Journal of General Virology (2001), 82, 373–378 Printed in Great Britain SHORT COMMUNICATION Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses Miriam Triyatni,1† Peter L Ey,1 Thien Tran,1 Marc Le Mire,1 Ming Qiao,2 Christopher J Burrell1, and Allison R Jilbert1, Hepatitis Virus Research Laboratory, Department of Molecular Biosciences, Adelaide University, North Terrace, Adelaide SA 5005, Australia Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia The genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the ‘ Chinese ’ DHBV branch of the avian hepadnaviruses Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the ‘ spacer ’ domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious The family Hepadnaviridae is divided into two genera, Orthohepadnavirus and Avihepadnavirus, each with restricted host specificity Ortho (mammalian) hepadnaviruses have been found in humans (hepatitis B virus, HBV), woodchucks Author for correspondence : Allison Jilbert (at Department of Molecular Biosciences) Fax j61 8303 7532 e-mail allison.jilbert!adelaide.edu.au † Present address : NIDDK/NIH, Liver Diseases Section, Building 10, Room 9B11, 10 Center Drive MSC 1800, Bethesda, MD 20892-1800, USA The EMBL/GenBank accession number of the AusDHBV sequence reported in this paper is AJ006350 0001-7253 # 2001 SGM (woodchuck hepatitis virus, WHV), ground squirrels (GSHV), arctic squirrels (ASHV) and woolly monkeys (WMHBV) The avian hepadnaviruses include duck hepatitis B virus (DHBV ; Mason et al., 1980), heron hepatitis B virus (HHBV ; Netter et al., 1997 ; Sprengel et al., 1988), Ross goose hepatitis B virus (RGHBV ; GenBank acc no M95589) and snow goose hepatitis B virus (SGHBV ; Chang et al., 1999) 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) We have previously reported that pooled serum from ducks congenitally infected with an Australian strain of DHBV (AusDHBV) had an infectivity (ID ) titre equivalent to the &! DHD M Triyatni and others Table Characterized avian hepadnavirus genomes Origin Accession no ‘ Chinese ’ DHBV M32990 M32991 X60213 AJ006350 M21953 X58568 X58569 – ‘ Western country’ DHBV K01834 M60677 X12798 X74623 AF047045 – X58567 AF110996 AF110997 AF110998 AF110999 AF111000 M95589 M22056 – Length (nt) Avian species Location Clone name (GenBank I D.) Reference 3027 3027 3027 3027 3024 3024 3024 Duck, brown Duck, white Duck, domestic Duck, Pekin Duck, domestic Duck, domestic Duck, domestic Duck, domestic Shanghai Shanghai Shanghai Australia Shanghai China Shanghai Shanghai DHBVS5cg (HPUS5CG) DHBVS31cg (HPUS31CG) DHBVQCA34 (DHVBCG) AusDHBV (DHV6350) DHBVS18-B (HPUGA) (DHBV22) (DHBV26) DHBVQ49* Uchida et al (1989) Uchida et al (1989) GenBank Current study Tong et al (1990) Sprengel et al (1991) Sprengel et al (1991) 3021 3021 3021 3021 3021 3021 3021 3024 3024 3024 3024 3024 3018 3027 3027 Duck, Pekin Duck, Pekin Duck Duck Duck Duck, Pekin Goose, domestic Snow goose Snow goose Snow goose Snow goose Snow goose Ross goose Grey heron Grey heron USA USA Germany India Canada Germany Germany Germany Germany Germany Germany Germany USA† Germany Germany DHBV16 (HPUCGD) DHBVp2–3 (HPUCGE) DHBVf1–6 (DHBVF16) (DHBVCG) (DHBV47045) DHBV3 (DHBV1) (SGHBV1–13) (SGHBV1–15) (SGHBV1–19) (SGHBV1–7) (SGHBV1–9) RGHBV (HPUGENM) HHBV4 (HPUCG) HHBV A, B, C and D Mandart et al (1984) GenBank Mattes et al (1990) GenBank GenBank Sprengel et al (1985) Sprengel et al (1991) Chang et al (1999) Chang et al (1999) Chang et al (1999) Chang et al (1999) Chang et al (1999) GenBank Sprengel et al (1988) Used by Chang et al (1999) * Tong & Mattes, unpublished ; cited by Wildner et al (1991) † John Newbold, personal communication number of DHBV genomes\ml (Jilbert et al., 1996) A similar result was obtained by Anderson et al (1997) where dilutions of serum containing approximately three AusDHBV genomes were infectious In both studies virus titration was performed in ducks, from the same commercial supplier, that were completely free of DHBV infection or anti-DHBV antibodies In recent experiments from our laboratory with the USA strain of DHBV (DHBV16 ; Mandart