Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis–splenomegaly syndrome in the United States

G. Haqshenas1, H. L. Shivaprasad2, P. R. Woolcock2, D. H. Read3 and X. J. Meng1

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Price’s Fork Road, Blacksburg, VA 24061-0342, USA1
California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California-Davis, 2789 South Orange Avenue, Fresno, CA 93725, USA2
California Veterinary Diagnostic Laboratory System, School of Veterinary Medicine, University of California-Davis, San Bernardino, CA 92408, USA3

Author for correspondence: X. J. Meng. Fax +1 540 231 3426. e-mail xjmeng{at}vt.edu


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis–splenomegaly (HS) syndrome is an emerging disease in chickens in North America; the cause of this disease is unknown. In this study, the genetic identification and characterization of a novel virus related to human hepatitis E virus (HEV) isolated from bile samples of chickens with HS syndrome is reported. Based upon the similar genomic organization and significant sequence identity of this virus with HEV, the virus has been tentatively named avian HEV in order to distinguish it from human and swine HEV. Electron microscopy revealed that avian HEV is a non-enveloped virus particle of 30–35 nm in diameter. The sequence of the 3' half of the viral genome (~4 kb) was determined. Sequence analyses revealed that this genomic region contains the complete 3' non-coding region, the complete genes from open reading frames (ORFs) 2 and 3, the complete RNA-dependent RNA polymerase (RdRp) gene and a partial helicase gene from ORF 1. The helicase gene is the most conserved gene between avian HEV and other HEV strains, displaying 58–61% aa and 57–60% nt sequence identities. The RdRp gene of avian HEV shares 47–50% aa and 52–53% nt sequence identities and the putative capsid gene (ORF 2) of avian HEV shares 48–49% aa and 48–51% nt sequence identities with the corresponding regions of other known HEV strains. Phylogenetic analysis indicates that avian HEV is genetically related to, but distinct from, other known HEV strains. This discovery has important implications for HEV animal models, nomenclature and natural history.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Human hepatitis E is an important public health disease in many developing countries and is endemic in some industrialized countries (Purcell, 1996 ; Harrison, 1999 ; Meng, 2000a , b). Hepatitis E virus (HEV), the causative agent of human hepatitis E, is a non-enveloped, single-stranded, positive-sense RNA virus (Purcell, 1996 ). The disease has a high mortality rate, reportedly up to 20%, in infected pregnant women (Purcell, 1996 ). The existence of a population of individuals who are positive for HEV antibodies (anti-HEV) in industrialized countries (Mast et al., 1997 ; Thomas et al., 1997 ; McCrudden et al., 2000 ) and the recent identification of numerous genetically distinct strains of HEV (Hsieh et al., 1998 , 1999 ; Schlauder et al., 1998 , 1999 , 2000 ; Erker et al., 1999 ; Zanetti et al., 1999 ; Wang et al., 1999 , 2000 ; Buisson et al., 2000 ; Pina et al., 2000 ) have led to a hypothesis that an animal reservoir for HEV exists. In 1997, we identified and characterized the first animal strain of HEV, swine HEV, from a pig in the US (Meng et al., 1997 ). We demonstrated that swine HEV is genetically related to human HEV (Meng et al., 1997 , 1998 b). Interspecies transmission of HEV has been documented previously: swine HEV was shown to infect non-human primates and a US strain of human HEV was shown to infect pigs (Meng et al., 1998 a, b , 1999 ; Meng, 2001 ).

Hepatitis–splenomegaly (HS) syndrome in chickens was first described in 1991 in western Canada (Ritchie & Riddell, 1991 ) and the disease has since been recognized in eastern Canada and the US (Riddell, 1997 ). HS syndrome is characterized by increased mortality in broiler breeder hens and laying hens of between 30 and 72 weeks of age. Affected chickens usually have regressive ovaries, red fluid in the abdomen and an enlarged liver and spleen. Microscopically, liver lesions vary from multifocal patches to areas of extensive hepatic necrosis and haemorrhage. Numerous other names have been used to describe this disease, such as necrotic haemorrhage hepatitis–splenomegaly syndrome, chronic fulminating cholangiohepatitis and necrotic haemorrhagic hepatomegalic hepatitis (Ritchie & Riddell, 1991 ; Tablante et al., 1994 ; Shivaprasad & Woolcock, 1995 ; Riddell, 1997 ).

