Species Association of Hepatitis B Virus (HBV) in Non-
Human Apes; Evidence for Recombination between
Gorilla and Chimpanzee Variants
Sine ´ad Lyons1*, Colin Sharp1,2, Matthew LeBreton5, Cyrille F. Djoko5, John A. Kiyang3, Felix Lankester3,
Tafon G. Bibila6, Ubald Tamoufe ´4, Joseph Fair4, Nathan D. Wolfe5, Peter Simmonds1
1Centre for Immunology, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom, 2Roslin Institute, University of Edinburgh, Midlothian, United
Kingdom, 3Limbe Wildlife Centre, Limbe, Cameroon, 4Global Viral Forecasting, San Francisco, California, United States of America, 5Global Viral Forecasting, Stanford
University, Program in Human Biology, Stanford, California, United States of America, 6Ape Action Africa, Yaounde, Cameroon
Hepatitis B virus (HBV) infections are widely distributed in humans, infecting approximately one third of the world’s
population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla),
Pan troglodytes (chimpanzee), Pongo pygmaeus (orang-utan), Nomascus nastusus and Hylobates pileatus (gibbons) and from
the New World monkey, Lagothrix lagotricha (woolly monkey). To investigate species-specificity and potential for cross
species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa) or
between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were
genetically characterised. 9 of 62 chimpanzees (11.3%) and two from 11 gorillas (18%) were HBV-infected (15% combined
frequency), while other Old world monkey species were negative. Complete genome sequences were obtained from six of
the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this
subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of
transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from
chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant
in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different
species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of
Citation: Lyons S, Sharp C, LeBreton M, Djoko CF, Kiyang JA, et al. (2012) Species Association of Hepatitis B Virus (HBV) in Non-Human Apes; Evidence for
Recombination between Gorilla and Chimpanzee Variants. PLoS ONE 7(3): e33430. doi:10.1371/journal.pone.0033430
Editor: Lark L. Coffey, Blood Systems Research Institute, United States of America
Received November 3, 2011; Accepted February 8, 2012; Published March 14, 2012
Copyright: ? 2012 Lyons et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NDW is supported by the National Institutes of Health Director’s Pioneer Award (DP1-OD000370). Global Viral Forecasting is supported by google.org,
http://google.org/, the Skoll Foundation, the Henry M. Jackson Foundation for the Advancement of Military Medicine, the United States Armed Forces Health
Surveillance Center Division of GEIS Operations, and the United States Agency for International Development Emerging Pandemic Threats Program, PREDICT
project, under the terms of Cooperative Agreement Number GHNA-OO-09-00010-00. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: S.M.K.Lyons@sms.ed.ac.uk
Hepatitis B virus (HBV) is a member of the Hepadnaviridae family
of viruses, containing a partially double-stranded DNA genome of
approximately 3182–3221 nucleotides . Human hepatitis B
virus is globally distributed, infecting approximately one third of
the world’s human population. A substantial proportion of liver
disease is attributable to HBV, killing over one million people each
year . In South and East Asia, Sub-Saharan Africa and South
and Central America populations show a particularly high
frequency of HBV infection which can be maintained by vertical
mother to child transmission or horizontal transmission during
HBV variants infecting humans show genetic and antigenic
heterogeneity and are currently classified into 7 or 8 genotypes (A–
H) with a nucleotide sequence divergence ranging from 9% to
13%. Two putative genotypes I and J have also been reported.
Genotype I was tentatively suggested for strains recovered in Laos
. A ninth genotype J was recovered from an 88-year-old
Japanese patient with hepatocellular carcinoma, with mean
sequence divergence between HBV/J and gibbon and orangutan
genotypes of 10.9% and 10.7% respectively .Both active and
resolved HBV infections are also found at high frequencies in
chimpanzees [6,7] and South Asian apes ; whose 10–12 host
taxa-associated variants are distinct from the human variants of
the virus. In addition to the current HBV genotypes, recombina-
tion between human genotypes, for example between genotypes A
and D [1,8,9,10] and B and C [8,9,11,12] can generate novel
variants, contributing to the genetic diversity of the virus.
