www.elsevier.com/locate/gene

Gene 349 (2005

Re-analysis of a human hepatitis B virus (HBV) isolate from an

East African wild born Pan troglodytes schweinfurthii: Evidence for

interspecies recombination between HBV infecting

chimpanzee and human

Emmanuil N. Magiorkinis, Gkikas N. Magiorkinis,

Dimitrios N. Paraskevis, Angelos E. Hatzakis*

National Retrovirus Reference Center, Department of Hygiene and Epidemiology, University of Athens, School of Medicine,

Mikras Asias 75, GR-11527, Athens, Greece

Received 9 September 2004; received in revised form 22 November 2004; accepted 15 December 2004

Available online 10 March 2005

Received by G. Pesole

Abstract

According to current estimates, hepatitis B virus (HBV) has infected 2 billion people worldwide and among them, 360 million suffer from

chronic HBV infection. Except humans, HBV or HBV-like viruses have also been isolated from different species of apes and mammals.

Although recombination has been described to occur extensively between different genotypes within the human HBV lineage, no

recombination event has ever been reported between human and non-human primate HBV sequences. It was our objective to perform an

exhaustive search for recombination between human and non-human primate HBV strains among all available full-length human and non-

human primate HBV sequences, using bootscanning and phylogenetic analyses. Intriguingly, we found that an HBV sequence isolated from a

wild born Pan troglodytes schweinfurthii in East Africa–FG–is a recombinant consisting of HBV infecting chimpanzee (ChHBV) and human

genotype C. More specifically, in a fragment of approximately 500 nt (positions 551–1050 spanning half of the RT domain of pol, which

overlaps with half of the coding region of the small surface protein), FG grouped with HBV genotype C, while in the rest of the genome it

grouped with ChHBV sequences. Phylogenetic analyses showed that in the latter region FG was more closely related to the Pan troglodytes

troglodytes subspecies, forming an outlier to this group. Moreover, we show evidence that the recombination event occurred after the initial

dispersion of HBV genotype C in humans. Finally, our findings point out that although rare recombination between HBV viruses infecting

different species occurs.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Hepadnaviruses; Mosaic; Primates; Phylogenetic analysis

0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.gene.2004.12.021

Abbreviations: ChHBV, chimpanzee hepatitis B virus; G, gamma;

GiHBV, gibbons hepatitis B virus; GTR, general time reversible model;

HBV, hepatitis B virus; HBV-DNA, hepatitis B virus DNA; HBsAg,

hepatitis B virus surface antigen; MC, Monte Carlo; ML, maximum

likelihood; OuHV, orang-utan hepatitis virus; pol, polymerase gene; RT,

reverse transcriptase; SHBs, small hepatitis B virus surface protein;

WMHBV, woolly monkey hepatitis B virus.

* Corresponding author. Tel.: +30 210 7462090; fax: +30 210 7462190.

E-mail address: ahatzak@med.uoa.gr (A.E. Hatzakis).

1. Introduction

Hepatitis B virus (HBV) is the prototype member of a

family of viruses designated as Hepadnaviridae. This

family consists of enveloped DNA viruses with a partially

double-stranded genome of approximately 3182–3221 nt in

length. HBV sequences isolated from humans have been

classified into eight genotypes A–H (Norder et al., 1994;

Stuyver et al., 2000; Okamoto et al., 1988; Arauz-Ruiz et

al., 2002). The distribution of human HBV genotypes

) 165–171

E.N. Magiorkinis et al. / Gene 349 (2005) 165–171166

differs greatly with the geographic origin: genotypes A, D

and G circulate globally (Norder et al., 1993a; Stuyver et al.,

2000), B and C prevail in East and South East Asia

(Okamoto et al., 1988), genotype E predominates in West

Africa (Norder et al., 1994), while genotypes F and H

circulate among native Amerindians in Central and South

America (Norder et al., 1993b; Arauz-Ruiz et al., 2002).

