Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices 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
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Abstract |
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Introduction |
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Hepatitissplenomegaly (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 hepatitissplenomegaly 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.
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Methods |
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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 0·31·0 ml deionized water and 23 µl was mixed with 100200 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.
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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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 manufacturers 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).
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.
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).
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Results |
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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 4750% aa and 5253% 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 5760% nt and 5861% aa sequence identities with the corresponding region of other HEV strains (data not shown). All three conserved motifs (IVVI) 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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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 RTPCR 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 RTPCR 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 RTPCR 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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Discussion |
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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, RTPCR 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 RTPCR 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 111112 (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 79·585·8% 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.
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Acknowledgments |
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Footnotes |
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References |
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Received 28 March 2001;
accepted 22 July 2001.