The cause of HS syndrome is not known; a viral aetiology for HS syndrome has been suspected, but attempts to propagate the virus in either cell culture or embryonated eggs were unsuccessful (Shivaprasad & Woolcock, 1995 ). As anti human HEV were detected in 44% of chickens in Vietnam (Tien et al., 1997 ), suggesting that the chickens had been infected with HEV (or a related agent), we therefore aimed to investigate a possible link between HEV infection and HS syndrome in chickens. In this study, we report the genetic identification and characterization of a novel HEV-related virus which is associated with HS syndrome in chickens. Based upon its genomic organization and significant sequence identity with human and swine HEV, we tentatively designated this novel virus avian HEV, consistent with the current trends of naming animal strains of HEV.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Clinical specimens.
Bile samples collected from a 42-week-old White Leghorn chicken (S93-4346) and a 56-week-old White Plymouth Rock chicken with HS syndrome (F93-5077) in California were used in this study. The flocks from which both chickens originated had a history of decreased egg production and increased mortality.

{blacksquare} Electron microscopy (EM).
Bile samples were diluted in PBS buffer and clarified by centrifugation at 1780 g for 10 min at 4 °C. The supernatant was centrifuged at 140000 g for 90 min to pellet virus. The virus pellet was then resuspended in 0·3–1·0 ml deionized water and 2–3 µl was mixed with 100–200 µl of 0·8% phosphotungstic acid. A drop of this mixture was placed onto a 200 mesh Formvar-coated grid for up to 3·5 min. After wicking away unabsorbed material with filter paper, the grid was examined on a Zeiss EM10A electron microscope.

{blacksquare} Amplification of the 3' half of the viral genome.
Based on the assumption that the putative virus shares nt sequence homology with human and swine HEV, we employed a modified 3' RACE (rapid amplification of cDNA ends) technique to amplify the 3' half of the viral genome. Briefly, the sense primer F4AHEV (Table 1) was chosen from a conserved region within open reading frame (ORF) 1 of known HEV strains and big liver and spleen disease virus (BLSV), which was identified from chickens in Australia (Payne et al., 1999 ). The antisense primers included two commercial primers, an anchored adapter primer (AP) with a poly(T) stretch and an abridged universal amplification primer (AUAP), both of non-viral origin (Gibco-BRL) (Table 1). Total RNA, extracted from 100 µl of the bile sample S93-4346 using TriZol reagent (Gibco-BRL), was resuspended in 11·5 µl of DNase-, RNase- and proteinase-free water (Eppendorf). Total RNA was then reverse-transcribed at 42 °C for 90 min in the presence of a reverse transcription reaction mixture containing 11·5 µl of total RNA, 1 µl (200 units/µl) of Superscript II reverse transcriptase (RT) (Gibco-BRL), 1 µl of 10 µM antisense primer, 20 units of RNase inhibitor (Gibco-BRL), 0·5 µl of 0·1 M DTT and 4 µl of 5x RT buffer.


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Table 1. Synthetic oligonucleotide primers used for PCR amplification and DNA sequencing of the avian HEV genome

 
PCR was performed using the Elongase kit (Gibco-BRL) with a mixture of Taq DNA polymerase and a proofreading Pfu polymerase, according to the manufacturer’s instructions. Alternatively, AmpliTaq Gold polymerase either with or without 5% DMSO was used. Conditions for PCR were as follows: 1 cycle of denaturation at 94 °C for 1 min; 5 cycles of denaturation at 94 °C for 40 s, annealing at 42 °C for 40 s and extension at 68 °C for 5 min; 16 cycles of touch-down PCR with an initial annealing temperature at 59  °C, which then decreased by 1 °C every 2 cycles; 11 cycles of amplification with an annealing temperature at 51 °C; and a final extension of 10 min at 74 °C. The resulting PCR product was analysed on an 0·8% agarose gel. When AmpliTaq Gold polymerase was used, the thermal cycle profile and parameters remained the same, except that the enzyme was first activated by incubation at 95 °C for 9 min.

