Hepatitis Virus Research Laboratory, Department of Molecular Biosciences, Adelaide University, North Terrace, Adelaide SA 5005, Australia1
Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia2
Author for correspondence: Allison Jilbert (at Department of Molecular Biosciences). Fax +61 8 8303 7532. e-mail allison.jilbert{at}adelaide.edu.au
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Abstract |
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Mammalian and avian hepadnaviruses show similarity in terms of genetic organization, virus replication and, to some extent, the outcome of infection in their respective hosts. Although there is 60% sequence divergence between HBV and DHBV (Orito et al., 1989
; Sprengel et al., 1985
), the latter have provided a useful animal model for HBV infection. Studies of DHBV infection in vitro and in Pekin ducks (Anas domesticus) have contributed significantly to our understanding of various aspects of the replication cycle of hepadnaviruses. To date, the nucleotide sequences of 20 avian hepadnaviruses originating from different geographical regions are available from the GenBank database, and the existence of an additional six sequences is known from published studies (Table 1
). In comparing the sequences of nine strains of DHBV (six from China, two from Germany, one from USA) with hepadnaviruses isolated from a domestic goose and a grey heron (Ardea cinerea), Sprengel et al. (1991)
defined a phylogenetic tree for the avian hepadnaviruses that consisted of three major branches: (i) Chinese DHBV, (ii) Western country DHBV, which included the domestic goose isolate, and (iii) the heron isolate (HHBV). More recently, Chang et al. (1999)
described a new strain of avian hepadnavirus, SGHBV, from snow geese (Anser caerulescens) and compared these sequences with other avian hepadnaviruses available from GenBank. Their analysis, using Splitstree (Huson, 1998
), supported the division of DHBV into the Chinese and Western country branches defined by Sprengel et al. (1991)
and further resolved SGHBV, HHBV and RGHBV into separate, highly distinct lineages. The RGHBV was isolated in the USA from a Ross goose (Anser rossi; Table 1
).
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To further characterize the AusDHBV strain we cloned and sequenced the 3027 nt genome, tested its infectivity in newly hatched ducks and defined its relationship with other avian hepadnaviruses. DHBV-negative and congenitally DHBV-infected ducks (Anas domesticus platyrhyncos) were obtained from two commercial suppliers. Virus particles were isolated from 20 ml of congenitally DHBV-infected-duck serum by sedimentation (230000 g, 4 h) through 20% (w/v) sucrose. Viral DNA was converted to the complete double-stranded form using the endogenous DNA polymerase reaction, followed by DNA extraction and treatment with T4 DNA polymerase as previously described (Uchida et al., 1989 ). The full-length genome of double-stranded viral DNA was digested and cloned into the EcoRI site of pBluescript IIKS(+). Following transformation of E. coli strain DH5
F', transformants were identified and recombinant plasmids containing DHBV genomic inserts were isolated. One of these (pBL4.8) was chosen for detailed examination. This clone contained a DHBV genome in the same orientation as the lacZ promoter, as shown by cleavage with (i) BglII+PvuI and (ii) BglII+EcoRI followed by Southern blot hybridization using a [32P]dCTP-labelled pSP.DHBV 5.1 DNA probe. The latter comprised the full-length genome of DHBV16 (Mandart et al., 1984
) within the pSP65 vector (Promega). On the basis of restriction analysis, the AusDHBV genome appeared more similar to the Chinese DHBVS31cg strain (Uchida et al., 1989
) than to DHBV16. The nucleotide sequence of AusDHBV was determined by primer walking from both strands, starting with the T3 and T7 primers which anneal to the vector at each end of the viral DNA insert. Clone pBL4.8 contained a full-length, double-stranded DHBV genome that was 3027 nt in length, the same as the Chinese DHBV strains S31cg, S5cg and QCA34 but 3 nt longer than other Chinese DHBV (strains 22, 26 and S18-B), and 6 nt longer than Western country DHBV isolates (Table 1
). These differences correspond to changes in the 3027 nt DHBV genome at 12361238 and 12781280, respectively, both sites occurring within the Pre-S domain of the Pre-S/S gene and the spacer domain of the POL gene.