HBV viruses have been also isolated from different

primate species, such as chimpanzees (Vaudin et al., 1988;

Hu et al., 2000; MacDonald et al., 2000; Takahashi et al.,

2000), lowland gorillas (Grethe et al., 2000), gibbons (Norder

et al., 1996; Grethe et al., 2000), orang-utans (Warren et al.,

1999; Verschoor et al., 2001), as well as from woolly

monkeys (Lanford et al., 1998). Chimpanzee subspecies

inhabit in West, West-Central and East Africa; gibbons and

orang-utans in East and South Eastern Asia, while woolly

monkeys inhabit in South America. The last group (woolly

monkey hepatitis B virus orWMHBV) is themost genetically

divergent among primate hepadnaviruses providing, thus, an

outlier to the other lineages. According to previous studies,

the prevalence of active HBV-infection (detection of both

HBV-DNA and HBsAg) is 10.5% in chimpanzees, 39% in

lowland gorillas, 26% in gibbons and 20% in orang-utans,

while the respective combined rates for woolly monkeys is

30% (Starkman et al., 2003). These findings are in

accordance with another study reporting a high prevalence

of active or past HBV infection in chimpanzees and gorillas

(32% and 30%, respectively) and the absence of HBV

infection in old world monkeys (Makuwa et al., 2003).

Until now, recombination has been documented in

several cases among HBV genotypes isolated from humans,

as for example between genotypes A and D, A and C, B and

C or between D and C (Bollyky et al., 1996; Morozov et al.,

2000; Owiredu et al., 2001; Hannoun et al., 2000; Cui et al.,

2002). Interestingly, HBV genotypes E and G are also

shown to be recombinant consisting partially of D and A,

respectively (Bowyer and Sim, 2000; Fares and Holmes,

2002). On the other hand, although several cross-species

transmission events have been reported which occurred

between different primate species, such as between gibbons

and chimpanzees, chimpanzees and gorillas (Norder et al.,

1996; Grethe et al., 2000) and between chimpanzees and

humans (Takahashi et al., 2000; Hu et al., 2000), no

recombination events have ever been documented between

human and non-human primate HBV sequences.

It was our objective to investigate all available full-length

HBV sequences for evidence of recombination between

human and non-human primate HBV sequences.

2. Materials and methods

2.1. Materials

Full-length HBV sequences (550 sequences) were down-

loaded from the GenBank database (http://www.ncbi.nih.

gov). The following sequences were used as reference panel

using the currently available HBV genotype nomenclature:

(a) human sequences: genotypes A (U87742, M57663), B

(AB010289, D00329), C (AY123041, AF068756), D

(AF151735, X02496), E (X75657, AB032431), F

(AY090455, X69798), G (AB064313, AF160501) and H

(AY090454, AB059659); (b) non-human primate sequen-

ces: HBV infecting chimpanzees (ChHBV) (D00220,

AF242586), gibbons (GiHBV) (AJ131568, AY077736),

orang-utans (OuHV) (AF193863, AF193864) and woolly

monkeys (WMHBV) (AY226578, AF046996). Additional

HBV sequences for human genotype C and ChHBV used

for phylogenetic were: genotype C (Y18858, X04615,

AF223960, AY306136), ChHBV from Pan troglodytes

troglodytes (AF242585, AF222322, AY330911) and Pan

troglodytes vellerosus (AY330912, AF305327). Both strains

D00220 and AF242586 have been isolated from Pan

troglodytes verus.

2.2. Methods

Full genomic sequences were aligned using the Clustal

W program version 1.81 (Higgins and Sharp, 1988;

Thompson et al., 1994). An exploratory bootscanning

analysis was performed using a sliding window of 400 nt

moving in steps of 50 nt and maximum likelihood

estimated distances (F84 model with transitions/trans-

versions=2, default value) as implemented in Simplot

(3.2 beta version) (http://sray.med.som.jhmi.edu/Raysoft/

Simplot). The F84 model was used since it is the most

complex model available in the current version of

Simplot. HBV genotypes E and G that have been referred

to be recombinant were included separately in the

bootscanning analysis of each of the rest 526 HBV

sequences (Bowyer and Sim, 2000; Fares and Holmes,

2002).