Within the past 10 years, both active and resolved HBV
infections have also been found in chimpanzees , gorillas ,
gibbons [13,14] and orang-utans  at infection frequencies
comparable to human rates in endemic regions [15,16] in addition
to a single isolate from a woolly monkey . Furthermore
tentative evidence for the occurrence of recombination has been
obtained between the human genotype C and the chimpanzee
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variant AF498266  and gibbon variants . Their occur-
rence demonstrates that despite their genetic divergence, human
and non-human associated variants of HBV can share hosts in
nature. A recently published study characterising HBV variants
infecting ape populations in Cameroon  demonstrated the
existence of a gorilla-specific HBV strain and evidence of
recombination between HBV strains circulating in chimpanzees.
This and previous studies of HBV nucleotide sequence similarity
 indicate non-human primates (NHP) have distinct species-
specific variants of HBV distinguishable both from each other and
from human HBV despite occupying overlapping geographical
Cameroon is within a region of endemic human HBV infection
with a hepatitis B surface antigen (HBsAg) prevalence in humans
of 8% or greater . Additionally, four different great ape taxa
also occur in Cameroon, providing the conditions for potential
inter-species transmission. Although no human-derived genotypes
of HBV were detected in non-human primates in the current
study, evidence for transmission of HBV between chimpanzee
subspecies and between chimpanzees and gorillas was obtained.
A total of 164 non-human primate plasma samples from 11
gorillas, 62 chimpanzees and 91 Old World Monkeys (OWM)
were screened for the presence of HBV DNA. PCR screening
showed 9/73 (12%) apes were positive corresponding to 2/11
gorillas (18%) and 7/62 chimpanzees (11.3%) and 2/91 (2.2%)
OWM. Complete HBV genomes were obtained from the isolates
of 2 Gorilla gorilla and 6 chimpanzees (4 P.t.ellioti, 2 P.t.troglodytes),
while both the Old World Monkey isolates (1 Grey cheeked
mangeby and 1 Mandrill) and 1 chimpanzee were positive only
with the screening primers originally used.
Phylogenetic analysis of the HBV strains using 415 bp S gene
fragments confirmed the grouping of the novel chimpanzee HBV
strains with previously published HBV chimpanzee sequences and
the grouping of the two novel gorilla HBV sequences with
previously published HBV strains AJ131657 and FJ98095-97
[19,21]. (The novel HBV sequence ECO50065LIP3J and FJ98095
were retrospectively identified as originating from the same gorilla
in Limbe Wildlife Centre) (Data not shown). Mitochondrial
sequencing confirmed that the ECO50083LIP5, ECO50210LIP4,
ECO51394CWP1.4, ECO51377CWP2 and ECO51109CWP4,
ECO51212CWP6 HBV variants originated from chimpanzee
subspecies P. t. ellioti and P. t. troglodytes respectively, while
ECO50003LIP3 and ECO50065LIP3 were identified as gorilla-
derived (Table 1). Complete genome sequencing of the eight study
isolates produced sequences of 3182-bp in length, comparable to
reference chimpanzee and gorilla strains (Fig 1). Phylogenetic
analysis based on the complete genome (Fig. 1a), demonstrated
monophyletic groupings for each human genotype (A–H), a clade
containing gibbon and orangutans variants and a third containing
chimpanzee and gorilla HBV sequences, each supported by high
bootstrap values. Sequences of all novel chimpanzee HBV
variants, irrespective of their sub species specific host, clustered
with HBV sequences previously obtained from P. t. ellioti . As
the study samples were obtained from captive settings where P. t.
troglodytes and P. t. ellioti are frequently co-housed, it remains
unclear whether these findings reflect the HBV genotype
distribution among wild chimpanzees in Cameroon or whether
infection of troglodytes by ellioti-derived strains occurred in captivity.