{blacksquare} Cloning of the amplified PCR product.
A PCR product of ~4 kb in length was amplified, purified from an agarose gel using the Concert Rapid Gel Extraction system (Gibco-BRL) and cloned into a TA vector. The recombinant plasmid was then used to transform the competent cells supplied in the Advantage PCR Cloning kit (Clontech), according to the manufacturer’s instructions. White colonies were selected and grown in LB broth containing 100 µg/ml of ampicillin. Recombinant plasmids containing the insert were isolated with a Plasmid DNA Isolation kit (Qiagen).

{blacksquare} DNA sequencing.
Three independent cDNA clones were selected and sequenced at Virginia Tech DNA Sequencing Facility (Corporate Research Center, Suite 1100, Blacksburg, VA, USA) using an automated DNA sequencer (Applied Biosystems). The primer walking strategy was employed to determine the sequence of both DNA strands of the three independent cDNA clones. The M13 forward and reverse primers as well as 16 avian HEV-specific primers were used (Table 1). To facilitate sequencing, we also utilized the unique EcoRI site present in this ~4 kb viral genome fragment. The recombinant plasmid containing the ~4 kb insert was digested with EcoRI and the resulting two EcoRI fragments were subcloned into pGEM-9zf(-) (Promega). The cDNA subclones were also sequenced using the primer walking strategy.

{blacksquare} Sequence and phylogenetic analyses.
The complete sequence of the ~4 kb viral genome fragment was assembled and analysed with the MacVector (Oxford Molecular) and DNASTAR computer programs. The consensus sequence was derived from at least three independent cDNA clones. The putative signal peptide of the ORF 2 protein was predicted with the SignalP program, version 1.1 (http://www.cbs.dtu.dk/services/SignalP). Phylogenetic analyses were conducted with the aid of the PAUP program (D. L. Swofford, Smithsonian Institution, Washington DC, USA). The branch-and-bound search and mid-point rooting options with 1000 replicates were used to produce the phylogenetic trees. Phylogenetic analyses were performed on three different regions for which the sequences of most HEV strains are available: a 148 bp fragment of the ORF 2 gene, a 196 bp fragment of the RNA-dependent RNA polymerase (RdRp) gene and a 439 bp fragment of the helicase gene, for which the sequence of BLSV is also known. A phylogenetic tree was also constructed using the entire 3' half of the avian HEV genome (~4 kb) and the corresponding region of other HEV isolates with known complete genomic sequences. The sequences of known HEV strains used in the sequence and phylogenetic analyses, which are either published or available in GenBank, are as follows: Nepal (Gouvea et al., 1997 ); Egypt93 and Egypt94 (Tsarev et al., 1999 ); Morocco (J. Meng et al., 1999 ); Pakistan strain Sar55 (Tsarev et al., 1992 ); Burma (Reyes et al., 1990 ); Myanmar (Tam et al., 1991 ); Vietnam (accession no. AF170450); Greek1 and Greek2 (Schlauder et al., 1999 ); Italy (Schlauder et al., 1999 ); Mexico (Huang et al., 1992 ); US1 (Schlauder et al., 1998 ) and US2 (Erker et al., 1999 ); the US strain of swine HEV (Meng et al., 1997 , 1998 b); the New Zealand strain of swine HEV (accession no. AF200704); the Indian strains Hyderabad (Panda et al., 2000 ), Madras (accession no. X99441), HEV037 (accession no. X98292; Donati et al., 1997 ), AKL90 (Arankalle et al., 1999 ) and U22532 (Panda et al., 1995 ); the Taiwanese strains TW4E, TW7E and TW8E (Wu et al., 1998 ); and the Chinese strains 93G (accession no. AF145208), L25547 (Yin et al., 1994 ), Hetian (Uchida et al., 1992 ), KS2-87 (Yin et al., 1994 ), D11093 strain Uigh 179 and D11092 (Aye et al., 1992 ), HEV-T1 (Wang et al., 2000 ), Ch-T11 (accession no. AF151962) and Ch-T21 (accession no. AF151963).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Electron microscopy
Negative-staining EM of bile samples from chickens with HS syndrome revealed 30–35 nm diameter, non-enveloped virus particles (Fig. 1). The virus particles were similar in size and morphology to those of human HEV (Purcell, 1996 ).



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Fig. 1. Electron micrograph of negatively stained 30–35 nm diameter virus particles in a bile sample from a chicken with HS. Bar, 100 nm.