et al., 1984), infectivity titres determined in ducks from the same source were also similar to the number of genomes\ml of serum (unpublished) These results suggest that differences in the infectivity of the USA strain observed by other laboratories may be related to the strain of duck used in the inoculation experiments and or to trace amounts of maternally transmitted anti-DHBV antibodies 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 DHBVinfected ducks (Anas domesticus platyrhyncos) were obtained from two commercial suppliers Virus particles were isolated DHE from 20 ml of congenitally DHBV-infected-duck serum by sedimentation (230 000 g, 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(j) Following transformation of E coli strain DH5αFh, 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) BglIIjPvuI and (ii) BglIIjEcoRI followed by Southern blot hybridization using a [$#P]dCTPlabelled 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 de- An Australian strain of duck hepatitis B virus Fig Phylogenetic relationship of avian hepadnaviruses, inferred from the fulllength (3018–3027 nt) sequences by neighbour-joining analysis (pairwisedeletion/Tamura–Nei distances/all sites ; Tamura & Nei, 1993) Distance values calculated using MEGA (Kumar et al., 1994) were used to construct a dendrogram file for display by Treeview (Page, 1996) Bootstrap values for the major nodes ( % support ; 2000 iterations) are indicated termined 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 nt longer than other Chinese DHBV (strains 22, 26 and S18-B), and nt longer than ‘ Western country’ DHBV isolates (Table 1) These differences correspond to changes in the 3027 nt DHBV genome at 1236–1238 and 1278–1280, 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 490–1600 (1110 nt) of the DHBV genome encompassing overlapping sections of the POL (nt 170–2536) and PreS\S (nt 801–1793) 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(j) (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 weeks of inoculation, similar to the findings of Tagawa et al (1996) using the same method of inoculation DHF M Triyatni and others The relationship of AusDHBV to the other known avian hepadnaviruses was investigated by phylogenetic analysis of the 3018–3027 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 (3n1–7n5 % and 4n0–5n2 % 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) The ‘ Western country ’ branch is defined by seven strains of DHBV, of which six are available from GenBank (Table 1, Fig 1) Five of these (derived from ducks) are very closely related (0n7–1n7 % sequence differences) The sixth, DHBV1 isolated from a domestic goose, differs by 6n1–6n8 % from the other five members of this group but, despite its obvious divergence, the evolutionary distance involved lies within the range that separates the DHBV strains within the ‘ Chinese ’ branch (3n1–9n5 % difference) but is less than the distance separating the ‘ Chinese ’ from the ‘ Western country ’ DHBV strains (9n4–10n5 % difference) Analysis of additional virus strains from domestic geese is needed to determine whether DHBV1 represents a virus strain which can infect both ducks and geese or a variant that infects geese preferentially In comparison to the DHBV strains (which have geographically diverse origins), the five isolates of SGHBV differ by only 0n4–0n5 % These were isolated from a single flock of geese (Hans Will, personal communication) and form a tight cluster that is well resolved from all of the DHBV strains by sequence differences of 11–11n8 % The RGHBV and HHBV isolates are the most divergent, differing from each other by 22n4 %, and from the DHBV and SGHBV strains by 17n3–19n1 % and 18n9–19 % (RGHBV), and 22n4–24 % and 22n7–23 % (HHBV), respectively The larger evolutionary distance between HHBV and DHBV may reflect their distinct host ranges (HHBV appears to infect only grey herons and not ducks ; Ishikawa & Ganem, 1995 ; Sprengel et al., 1988), and may have resulted from coevolution of each virus in its respective host 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 