Phylogenetic trees of 200 bp fragments incrementing by 50 bp
across the entire genome were constructed to identify changes in
phylogeny potentially indicative of recombination events. This
procedure was automated by the program TreeOrder Scan  in
SSE v1.0 [Manuscript in preparation]. The tree position of each
sequence across the genome is recorded (y-axis) and colour-coded
by HBV genotype and species (Fig. 2a) and by sub-species (Fig. 2b).
Changes in the tree order of individual sequences or genotypes
with 70% or greater bootstrap support are indicative of alterations
in the phylogenetic relationships of clades and identify potential
Consistent with previous findings , phylogenetic relation-
ships between human genotypes changed between genome
regions, leading to alterations in the branching order. For
example, genotypes D and E were largely phylogenetically distinct
across the genome, but between position 1950 and 2500 (the core
gene); genotype E falls within the genotype D clade. Excluding
gorilla and chimpanzee sequences, recombination events typically
occur around positions 750, 900, 1600, 1950, 2500, 2650 and
Table 1. Specimen isolation data.
Specimen Number Mito HBV VariantLocation
collection Previous holder Likely Wild Origin
Gorilla ECO50003LIP3G.g. Gorilla gorillaLimbe Wildlife
13-Sep-039 years19-Aug-04 Nkoma, Mvangan, South
Gorilla ECO50065LIP3 G.gGorilla gorillaLimbe Wildlife
17-Dec-96 9 months22-Jun-05 Bertoua, East Region East Region
04-Oct-05 1.5 years12-Oct-05 Garoua Zoo (North Region) Adamaoua or East
P.t.e. Pan troglodytes
31-Dec-04 8 months05-Dec-06Bachou, Manyu, South
Banyang Mbo Widlife
P.t.t. Pan troglodytes
25-May-00 1.25 years 08-Aug-06Mfou, Centre Region Akom II, South Region
P.t.t. Pan troglodytes
08-Aug-061 years 10-Mar-08Unknown East Region
13-Sep-05 1.5 26-Aug-09Unknown Centre Region
P.t.e. Pan troglodytes
01-Oct-974.6 yrs 26-Jul-09Unknown Unknown
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2750, frequently coinciding with gene boundaries as previously
In order to detect recombination events between HBV variants
from different ape taxa, a Tree Order Scan was performed with all
HBV reference and study sequences from gorillas and chimpan-
zees with a Hylobates pileatus sequences as an out-group (Fig. 2b).
Outside of the core gene region, gorilla-derived variants were
phylogenetically distinct from other ape-associated and human
genotypes. However, between positions 1560 and 2120 (in the X
and pre/core gene), the gorilla isolate ECO50003LIP3 sequence
grouped within the chimpanzee clade, indicative of a recombina-
tion event. ECO50003LIP3 showed 99.45% (99.0–99.9%) simi-
larity with other gorilla variants between positions 1 and 1559 and
99.65% (99.5–99.8%) similarity between positions 2121–3272,
and an average 95.5% and 97.4% similarity with P.t.ellioti variants
across the same respective regions.
This analysis also identified recombination in the sequence
A498266 (Fig 2b), a P. t. schweinfurhii HBV isolate previously
identified as a recombinant between human genotype C and
chHBV . For this sequence, a 500 nt region between positions
550–1050 nt grouped with species C while the remainder of the
genome grouped with P.t.troglodytes sequences, consistent with
previous findings . The recently described HBV sequence
from P.t.schweinfurthii  may therefore represent the ‘‘original’’
P. t. schweinfurthii sequence, from which the recombinant arose
. The inclusion of these sequenced isolates described in our
investigation supports the formation of a P. t. schweinfurthii species-
specific clade (Fig. 1) which includes the genotype C/chHBV
recombinant. Corroborating evidence of recombination was
identified in Cameroon chimpanzee sequences, FJ09898.1 and
FJ09899.1; between positions 820 and 1300 nt, traversing a partial
region of the polymerase gene confirming previous findings on
recombinant P.t.t/P.t.e HBV variants .
