Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses

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1 Journal of General Virology (2001), 82, 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. Burrell 1, 2 and Allison R. Jilbert 1, 2 1 Hepatitis Virus Research Laboratory, Department of Molecular Biosciences, Adelaide University, North Terrace, Adelaide SA 5005, Australia 2 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 allison.jilbert adelaide.edu.au Present address: NIDDK/NIH, Liver Diseases Section, Building 10, Room 9B11, 10 Center Drive MSC 1800, Bethesda, MD , USA. The EMBL/GenBank accession number of the AusDHBV sequence reported in this paper is AJ (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 SGM DHD

2 M. Triyatni and others Table 1. Characterized avian hepadnavirus genomes Origin Length Accession no. (nt) Avian species Location Clone name (GenBank I. D.) Reference Chinese DHBV M Duck, brown Shanghai DHBVS5cg (HPUS5CG) Uchida et al. (1989) M Duck, white Shanghai DHBVS31cg (HPUS31CG) Uchida et al. (1989) X Duck, domestic Shanghai DHBVQCA34 (DHVBCG) GenBank AJ Duck, Pekin Australia AusDHBV (DHV6350) Current study M Duck, domestic Shanghai DHBVS18-B (HPUGA) Tong et al. (1990) X Duck, domestic China (DHBV22) Sprengel et al. (1991) X Duck, domestic Shanghai (DHBV26) Sprengel et al. (1991) Duck, domestic Shanghai DHBVQ49* Western country DHBV K Duck, Pekin USA DHBV16 (HPUCGD) Mandart et al. (1984) M Duck, Pekin USA DHBVp2 3 (HPUCGE) GenBank X Duck Germany DHBVf1 6 (DHBVF16) Mattes et al. (1990) X Duck India (DHBVCG) GenBank AF Duck Canada (DHBV47045) GenBank 3021 Duck, Pekin Germany DHBV3 Sprengel et al. (1985) X Goose, domestic Germany (DHBV1) Sprengel et al. (1991) AF Snow goose Germany (SGHBV1 13) Chang et al. (1999) AF Snow goose Germany (SGHBV1 15) Chang et al. (1999) AF Snow goose Germany (SGHBV1 19) Chang et al. (1999) AF Snow goose Germany (SGHBV1 7) Chang et al. (1999) AF Snow goose Germany (SGHBV1 9) Chang et al. (1999) M Ross goose USA RGHBV (HPUGENM) GenBank M Grey heron Germany HHBV4 (HPUCG) Sprengel et al. (1988) 3027 Grey heron Germany HHBV A, B, C and D 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 from 20 ml of congenitally DHBV-infected-duck serum by sedimentation ( 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 DH5αF, 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 [ 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- DHE

3 An Australian strain of duck hepatitis B virus Fig. 1. Phylogenetic relationship of avian hepadnaviruses, inferred from the fulllength ( 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 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 and , 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 (1110 nt) of the DHBV genome encompassing overlapping sections of the POL (nt ) and PreS S (nt ) 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 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. DHF

4 M. Triyatni and others The relationship of AusDHBV to the other known avian hepadnaviruses was investigated by phylogenetic analysis of the 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 ( % and % 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 ( % sequence differences). The sixth, DHBV1 isolated from a domestic goose, differs by % 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 ( % difference) but is less than the distance separating the Chinese from the Western country DHBV strains ( % 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 %. 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 %. The RGHBV and HHBV isolates are the most divergent, differing from each other by 22 4%, and from the DHBV and SGHBV strains by % and % (RGHBV), and % and % (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 the circular conformation of the genome and contains a pair of 12 nt direct repeat (DR) sequences, DR1 (nt ) and DR2 (nt ); (ii) the polyadenylation signal sequence (nt ) 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 ), 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 ) and the Pre-C segment (nt ), 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 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. In comparing all 20 avian hepadnavirus sequences within the current data set, we have depicted graphically in Fig. 2 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 Pre- S S gene of AusDHBV (nt ) exhibits 99 6 and 95 4% nt sequence identity with DHBV strains S31cg and 16, respectively, whereas the Pre-S segment (nt ) is less conserved (93 5 and 80 5% identity, respectively). The greater conservation of the S segment (compared with the Pre-S region) is evident in Fig. 2. 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. 2. 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 % and % respectively, while across all 20 strains the maximum differences were 52 4 and 16 8%. DHG

5 An Australian strain of duck hepatitis B virus Fig. 2. 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 % and % respectively for the DHBV strains, while across all strains the maximum differences were 71 8 and 10 8%. 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 Pre-S segment. These substitutions may confer selective advantages to the virus including altered antigenicity or target cell specificity. In contrast, it appears that substitutions within the adjacent segment that encodes both the reverse transcriptase domain of the POL protein and the S domain of the envelope protein are highly deleterious as very few substitutions are detected. This research was supported by the National Health and Medical Research Council of Australia (NHMRC) and by a postgraduate scholarship (M. T.) from the Australian Government (AusAID). All animal handling procedures were approved by the IMVS and University of Adelaide Animal Ethics Committees and followed NHMRC guidelines. We are grateful to Darren Miller for technical advice and to Professor Ieva Kotlarski for critical reading of the manuscript. References Anderson, D. A., Grgacic, E. V., Luscombe, C. A., Gu, X. & Dixon, R. (1997). Quantification of infectious duck hepatitis B virus by radioimmunofocus assay. Journal of Medical Virology 52, Chang, S. F., Netter, H. J., Bruns, M., Schneider, R., Froelich, K. & Will, H. (1999). A new avian hepadnavirus infecting snow geese (Anser caerulescens) produces a significant fraction of virions containing singlestranded DNA. Virology 262, Huson, D. H. (1998). Splitstree: analyzing and visualizing evolutionary data. Bioinformatics 14, Ishikawa, T. & Ganem, D. (1995). The pre-s domain of the large viral envelope protein determines host range in avian hepatitis B viruses. Proceedings of the National Academy of Sciences, USA 92, Jilbert, A. R., Miller, D. S., Scougall, C. A., Turnbull, H. & Burrell, C. J. (1996). Kinetics of duck hepatitis B virus infection following low dose virus inoculation: one virus DNA genome is infectious in neonatal ducks. Virology 226, DHH

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