 
Amplification and sequence determination of the 3' half of the avian HEV genome
We attempted to amplify a ~4 kb fragment at the 3' half of the avian HEV genome. In the presence of 5% DMSO, a weak PCR product of ~4 kb in length was generated using AmpliTaq Gold polymerase (Fig. 2A). To increase the efficiency of amplification, we performed the PCR with a mixture of Pfu and Taq DNA polymerase using the Elongase kit. After 32 cycles of amplification, an abundant amount of PCR product of ~4 kb was generated (Fig. 2A). Due to the very limited number of positive bile samples, attempts to amplify the remaining 5' end of the viral genome were not possible. The resulting ~4 kb PCR product was subsequently cloned into a TA vector. Three independent cDNA clones were selected and sequenced for both DNA strands. The number of A residues at the 3' end of each of the three cDNA clones (19, 23 and 26 residues, respectively) was different, indicating that these three clones represent independent cDNA clones. GenBank BLAST sequence similarity searches revealed that the novel virus associated with HS syndrome in chickens is genetically related to both human and swine HEV, but has no significant sequence identity with other viruses, such as caliciviruses.



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Fig. 2. (A) Amplification of the 3' half (~4 kb) of the avian HEV genome by RT–PCR. Lane M, 1 kb ladder; lanes 1 and 2, PCR using AmpliTaq Gold polymerase; lanes 3 and 4, PCR using AmpliTaq Gold polymerase in the presence of 5% DMSO; lane 5 and 6, PCR amplification with a mixture of Taq and Pfu DNA polymerase. (B) RT–PCR amplification of the avian HEV genomic region with a major deletion. Lane M, 1 kb plus ladder; lanes 1 and 2, PCR amplification without DMSO; lane 3, PCR amplification in the presence of 5% DMSO; lane 4, PCR amplification in the presence of 5% formamide.

 
Genomic organization of avian HEV
The sequences of the three independent cDNA clones, excluding the poly(A) tract, are all equal in length, but differ in 16 nt. However, at any given position, two of the three cDNA clones have the same nt. The resulting consensus sequence of the 3' half of the avian HEV genome is 3931 nt in length, excluding the poly(A) tract. The genomic organization of the ~4 kb viral genome fragment of avian HEV is very similar to that of human and swine HEV in that it comprises (5'->3') an incomplete ORF 1, which contains a partial helicase gene and the complete RdRp gene, complete ORFs 2 and 3 and the complete 3' non-coding region (NCR). ORF 3 of other known HEV strains overlap with ORF 1, with the exception of a newly identified Chinese strain of human HEV, HEV-T1 (Wang et al., 2000 ). ORF 3 of the HEV-T1 strain does not overlap with ORF 1, but with ORF 2. Like the human HEV-T1 strain, ORF 3 of avian HEV also does not overlap with ORF 1. Upon further analysis of the published sequence of HEV-T1 (Wang et al., 2000 ), it was found that if HEV-T1 uses the third AUG codon for ORF 2, which is used in other HEV strains, HEV-T1 would have a very similar genomic organization to avian HEV in that neither ORF 2 nor ORF 3 would overlap with ORF 1.

Sequence analysis of ORF 1
Significant sequence identities were found in ORF 1 between avian HEV and other HEV strains (Table 2). Avian HEV ORF 1 region sequenced to date contains the complete RdRp gene and a partial helicase gene. The RdRp gene of avian HEV encodes 483 aa. A GDD motif, which is believed to be critical for virus replication, was identified and this motif was found in all RdRps (Kamer & Argos, 1984 ). The RdRp gene of avian HEV is 4 aa shorter than those of other known HEV strains and shares 47–50% aa and 52–53% nt sequence identities with those of other known HEV strains (Table 2). The C-terminal 146 aa of the partial helicase gene of avian HEV shares approximately 57–60% nt and 58–61% aa sequence identities with the corresponding region of other HEV strains (data not shown). All three conserved motifs (IV–VI) present at the C terminus of the helicase genes of positive-stranded RNA viruses (Koonin et al., 1992 ) were also conserved in the helicase gene of avian HEV. Also, avian HEV shares about 80% nt sequence identity with BLSV in the helicase gene region.


