which in mammalian HEVs are the most divergent regions. Therefore, deletions and extensive sequence variations in this region of the avian HEV genome are not surprising. Taken together, the evidence suggests that this region may not play a functional role in HEV replication. Importantly, the deletions and the insertions did not cause a frame-shift in ORF1 of avian HEV as might have happened if they were errors caused by PCR. It is known that strong secondary structures in GC-rich regions can produce artificial deletions during RT-PCR amplification. To rule out this possibility, six overlapping fragments within ORF1 were directly amplified from the virus stock by RT-PCR in the presence of DMSO. No additional sequence was identified by direct sequencing of RT-PCR products. For these reasons, we concluded that the observed deletions in ORF1 were not artefacts. The biological significance of these deletions is not known.

Thus far, four major genotypes of HEV have been identified (Emerson & Purcell, 2003): the Burmese-like genotype 1 consisting of Asian and African strains (Arankalle et al., 2002; Tam et al., 1991; Tsarev et al., 1999), the Mexican genotype 2 consisting of a Mexican strain and possibly Nigerian strains (Buisson et al., 2000; Huang et al., 1992), the USA genotype 3 consisting of both human and swine HEV strains identified in countries where hepatitis E is rare (Erker et al., 1999; Meng et al., 1997; Schlauder et al., 1998, 1999; Takahashi et al., 2003a, b) and the new genotype 4 consisting of strains of both human and swine HEV from Asia (Takahashi et al., 2002; Wang et al., 1999). Phylogenetic analysis based on the full-length genomic sequences of 30 HEV strains including human, swine and avian HEV showed that avian HEV belongs to a distinct branch separate from those of genotypes 1–4 of human and swine HEV strains. It is not known whether avian HEV represents a fifth genotype of HEV or belongs to a separate genus. We have previously shown that avian HEV shares common antigenic epitope(s) in the capsid protein with human and swine HEVs (Haqshenas et al., 2002) and thus it is possible that avian HEV may belong to a new genotype 5 of HEV. However, additional studies such as cross-neutralization experiments are needed to classify avian HEV definitively.

The seroprevalence of HEV antibodies in different animal species has been widely reported (Chandler et al., 1999; Kabrane-Lazizi et al., 1999a; Meng et al., 1999). However, the viruses responsible for the seropositivity in these animal species have not been definitively identified except in pigs and chickens (Haqshenas et al., 2001; Meng et al., 1997; Payne et al., 1999). Accumulated evidence suggests that swine HEV infection may be zoonotic (Meng, 2000a, b; Tei et al., 2003). Therefore, in this study we attempted to infect rhesus monkeys with avian HEV. Unlike swine HEV, however, avian HEV was not transmitted to rhesus monkeys. The negative result was not surprising since avian HEV shared only approximately 50 % nucleotide sequence identity with human and swine HEVs. The availability of the complete genomic sequence of avian HEV from this study now affords us an opportunity to construct an infectious cDNA clone of avian HEV and to study the molecular mechanism of HEV pathogenesis using chickens as a model.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Institutes of Health (AI01653, AI46505 and AI50611) and from the US Department of Agriculture's National Research Initiative Competitive Grant Program (NRI 2004-35204-12531). We thank Dr Zhijian Tu for his expert assistance in colony hybridization and sequence analysis.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Arankalle, V. A., Chobe, L. P., Joshi, M. V., Chadha, M. S., Kundu, B. & Walimbe, A. M. (2002). Human and swine hepatitis E viruses from Western India belong to different genotypes. J Hepatol 36, 417–425.[CrossRef][Medline]

Buisson, Y., Grandadam, M., Nicand, E. & 7 other authors (2000). Identification of a novel hepatitis E virus in Nigeria. J Gen Virol 81, 903–909.[Abstract/Free Full Text]

