Sequence comparison of an australian duc

Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses Miriam Triyatni,1† Peter L.. Jilbert1, 2 1 Hepatitis Virus Research Laboratory, Departm

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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, 2 and Allison R Jilbert1, 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

se-quenced 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

com-pared, substitutions or deletions in the polymerase

(POL) gene were most frequent in the 500 nt

segment encoding the ‘ spacer ’ domain that

over-laps 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

seg-ment that encodes the reverse transcriptase

do-main of the POL protein and the S dodo-main 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 8 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.

(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

hepadna-viruses 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

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M Triyatni and others

Table 1 Characterized avian hepadnavirus genomes

Length

Origin Accession no (nt) Avian species Location Clone name (GenBank I D.) Reference

‘ Chinese ’ DHBV

M32990 3027 Duck, brown Shanghai DHBVS5cg (HPUS5CG) Uchida et al (1989)

M32991 3027 Duck, white Shanghai DHBVS31cg (HPUS31CG) Uchida et al (1989)

X60213 3027 Duck, domestic Shanghai DHBVQCA34 (DHVBCG) GenBank

AJ006350 3027 Duck, Pekin Australia AusDHBV (DHV6350) Current study

M21953 3024 Duck, domestic Shanghai DHBVS18-B (HPUGA) Tong et al (1990)

X58568 3024 Duck, domestic China (DHBV22) Sprengel et al (1991)

X58569 3024 Duck, domestic Shanghai (DHBV26) Sprengel et al (1991)

‘ Western country’ DHBV

K01834 3021 Duck, Pekin USA DHBV16 (HPUCGD) Mandart et al (1984)

M60677 3021 Duck, Pekin USA DHBVp2–3 (HPUCGE) GenBank

X12798 3021 Duck Germany DHBVf1–6 (DHBVF16) Mattes et al (1990)

X58567 3021 Goose, domestic Germany (DHBV1) Sprengel et al (1991)

AF110996 3024 Snow goose Germany (SGHBV1–13) Chang et al (1999)

M22056 3027 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 DHBV

anti-bodies

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 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 (230 000 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(j) Following transformation of E coli strain DH5αFh,

trans-formants 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]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

de-DHE

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Fig 1 Phylogenetic relationship of avian hepadnaviruses, inferred from the full-length (3018–3027 nt) sequences by neighbour-joining analysis (pairwise-deletion/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 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

ex-periment, 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 nucleo-tides 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 inocu-lation of plasmid DNA (total of 50µg 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

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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

charac-terized 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,

deter-mined 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

co-evolution of each virus in its respective host

Three important features associated with virus replication

are well conserved in AusDHBV and other avian

hepadna-viruses : (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 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

Tyrosine-96 serves as the binding site to the RNA encapsidation signal sequence (nt 2566–2622), known as epsilon (ε), in the pre-genomic 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 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 dele-tions 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 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 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 0n6–11n7% and 0n6–7n8% respectively, while across all 20 strains the maximum differences were 52n4 and 16n8%

DHG

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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

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 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.

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Received 3 July 2000 ; Accepted 9 November 2000

DHI

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