The pBL4.8 clone represents the predominant strain of DHBV within the pool of congenitally DHBV-infected-duck serum. This was determined by restriction fragment analysis of 24 additional DHBV clones from the original cloning experiment, and by cloning of four additional DHBV genomes by long-range PCR as described by Netter et al. (1997) . All four clones were sequenced from nt 4901600 (1110 nt) of the DHBV genome encompassing overlapping sections of the POL (nt 1702536) and PreS/S (nt 8011793) ORFs. Within this 1110 nt fragment 13 sites contained substituted nucleotides in one (8/13), two (4/13) or four (1/13) clones. The consensus sequence generated from analysis of pBL4.8 and the four new clones matched the pBL4.8 clone in 12/13 sites. The infectivity of the AusDHBV genome was confirmed by cloning of a head-to-tail dimer in plasmid pBluescript IIKS(+) (pBL4.8x2) followed by intravenous and intrahepatic inoculation of plasmid DNA (total of 50 µg per duck) into a group of four newly hatched ducks. Two out of four inoculated ducks developed detectable serum DHBsAg within 2 weeks of inoculation, similar to the findings of Tagawa et al. (1996)
using the same method of inoculation.
The relationship of AusDHBV to the other known avian hepadnaviruses was investigated by phylogenetic analysis of the 30183027 nt sequences using MEGA (Kumar et al., 1994 ) and Splitstree (Huson, 1998
). Consistent and unambiguous support was found for placing AusDHBV on the Chinese branch of DHBV (Fig. 1
), now represented by seven characterized strains. These fall into two distinct subsets, consisting of viruses with genome lengths of either 3024 or 3027 nt (Fig. 1
, Table 1
). Both subsets exhibit similar levels of sequence polymorphism (3·17·5% and 4·05·2% respectively, determined by pairwise FASTA analyses; Pearson & Lipman, 1988
) and within the 3027 nt subset, AusDHBV is most closely related to the Shanghai DHBV strain S31cg (Fig. 1
). Its unambiguous identification as a member of the Chinese branch and its presence within Pekin ducks in Australia suggests that AusDHBV was introduced into Australia from China. Importation of live ducks into Australia has been banned since 1949 although the original source of the AusDHBV-infected ducks is unknown. Strains of DHBV have also been detected in two species of Australian wild duck, the grey teal and maned duck, and phylogenetic analysis of their genomes is in progress (Robert Dixon & Lun Li, personal communication).
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Three important features associated with virus replication are well conserved in AusDHBV and other avian hepadnaviruses: (i) the 69 nt cohesive overlap region which maintains the circular conformation of the genome and contains a pair of 12 nt direct repeat (DR) sequences, DR1 (nt 25412552) and DR2 (nt 24832494); (ii) the polyadenylation signal sequence (nt 27782783) that is necessary for termination of viral mRNA transcription; and (iii) the tyrosine residue at position 96 within the N-terminal domain of the POL protein. Tyrosine-96 serves as the binding site to the RNA encapsidation signal sequence (nt 25662622), known as epsilon (), in the pregenomic RNA used for negative-strand DNA synthesis. Also highly conserved amongst all the DHBV strains sequenced so far is the S segment of the Pre-S/S gene (nt 12901793) and the Pre-C segment (nt 25242652), which encodes the signal sequence for secretion of DHBeAg (Schlicht et al., 1987
).
The DHBV genome, in contrast to the genomes of HHBV (Sprengel et al., 1988 ), RGHBV (GenBank acc. no. M95589) and SGHBV (Chang et al., 1999
), does not contain an X-like gene with a conventional ATG start codon. However, all published DHBV genomes contain an X-like ORF (nt 22952639 in AusDHBV) which begins with an alternative initiation codon, TTA. This putative X-like ORF is in the same reading frame as the Pre-S/S gene and overlaps the 3' end of the POL gene and the 5' end of the Pre-C/C gene. Evidence for synthesis of an X-like protein in DHBV-infected liver and in LMH cells transfected with DHBV DNA has been obtained recently by Hans Will (personal communication). The four overlapping genes (P, Pre-S/S, X-like, Pre-C/C) identified in the avian hepadnaviruses are depicted in Fig. 2
.
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Acknowledgments |
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Footnotes |
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b Present address: NIDDK/NIH, Liver Diseases Section, Building 10, Room 9B11, 10 Center Drive MSC 1800, Bethesda, MD 20892-1800, USA.
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Received 3 July 2000;
accepted 9 November 2000.