DHG the circular conformation of the genome and contains a pair of 12 nt direct repeat (DR) sequences, DR1 (nt 2541–2552) and DR2 (nt 2483–2494) ; (ii) the polyadenylation signal sequence (nt 2778–2783) 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 Tyrosine96 serves as the binding site to the RNA encapsidation signal sequence (nt 2566–2622), known as epsilon (ε), 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 1290–1793) and the Pre-C segment (nt 2524–2652), 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 2295– 2639 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 3h end of the POL gene and the 5h 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 In comparing all 20 avian hepadnavirus sequences within the current data set, we have depicted graphically in Fig the percentage of sites in each 50 nt segment of the genome and in each 10 or 20 amino acid segment of the deduced polypeptides (POL, Pre-S\S, Pre-C\C) that contain substitutions or deletions Substitutions or deletions in the POL gene occur predominantly in the segment that overlaps the Pre-S domain of the Pre-S\S gene and which encodes the so-called ‘ spacer ’ domain of the POL protein This latter domain is not highly conserved among different isolates (Sprengel et al., 1991) and has no known enzymatic function The S domain of the PreS\S gene of AusDHBV (nt 1290–1793) exhibits 99n6 and 95n4 % nt sequence identity with DHBV strains S31cg and 16, respectively, whereas the Pre-S segment (nt 801–1289) is less conserved (93n5 and 80n5 % identity, respectively) The greater conservation of the S segment (compared with the Pre-S region) is evident in Fig These differences in the frequency of sites containing nucleotide substitutions or deletions are reflected by similar marked differences in the number of sites with amino acid substitutions or deletions in the Pre-S and S domains of the Pre-S\S protein and the ‘ spacer ’ and reverse transcriptase domains of the POL protein, as highlighted in Fig Using pairwise FASTA analysis of the ‘ Chinese ’ and ‘ Western Country ’ DHBV strains, the Pre-S and S domains yielded amino acid sequence differences (non-identities) of 0n6–11n7 % and 0n6–7n8 % respectively, while across all 20 strains the maximum differences were 52n4 and 16n8 % An Australian strain of duck hepatitis B virus Fig Percentage of sites per segment of viral DNA or deduced polypeptide at which a substitution or deletion was observed in alignments across the entire panel of 20 avian hepadnavirus genomes The DNA, POL protein, Pre-S/S and Pre-C/C polypeptides were analysed by calculating the number of substituted sites in sequential segments of 50 nt (viral DNA), 20 aa (POL protein) or 10 aa (Pre-S/S, Pre-C/C) polypeptides The respective genes, together with the X-like ORF, are depicted in the lower section by bold arrows (drawn to scale, with nucleotide positions indicated) Vertical arrowheads depict the Pre-S and S (Pre-C and C) domain boundaries Segments corresponding to functional domains of the POL protein [terminal protein (TP), ‘ spacer ’, reverse transcriptase (RT), RNase H] are also indicated Similarly the ‘ spacer ’ and reverse transcriptase domains yielded amino acid sequence differences (non-identities) of 18n4–47n2 % and 0n6–4n8 % respectively for the DHBV strains, while across all strains the maximum differences were 71n8 and 10n8 % The polymorphic nature of the segment encoding the Pre-S domain is surprising, as it represents two overlapping reading frames – just like the more-highly conserved adjacent segment encoding the S domain – which could be expected to confer greater selective pressure against substitutions It is tempting, therefore, to speculate that a lack of specific function (and thus selective constraints) of the ‘ spacer ’ domain of the POL protein has allowed the emergence of substitutions to accumulate within the 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Characterization of infectious and defective cloned avian hepadnavirus genomes Virology 185, 345–353 Received July 2000 ; Accepted November 2000