Chandler, J. D., Riddell, M. A., Li, F., Love, R. J. & Anderson, D. A. (1999). Serological evidence for swine hepatitis E virus infection in Australian pig herds. Vet Microbiol 68, 95–105.[CrossRef][Medline]

Choi, I. S., Kwon, H. J., Shin, N. R. & Yoo, H. S. (2003). Identification of swine hepatitis E virus (HEV) and prevalence of anti-HEV antibodies in swine and human populations in Korea. J Clin Microbiol 41, 3602–3608.[Abstract/Free Full Text]

Drobeniuc, J., Favorov, M. O., Shapiro, C. N. & 7 other authors (2001). Hepatitis E virus antibody prevalence among persons who work with swine. J Infect Dis 184, 1594–1597.[CrossRef][Medline]

Emerson, S. U. & Purcell, R. H. (2003). Hepatitis E virus. Rev Med Virol 13, 145–154.[CrossRef][Medline]

Erker, J. C., Desai, S. M., Schlauder, G. G., Dawson, G. J. & Mushahwar, I. K. (1999). A hepatitis E virus variant from the United States: molecular characterization and transmission in cynomolgus macaques. J Gen Virol 80, 681–690.[Abstract]

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

Halbur, P. G., Kasorndorkbua, C., Gilbert, C., Guenette, D., Potters, M. B., Purcell, R. H., Emerson, S. U., Toth, T. E. & Meng, X. J. (2001). Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. J Clin Microbiol 39, 918–923.[Abstract/Free Full Text]

Haqshenas, G., Shivaprasad, H. L., Woolcock, P. R., Read, D. H. & Meng, X. J. (2001). Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis–splenomegaly syndrome in the United States. J Gen Virol 82, 2449–2462.[Abstract/Free Full Text]

Haqshenas, G., Huang, F. F., Fenaux, M., Guenette, D. K., Pierson, F. W., Larsen, C. T., Shivaprasad, H. L., Toth, T. E. & Meng, X. J. (2002). The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus. J Gen Virol 83, 2201–2209.[Abstract/Free Full Text]

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

Huang, C. C., Nguyen, D., Fernandez, J., Yun, K. Y., Fry, K. E., Bradley, D. W., Tam, A. W. & Reyes, G. R. (1992). Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology 191, 550–558.[Medline]

Huang, F. F., Haqshenas, G., Guenette, D. K., Halbur, P. G., Schommer, S. K., Pierson, F. W., Toth, T. E. & Meng, X. J. (2002a). Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol 40, 1326–1332.[Abstract/Free Full Text]

Huang, F. F., Haqshenas, G., Shivaprasad, H. L. & 7 other authors (2002b). Heterogeneity and seroprevalence of a newly identified avian hepatitis E virus from chickens in the United States. J Clin Microbiol 40, 4197–4202.[Abstract/Free Full Text]

Kabrane-Lazizi, Y., Fine, J. B., Elm, J. & 7 other authors (1999a). Evidence for widespread infection of wild rats with hepatitis E virus in the United States. Am J Trop Med Hyg 61, 331–335.[Abstract/Free Full Text]

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

Kabrane-Lazizi, Y., Zhang, M., Purcell, R. H., Miller, K. D., Davey, R. T. & Emerson, S. U. (2001). Acute hepatitis caused by a novel strain of hepatitis E virus most closely related to United States strains. J Gen Virol 82, 1687–1693.[Abstract/Free Full Text]

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

Meng, X. J. (2000a). Novel strains of hepatitis E virus identified from humans and other animal species: is hepatitis E a zoonosis? J Hepatol 33, 842–845.[CrossRef][Medline]

Meng, X. J. (2000b). Zoonotic and xenozoonotic risks of hepatitis E virus. Infect Dis Rev 2, 35–41.