For a single HBV-sequence (isolate name FG, accession

number AF498266) for which according to bootscanning

plots there was evidence for recombination between human

and non-human primates, separate phylogenetic analyses

were performed for the putative non-recombinant fragments,

based on maximum likelihood using Tamura-Nei substitu-

tion model (Tamura and Nei, 1993) including a G-

distributed rates heterogeneity among sites, as implemented

in Treepuzzle (Schmidt et al., 2002). Phylogenetic trees

were also reconstructed using Bayesian inference with

the GTR model and a G-distributed rates heterogeneity

among sites as implemented in MrBayes (v 3.0)

(Huelsenbeck and Ronquist, 2001). Four Metropolis

Coupled Markov chains Monte Carlo (MC3) were run

for 106 generations with a burnin of 105 generations. An

intergenotype recombination breakpoint was defined as a

region of approximately 100–200 nt in length in which

the HBV genotype classification was different in the

adjacent pieces (supported by N0.9 posterior probability

by Bayesian inference of phylogeny and N75% ML

E.N. Magiorkinis et al. / Gene 349 (2005) 165–171 167

puzzling steps). To assess for any statistically significant

differences of alternative topologies in different frag-

ments, we used the Shimodaira test as implemented in

Treepuzzle.

3. Results

3.1. Results of the exploratory analysis: FG sequence is a

recombinant consisting of ChHBV and human HBV

genotype C

Exploratory bootscanning analysis, including 550 full-

length available HBV sequences isolated from humans and

primates, showed evidence that an HBV sequence isolated

from Pan troglodytes schweinfurthii (Vartanian et al.,

2002) (isolate name FG, accession number AF498266)

was potentially recombinant consisting of HBV sequences

from chimpanzees (ChHBV) and humans (HBV genotype

C) (Fig. 1). More specifically, sequence FG clustered

significantly within the ChHBV lineage in two different

regions spanning nucleotides 1–550 and 1051–3182. On the

other hand, the region between nucleotides 551–1050,

spanning half of the RT domain of pol (aa 139–306), which

overlaps with half of the coding region of the small surface

protein (SHBs) (aa 183–226), clustered significantly with

HBV genotype C (Fig. 1A). Phylogenetic analysis, using

ML and Bayesian methods (Fig. 1C), further confirmed the

previous findings about discordant phylogenetic clustering

in fragments 1–550, 1051–3182 and 551–1050. Moreover,

according to the Shimodaira test, there was evidence for

significant difference ( pb0.05) between candidate topolo-

gies in different pieces of the alignment (regions 1–550,

1051–3182 and 551–1050).

3.2. Analysis of the region clustering with ChHBV in

comparison with other chimpanzee subspecies

To examine in detail the phylogenetic relationship

between the FG sequence and the ChHBV subspecies,

we concatenated the genomic regions clustered with

ChHBV (1–550 and 1051–3182) and performed boot-

scanning and phylogenetic analyses using additional

ChHBV reference sequences isolated from different sub-

species (Pan troglodytes troglodytes, Pan troglodytes

vellerosus and Pan troglodyte verus). Bootscanning plot

showed that FG sequence was more closely related to the

ChHBV Pan troglodytes troglodytes group (genomic

regions 1051–16401 and 1951–3182) for the longest part

of the alignment, whereas in two short fragments (111–550

and 1641–1950) no significant clustering (bootstrap support

b75%) was found between FG and any of the other ChHBV

groups (data not shown). Phylogenetic analyses in the latter

1 Nucleotide positions correspond to the original FG sequence.

regions indicated that ChHBV sequences from the different

chimpanzee subspecies did not form monophyletic clusters,

probably due to limited phylogenetic signal in the align-

ment, thus suggesting that in these regions further classi-

fication of the HBV sequences from chimpanzees is not

possible (data not shown).

Phylogenetic analysis in the concatenated region (1–550

and 1051–3182) showed that FG clustered with the ChHBV

sequences from Pan troglodytes troglodytes subspecies,

albeit forming an outlier to the latter group (Fig. 2). The

evolutionary distances between the FG and the Pan

troglodytes troglodytes group were comparable to the

distances between different chimpanzee subspecies (e.g.

between Pan troglodytes verus and Pan troglodytes

vellerosus) (Fig. 2). These findings suggest that the partial

FG sequence belongs to a potentially new ChHBV

subgroup, which may provide a subspecies specific group

for Pan troglodytes schweinfurthii. However, given the

limited sequence information available for this subspecies, it

is not possible to identify the precise origin of the partial FG

sequence.