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Table 2. Pairwise comparison of the RdRp gene of avian HEV with those of other known HEV strains

 
Sequence analysis of the complete ORFs 2 and 3 of avian HEV
The ORF 2 gene of avian HEV comprises 1821 nt. Similar to swine and human HEV, the ORF 2 gene of avian HEV also overlaps with the ORF 3 gene (Fig. 3) and terminates at the UAA stop codon located 130 bases upstream of the poly(A) tract. The predicted aa sequence of ORF 2 contains a typical signal peptide; the sequence of the avian HEV signal peptide is distinct from those of other known HEV strains (Fig. 4). However, it contains common signal peptide features that are necessary for the translocation of the peptide into the endoplasmic reticulum: a positively charged aa (Arg) at its N terminus, a core of highly hydrophobic residues (Lys-rich) and a cleavage site (SRG/SQ) between aa 19 and aa 20 (Fig. 4). Sequence analysis of ORF 2 revealed that the N-terminal region before the conserved tetrapeptide APLT (residues 108–111) is hypervariable and 54 aa of this region are deleted from the avian HEV genome when compared to other HEV strains (Fig. 4). Three putative N-linked glycosylation sites were identified in avian HEV ORF 2: NLS (residues 255–257), NST (residues 510–512) and NGS (residues 522–524). Three N-linked glycosylation sites were also identified in other known HEV strains, but the locations are different from those of avian HEV (Fig. 4). The first glycosylation site in other known HEV strains is absent in avian HEV and the third glycosylation site in avian HEV is absent in other known HEV strains.



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Fig. 3. Sequence alignment of the overlapping regions of ORFs 1, 2 and 3. The sequence of the prototype Burmese strain is shown at the top and only differences are indicated in other HEV strains. The sequence of avian HEV is shown at the bottom. Start codons are indicated by arrows and stop codons are indicated by three asterisks (***). The location of the two PCR primers FdelAHEV and RdelAHEV, which were used to amplify the region flanking the deletion, are indicated. Deletions are indicated by dashes.

 


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Fig. 4. Amino acid sequence alignment of the putative capsid gene (ORF 2) of avian HEV with those of other known HEV strains. The putative signal peptide sequence is highlighted in grey and the predicted cleavage site is indicated by an arrowhead. N-linked glycosylation sites are underlined and in bold. The sequence of the prototype Burmese strain is shown at the top and only differences are indicated in other HEV strains. Asterisks indicate the conserved tetrapeptide APLT and deletions are indicated by dashes.

 
The ORF 2 gene of known HEV strains varies slightly in size and ranges from 655 to 672 aa. ORF 2 of avian HEV is 606 aa in length. Deletions are largely due to a shift in the ORF 2 start codon of avian HEV to 80 nt downstream from those of other known HEV strains (Fig. 3). ORF 2 of avian HEV shares 48–49% aa and 49–51% nt sequence identities with those of other HEV strains, when the N-terminal deletion is excluded in the comparison (Table 3).


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Table 3. Pairwise comparison of the putative capsid gene (ORF 2) of avian HEV with those of other known HEV strains

 
Multiple sequence alignment revealed that the start codon of the ORF 3 gene in other known HEV strains does not exist in avian HEV due to base substitutions (Fig. 3). Instead, ORF 3 of avian HEV starts 41 nt downstream of the ORF 3 start codon of other known HEV strains. Like the human HEV-T1 strain (Wang et al., 2000 ), the ORF 3 gene of avian HEV does not overlap with ORF 1 and locates 33 nt downstream of the ORF 1 stop codon (Fig. 3). ORF 3 of avian HEV comprises 264 nt with a coding capacity of 87 aa, which is 24–37 aa shorter than those of other known HEV strains.

Sequence analysis of the 3' NCRs
The 3' NCR of avian HEV is 130 nt in length, the longest among all known HEV strains; the 3' NCRs of other HEV strains range from 65 to 74 nt. Multiple sequence alignment indicated that the 3' NCRs of HEV are highly variable, although a stretch of sequence immediately proceeding the poly(A) tract is relatively conserved (data not shown).