Meng, X. J. (2003). Swine hepatitis E virus: cross-species infection and risk in xenotransplantation. Curr Top Microbiol Immunol 278, 185–216.[Medline]

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

Meng, X. J., Halbur, P. G., Haynes, J. S., Tsareva, T. S., Bruna, J. D., Royer, R. L., Purcell, R. H. & Emerson, S. U. (1998a). Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Arch Virol 143, 1405–1415.[CrossRef][Medline]

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

Meng, X. J., Dea, S., Engle, R. E. & 10 other authors (1999). Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. J Med Virol 59, 297–302.[CrossRef][Medline]

Meng, X. J., Wiseman, B., Elvinger, F., Guenette, D. K., Toth, T. E., Engle, R. E., Emerson, S. U. & Purcell, R. H. (2002). Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. J Clin Microbiol 40, 117–122.[Abstract/Free Full Text]

Okamoto, H., Takahashi, M., Nishizawa, T., Fukai, K., Muramatsu, U. & Yoshikawa, A. (2001). Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochem Biophys Res Commun 289, 929–936.[CrossRef][Medline]

Payne, C. J., Ellis, T. M., Plant, S. L., Gregory, A. R. & Wilcox, G. E. (1999). Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus. Vet Microbiol 68, 119–125.[CrossRef][Medline]

Pei, Y. & Yoo, D. (2002). Genetic characterization and sequence heterogeneity of a Canadian isolate of swine hepatitis E virus. J Clin Microbiol 40, 4021–4029.[Abstract/Free Full Text]

Schlauder, G. G., Dawson, G. J., Erker, J. C., Kwo, P. Y., Knigge, M. F., Smalley, D. L., Rosenblatt, J. E., Desai, S. M. & Mushahwar, I. K. (1998). The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. J Gen Virol 79, 447–456.[Abstract]

Schlauder, G. G., Desai, S. M., Zanetti, A. R., Tassopoulos, N. C. & Mushahwar, I. K. (1999). Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV. J Med Virol 57, 243–251.[CrossRef][Medline]

Takahashi, M., Nishizawa, T., Yoshikawa, A., Sato, S., Isoda, N., Ido, K., Sugano, K. & Okamoto, H. (2002). Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad. J Gen Virol 83, 1931–1940.[Abstract/Free Full Text]

Takahashi, M., Nishizawa, T. & Okamoto, H. (2003a). Identification of a genotype III swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. J Clin Microbiol 41, 1342–1343.[Free Full Text]

Takahashi, M., Nishizawa, T., Miyajima, H., Gotanda, Y., Iita, T., Tsuda, F. & Okamoto, H. (2003b). Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. J Gen Virol 84, 851–862.[Abstract/Free Full Text]

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

Tei, S., Kitajima, N., Takahashi, K. & Mishiro, S. (2003). Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362, 371–373.[CrossRef][Medline]

Tsarev, S. A., Binn, L. N., Gomatos, P. J., Arthur, R. R., Monier, M. K., van Cuyck-Gandre, H., Longer, C. F. & Innis, B. L. (1999). Phylogenetic analysis of hepatitis E virus isolates from Egypt. J Med Virol 57, 68–74.[CrossRef][Medline]

Tyagi, S., Korkaya, H., Zafrullah, M., Jameel, S. & Lal, S. K. (2002). The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. J Biol Chem 277, 22759–22767.[Abstract/Free Full Text]

van der Poel, W. H., Verschoor, F., van der Heide, R., Herrera, M. I., Vivo, A., Kooreman, M. & de Roda Husman, A. M. (2001). Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerg Infect Dis 7, 970–976.[Medline]

Wang, Y., Ling, R., Erker, J. C., Zhang, H., Li, H., Desai, S., Mushahwar, I. K. & Harrison, T. J. (1999). A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. J Gen Virol 80, 169–177.[Abstract]

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

Zhang, M., Purcell, R. H. & Emerson, S. U. (2001). Identification of the 5' terminal sequence of the SAR-55 and MEX-14 strains of hepatitis E virus and confirmation that the genome is capped. J Med Virol 65, 293–295.[CrossRef][Medline]

Received 24 November 2003; accepted 16 February 2004.