3.3. Analysis of the region clustering with human HBV

genotype C

In contrary to the phylogenetic clustering of FG sequence

within the ChHBV group, where it appeared to be an

outgroup to the Pan troglodytes troglodytes cluster, in

region (551–1050 nt) FG fell within the HBV genotype C

group (Fig. 1C), thus suggesting that the recombination

event occurred after the initial spread of human HBV

genotype C.

4. Discussion

4.1. Summary of analysis

Throughout an exhaustive search of all full-length

available HBV sequences, we found evidence for

recombination between human and non-human primate

HBV strains, in an HBV sequence isolated from a

chimpanzee which belongs to the Pan troglodytes

schweinfurthii subspecies. Further analysis showed that

the HBV isolate consisted of partial human HBV

genotype C and ChHBV, which was more closely related

to the Pan troglodytes troglodytes group. We should note

that the recombinant HBV sequence, FG, was isolated

from a chimpanzee living in the wild without co-culturing

the virus, in vitro (Vartanian et al., 2002), thus suggesting

that recombination could neither be due to laboratory

contamination nor to any human intervention. Although

the latter hypothesis cannot be entirely excluded, it

appears as a less plausible explanation given that the

HBV strain was isolated from a chimpanzee living in the

wild.

WMHBV

F

H

C

OuHV

GiHBV

B

D

ChHBV

G

A

E

WMHBV

F

H

C

OuHV

GiHBV

B

D

ChHBV

G

A

E1-550 551-1050 1051-3182

0

20

40

60

80

100

0 500 1000 1500 2000 2500 3000

WMHBV

F

H

C

OuHBV

GiHBV

B

D

ChHBV

G

A

E

C

A B

WMHBV

F

H

C

OuHV

GiHBV

B

D

ChHBV

G

A

E

Nucleotides

Boo

stra

p va

lues

0.1

X75657

AB032431

AF160501

AB064313

M57663

U87742

AAF498266F498266

AF242586

D00220

X02496

AF151735

D00329

AB010289

AY123041

AF068756

AY077736

HB131568

AF193864

AF193863

X69798

AY090455

AB059659

AY090454

AF046996

AY226578 0.1

X75657

AB032431

AF160501

AB064313

D00329

AB010289

M57663

U87742

AF242586

D00220

AY077736

HB131568

AF193864

AF193863

AF068756

AF498266AF498266

AY123041

X02496

AF151735

X69798

AY090455

AB059659

AY090454

AF046996

AY226578 0.1

X75657

AB032431

X02496

AF151735

D00329

AB010289

M57663

U87742

AY123041

AF068756

AF160501

AB064313

X69798

AY090455

AB059659

AY090454

AF498266AF498266

AF242586

D00220

AY077736

HB131568

AF193864

AF193863

AF046996

AY226578

Pres1 PreS2

S

PreC

P

C

X

Fig. 1. (A) Schematic representation of the HBV-genome showing regions with discordant phylogenetic clustering of the FG recombinant sequence. (B) Bootscanning plot of the FG sequence against representative

HBV sequences from humans and primates. (C) Midpoint phylogenetic trees. Bullets indicate clusters supported by higher than 75% ML puzzling steps using Treepuzzle and 0.9 posterior probability using

Bayesian inference of phylogeny (see Results, Section 3.1).

E.N.Magiorkin

iset

al./Gene349(2005)165–171

168

E

D

C

ChHBV

Pan t. verusPan t. verus

Pan t. vellerosusPan t. vellerosus

Pan t. troglodytesPan t. troglodytes

OuHV

GiHBV

F

H

B

G

WMHBV

A

0.1

AB032431

X75657

X02496

AF151735

AF160501

AB064313

D00220

AF242586

AF305327

AY330912

AF498266AF498266

AF222322

AY33091 1

AF242585

AJ131569

U46935

AF193863

AF193864

AY090455

X69798

AY090454

AB059659

AB010289

D00329

U87742

M57663

AY123041

Y18858

AY306136

X04615

AF068756

AF223960

AY226578

AF046996

Fig. 2. Midpoint phylogenetic tree in region spanning nucleotides 1050–3182 and 1–550 that clusters with ChHBV group. Bullets show clusters

supported by higher than 75% ML puzzling steps using Treepuzzle and 0.9 posterior probability using Bayesian inference of phylogeny (see Results,

Section 3.2).