Identification of a major deletion in the overlapping region of ORFs 2 and 3 of avian HEV
Sequence analyses revealed a major deletion of 54 aa in the N-terminal region of ORF 2 of avian HEV (Fig. 4). To rule out the possibility of RT–PCR artefacts, a pair of avian HEV-specific primers flanking the deleted region, FdelAHEV (5' sense primer) and RdelAHEV (3' antisense primer), was designed (Table 1 and Fig. 3). To minimize problems of potential secondary structure, reverse transcription was performed at 60 °C with a One Step RT–PCR kit (Qiagen). In addition, PCR was also performed with a shorter annealing time and a higher denaturation temperature. The PCR reaction comprised an initial enzyme activation step at 95 °C for 13 min, followed by 35 cycles of denaturation at 98 °C for 20 s, annealing at 55 °C for 5 s and extension at 73 °C for 1 min. It has been reported that formamide or DMSO can enhance the capability of PCR to amplify certain genomic regions of HEV (Yin et al., 1994 ). Therefore, 5% formamide or DMSO was added to the PCR reaction mixtures. An expected PCR product of the same size (502 bp) as that observed in conventional PCR was produced under various different RT–PCR parameters, including the addition of 5% formamide or DMSO, the use of higher denaturation temperatures and short annealing times and the synthesis of cDNA at 60 °C (Fig. 2B). The deletion was confirmed further by directly sequencing the 502 bp PCR product.

Phylogenetic evidence that avian HEV is related to, but distinct from, human and swine HEV
The topology of the four trees based on the entire 3' half of the HEV genome (~4 kb) and three other different genomic regions, for which the sequences of most HEV strains are available, is similar (Fig. 5). Most Asian strains of HEV are related to the prototype Burmese strain and cluster together. The African strains of HEV (Egypt93, Egypt94 and Morocco) were related to, but distinct from, the Burmese-like strains. The single Mexican strain of HEV forms a distinct branch. Based upon the limited length of sequences available, the two US strains of human HEV (US1 and US2), a US strain of swine HEV, a New Zealand strain of swine HEV and several novel European strains of human HEV (Greek1, Greek2 and Italy) were found to cluster together. Several variant strains of HEV identified from patients in China (HEV-T1, Ch-T11, Ch-T21 and 93G) and Taiwan (TW7E, TW4E and TW8E) also clustered together. Avian HEV was found to cluster with the Australian BLSV identified from chickens and formed a distinct branch. It is not clear whether avian HEV represents a new genotype of HEV or belongs to a separate genus of the unclassified HEV family.



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Fig. 5. Phylogenetic trees based on the sequences of different genomic regions of HEV. The branch-and-bound search and mid-point rooting options with 1000 replicates were used to produce the phylogenetic trees. (A) The 439 bp sequence of the helicase gene, (B) the 196 bp sequence of the RdRp gene, (C) the 148 bp sequence of the ORF 2 gene and (D) the 3' half genomic sequence (~4 kb) of avian HEV and other HEV isolates with known complete genomic sequence. Bootstrap values of more than 75% are indicated. Meaningful bootstrap values cannot be obtained for the tree based on a short 148 bp sequence of the ORF 2 gene (C). Scale bars representing the numbers of character state changes are shown. Branch lengths are proportional to the numbers of character state changes.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Evidence of HEV infection of domestic and farm animals has been well documented (Purcell, 1996 ; Meng, 2000a , b , 2001 ). Anti-HEV were detected in pigs from developing countries, such as Nepal (Clayson et al., 1995 ), China and Thailand (Meng et al., 1999 ), and from industrialized countries, such as the US, Canada, Korea (Meng et al., 1999 ), Taiwan (Hsieh et al., 1999 ), Spain (Pina et al., 2000 ) and Australia (Chandler et al., 1999 ). In addition to pigs, anti-HEV have also been detected from rodents caught in the wild in the US (Kabrane-Lazizi et al., 1999b ; Favorov et al., 2000 ). In Vietnam, anti-HEV was reportedly detected in 44% of chickens, 36% of pigs, 27% of dogs and 9% of rats (Tien et al., 1997 ). About 29–62% of cows from Somali, Tajikistan and Turkmenistan and about 42–67% of the sheep and goats from Turkmenistan were found to be positive for anti-HEV (Favorov et al., 1998 ). Naturally acquired anti-HEV have also been detected in rhesus monkeys (Tsarev et al., 1995 ). These serological data strongly suggest that these animal species are infected with either HEV or a related agent. However, until recently, the source of seropositivity in these animals could not be definitively demonstrated. The first animal strain of HEV that has been genetically identified and characterized is swine HEV (Meng et al., 1997 ). In the present study, we report the genetic identification and characterization of yet another animal strain of HEV, avian HEV. Like swine HEV, the avian HEV strain identified in this study is genetically related to human HEV strains. Unlike swine HEV, which causes only subclinical infection in pigs (Halbur et al., 2000 ; Meng et al., 1998 a, b ), avian HEV could be associated with a disease (HS syndrome) in chickens. However, a definitive causal relationship between avian HEV and HS syndrome is still lacking.

The sequences of the complete 3' NCR, the complete ORFs 2 and 3 genes, the complete RdRp gene and a partial helicase gene of avian HEV have been determined. Sequence analyses revealed that avian HEV shares significant sequence identity with swine and human HEV. The genomic organization of avian HEV is also similar to that of human and swine HEV. ORF 2 of avian HEV is relatively conserved at its N-terminal region (excluding the signal peptide), but is less conserved at its C-terminal region. The ORF 3 gene of avian HEV is very divergent compared to those of other known HEV strains. However, the C terminus of the ORF 3 gene of avian HEV is relatively conserved; this region is believed to be the immuno-dominant portion of the ORF 3 protein (Zafrullah et al., 1999 ). Unlike most known HEV strains, ORF 3 of avian HEV does not overlap with ORF 1. Also, ORF 3 of a novel strain of human HEV, HEV-T1, recently identified from a patient in China does not overlap with ORF 1 and its ORF 3 start codon locates 28 nt downstream of the ORF 1 stop codon (Wang et al., 2000 ). The unique difference in the avian HEV genome is not unexpected, since chickens are very different from mammalian species. The biological significance of these differences remains to be determined.

A major deletion located in the N-terminal region of ORF 2, which overlaps with ORF 3, was identified in avian HEV. It has been shown that, in certain HEV strains, this genomic region is difficult to amplify by conventional PCR methods (Yin et al., 1994 ; Wang et al., 2000 ) and that the addition of 5% formamide or DMSO, or combination of DMSO and GC Melt, to the PCR reaction mixture often resulted in successful PCR amplification. In this study, RT–PCR was carried out under various different parameters and conditions, including cDNA synthesis at 60 °C, PCR amplification with higher denaturation temperatures and shorter annealing times and PCR with the addition of 5% formamide or DMSO. No additional sequence was identified and the deletion was verified further by direct sequencing of the amplified PCR product flanking the deletion region. Thus, we conclude that the observed deletion in the avian HEV genome is not due to RT–PCR artefacts. Interestingly, Ray et al. (1992) also reported a major deletion in the overlapping region of ORFs 2 and 3 of an Indian strain of human HEV. The biological significance of this deletion is not known. It has been shown that, when ORF 2 is expressed in the baculovirus system, a truncated version of the ORF 2 protein lacking the N-terminal 111 aa is produced (Li et al., 1997 ; Zhang et al., 1997 ). The truncated ORF 2 protein was cleaved at aa 111–112 (Zhang et al., 1997 ), but was still able to form virus-like particles (Li et al., 1997 ). Avian HEV lacks most of the N-terminal 100 aa of ORF 2. It is possible that this genomic region corresponding to the avian HEV aa deletion might be dispensable for HEV replication.

So far, HS syndrome has only been reported in Canada and the US (Ritchie & Riddell, 1991 ; Tablante et al., 1994 ; Julian, 1995 ; Riddell, 1997 ; Shivaprasad & Woolcock, 1995 ; Jeffrey & Shivaprasad, 1998 ). In Australia, chicken farms have been experiencing outbreaks of big liver and spleen disease (BLS) for many years (Handlinger & Williams, 1988 ). However, there has been no report regarding a possible link between HS syndrome in North America and BLS in Australia. A virus designated BLSV was recently isolated and identified from chickens with BLS in Australia. BLSV was shown (based on a very short stretch of available sequence) to be genetically related to HEV (Payne et al., 1999 ). It appears that the avian HEV identified from US chickens associated with HS syndrome is genetically related to, but different from, the BLSV associated with BLS in Australian chickens, displaying about 80% nt sequence identity.