E.N. Magiorkinis et al. / Gene 349 (2005) 165–171 169

4.2. Assumptions about the recombination and the trans-

mission event of ChHBV and human HBV genotype C to

Pan troglodytes schweinfurthii

This finding implies for at least one cross-species

transmission event between humans (carrying HBV

genotype C) and chimpanzees (Pan troglodytes troglodytes

or Pan troglodytes schweinfurthii). In case that the

recombination event occurred between humans and Pan

troglodytes troglodytes, it implies for an additional cross-

species transmission event between different chimpanzee

subspecies.

According to the results of phylogenetic analyses, FG

probably belongs to a distinct lineage that may provide a

subspecies specific group. In case that the latter hypothesis

is true, it implies that the Pan troglodytesschweinfurthii

E.N. Magiorkinis et al. / Gene 349 (2005) 165–171170

subspecies is also infected with ChHBV virus in the wild

probably introduced at a similar time frame as in the other

chimpanzee subspecies, whereas we should note that, given

the limited ChHBV sequence information available until

now, we cannot estimate the precise origin of the partial FG

sequence.

Interestingly, the recombination breakpoints in FG were

mapped within the overlapping region of the RT domain of

pol and the small surface protein and in the non-overlapping

region of pol. Recombination breakpoints have also been

reported similarly for other intergenotypic HBV recombi-

nants in humans (Owiredu et al., 2001; Fares and Holmes,

2002; Cui et al., 2002).

4.3. The HBV genotype distribution in Africa indicates that

recombination between ChHBV and human HBV genotype

C is an extremely rare event—lack of data concerning

genotypic diversity accounts for the limited knowledge of

recombination events

Recombination between human HBV genotype C and

ChHBV seems to be a rare event, since chimpanzees inhabit

in Africa, where only HBV genotypes A, D and E are

reported to circulate (Norder et al., 1993a, 1994). Interest-

ingly, Hu et al. (2000) recently reported the infection of a

wild-caught chimpanzee with HBV genotype C, while it

was not possible to clarify whether the infection occurred in

the wild, or if it was due to the injection with contaminated

human serum. Moreover, Kramvis et al. (2002) reported

similarly an HBV sequence belonging to genotype C

isolated from a patient in South Africa, who according to

the designation of the HBV sequence in the GenBank

database probably originated from Eastern Asia. We should

note here that only a small fraction of the full-length HBV

sequences available worldwide originated from Africa and,

moreover, that only a small number of ChHBV sequences is

available so far from the troglodytes troglodytes and Pan

troglodytes schweinfurthii chimpanzees subspecies. Thus,

we should expect more recombinants and especially

between ChHBV and HBV genotypes A, D and E

circulating in Africa, as soon as more HBV and ChHBV

sequences are available in the future.

Our findings, therefore, show evidence about the first

interspecies recombinant, for which in contrast to the HBV

recombinants reported frequently among humans (Owiredu

et al., 2001; Fares and Holmes, 2002; Cui et al., 2002),

parental viruses are not circulating in the same geographic

area, thus suggesting that it might have been generated by

occasional exposure of humans in ChHBV from either Pan

troglodytes troglodytes or Pan troglodytes schweinfurthii, or

most possibly vice versa (chimpanzees got transmitted from

humans with HBV genotype C). Interestingly, the partial FG

sequence fell within genotype C suggesting that the

recombination event between human HBV and ChHBV

occurred after the initial disperse of the genotype C in

humans.

4.4. Conclusions—future perspectives

In conclusion, the only currently available ChHBV

virus from Pan troglodytes schweinfurthii was found to be

recombinant consisting of HBV genotype C and ChHBV

sequences. Our results are in favour for a subspecies

specific ChHBV infection in Pan troglodytes schweinfur-

thii. However, this issue will be clarified when the

prevalence of ChHBV infection is estimated and when

additional virus sequences become available from this

chimpanzee subspecies.

Acknowledgements

This work was supported by the Hellenic Scientific

Society for the Study of AIDS and Sexually Transmitted

Diseases. DP was supported by the Hellenic Center for

Infectious Diseases Control (HCIDC) of the Ministry of

Health and Welfare. MM and GM were supported by the

Hellenic Scientific Society for the Study of AIDS and

Sexually Transmitted Diseases.

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