Recently, numerous genetically distinct strains of HEV have been identified from patients with acute hepatitis in both developing and industrialized countries (Hsieh et al., 1998 ; Wu et al., 1998 ; Schlauder et al., 1998 , 1999 , 2000 ; Erker et al., 1999 ; Zanetti et al., 1999 ; Wang et al., 1999 , 2000 ; Buisson et al., 2000 ; Pina et al., 2000 ). The two US strains of human HEV (US1 and US2) are genetically distinct from other known HEV strains worldwide, but are closely related to each other and to the US strain of swine HEV (Schlauder et al., 1998 ; Erker et al., 1999 ; Meng et al., 1998 b). Similarly, several novel strains of HEV have been identified from patients in Taiwan who have no history of travel to endemic regions (Hsieh et al., 1998 ; Wu et al., 1998 ). A novel Italian strain of human HEV was found to share only about 79·5–85·8% nt sequence identity with other known strains of HEV (Zanetti et al., 1999 ). Schlauder et al. (1999) recently identified another Italian and two Greek novel strains of HEV. The sequences of the Greek and Italian strains of HEV differed significantly from other known strains of HEV. In endemic regions, novel strains of HEV, distinct from the known epidemic strains, have also been identified in Pakistan (van Cuyck-Gandre et al., 2000 ), Nigeria (Buisson et al., 2000 ) and China (Wang et al., 1999 , 2000 ). The intriguing fact is that these recently identified novel strains of HEV are genetically distinct from each other and from other known strains of HEV. Although the source of these novel human HEV strains is not clear, it is plausible that they may be of animal origin.

Since the identification of the first animal strain of HEV, swine HEV, in the US in 1997 (Meng et al., 1997 ), several other HEV strains of animal origin have been genetically identified. Hsieh et al. (1999) identified a second strain of swine HEV from a pig in Taiwan. This Taiwanese strain of swine HEV shares 97·3% nt sequence identity with a human strain of HEV isolated from a retired Taiwanese farmer, but is genetically distinct from other known strains of HEV (about 70% nt sequence identity), including the US strain of swine HEV. Recently, Pina et al. (2000) identified another novel strain of HEV (E11) from sewage samples of animal origin from a slaughterhouse in Spain. The E11 strain of possible animal origin is most closely related to two Spanish strains of human HEV and is more closely related to the US swine and human HEV strains than to other HEV strains. In addition to pigs, a strain of HEV was reportedly identified in rodents caught in the wild from Nepal (Tsarev et al., 1998 ). Sequence analyses revealed that the HEV sequence recovered from the Nepalese rodents is most closely related to the HEV isolates from patients in Nepal. Increasing data show that pig handlers are at risk of zoonotic HEV infection (Meng et al., 1999 ; Meng, 2000a , b ). Karetnyi et al. (1999) reported that human populations with occupational exposure to wild animals also have increased risks of HEV infection. Therefore, it will be important to determine if the novel avian HEV identified in this study infects humans or vice versa.

HEV was classified as a member of the family Caliciviridae (Purcell, 1996 ). The lack of common features between HEV and caliciviruses (Koonin et al., 1992 ; Kabrane-Lazizi et al., 1999a ) has led to the recent removal of HEV from the family Caliciviridae (Pringle, 1998 ). The seropositivity detected in a variety of animal species suggests that there exists a family of viruses that are related to HEV (Meng, 2000a , b , 2001 ). The identification of an avian HEV strain in this study prompted us to propose a tentative classification for HEV: hepatitis E and related viruses or Hearviridae. However, a definitive taxonomic classification awaits the identification and characterization of additional HEV-related viruses from humans and other animal species.


   Acknowledgments
 
This project is supported by an Innovation Grant Award from Fort Dodge Animal Health Inc., Fort Dodge, Iowa, USA and in part by grants from the National Institutes of Health (grant nos AI01653 and AI46505). We thank Lee Weigt of Virginia Tech DNA Sequencing Facility for assistance with sequencing, Dr Mahesh Kumar of Fort Dodge Animal Health Inc., Dr John Barnes of North Carolina State University and Dr Thomas Toth of Virginia-Maryland Regional College of Veterinary Medicine for their support and helpful discussion.


   Footnotes
 
The GenBank accession no. of the sequence reported in this paper is AY043166.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 28 March 2001; accepted 22 July 2001.