To confirm the position and phylogenetic grouping of the
putative recombinant sequences identified by the TreeOrder scan,
a Grouping Scan was performed . This examines how deeply
embedded the test sequence is within clades formed by non-
recombinant control sequences assigned into species-associated
groups (P.t.e, P.t.t., P.t.v and Gorilla gorilla) (Fig. 3). This method
identified two changes in grouping of the query sequence,
ECO50003LIP3 at position 1560 where it changed grouping
from the gorilla HBV clade to the P. t. ellioti sub-species clade and
a reversion to the gorilla clade at position 2120. Grouping scan
analysis of recombinant sequences A498266 and FJ98098.1
provides substantial support for the formation of recombinant
regions between positions 550–1050 nt and 820–1300 nt respec-
tively. However, the Tree Order or grouping scan methods
provided no evidence for recombination in the P.t.troglodytes
derived sequence, AM117396 (based on its grouping in Fig. 1a)
between chimpanzee sub-species.
Sequence AB046525 from P.t.troglodytes in Central Africa
grouped separately from other P.t.t variants in core gene region,
consistent with past recombination with a divergent and currently
uncharacterised genotype of HBV . Both TreeOrder scan
analysis and Grouping Scan analysis confirmed the rest of the
genome groups consistently with P.t.troglodytes, while adopting an
outlier position to all other chimpanzee and gorilla isolates in the
In this study a large number plasma samples from great apes
and monkeys from Cameroon were screened for HBV-DNA. The
prevalence amongst chimpanzees in our study was found to be
9.7% (6/62), 18% (2/11) in gorillas. This confirms previous
findings on the existence of HBV in great apes in the wild [16,22]
and the rates are similar to those observed in human populations
in areas of endemic infection, such as Central Africa and South
East Asia. In a recent study, the prevalence of active HBV
infection (DNA-positive in plasma) was 15% (8/53) in gorillas and
18% (40/205) in chimpanzees [15,24].
Eight new complete HBV genomes were obtained in the current
study from two gorillas and six chimpanzees born in the wild.
Gorilla sequence ECO50065LIP3 was almost identical to the
previously described sequence, FJ798095 , and retrospective
analysis revealed that these sequences originated from the same
animal in Limbe Wildlife Centre. The six complete chimpanzee
HBV sequences all grouped with previously identified P.t.ellioti
variants  although two were recovered from P.t.troglodytes.
Current analyses cannot determine whether these two cross species
infections occurred in the wild or through contact with infected
P.t.ellioti chimpanzees while in captivity although the latter is
highly likely given the mixing of chimpanzee subspecies in
sanctuaries. The existence of P.t.troglodytes and P.t.ellioti associated
variants of HBV, as is the case for other chimpanzee subspecies
(P.t.verus and P.t.schweinfurthii) requires further investigation of
variants infecting chimpanzees in the wild in Cameroon, in
particular in regions where these sub-species may converge, for
example around the confluence of the Mbam and Sanaga Rivers
. The observation of cross-species infections and recombina-
tion events for HBV infections also provides an additional reason
for ensuring that captive chimpanzees are correctly identified to
subspecies and segregated appropriately to avoid the creation of
recombinant HBV variants with potentially different pathogenic-
ities and transmission patterns.
The phylogenetic tree comparing these eight sequences to
previously recorded HBV sequences in non-human primates
confirms that variants recovered from chimpanzees and gorillas in
Africa are distinct from those reported in Asian gibbons and from
all human HBV sequences that cluster separately into genotypes
A–H. This is consistent with the geographical association of HBV
in NHP previously reported . Phylogenetic analysis based on
complete genome found significant bootstrap support for the
formation of four HBV clusters that, excluding likely cross-species
transmissions, corresponded with P. troglodytes subspecies: P. t.
troglodytes (81% bootstrap), P. t. verus (100% bootstrap), P. t. ellioti
(100% bootstrap) and P. t. schweinfurthii (100% bootstrap). Recent
phylogenetic analysis of the P. t. schweinfurthii HBV strain;
confirmed by our GroupScan analysis; (Fig. 3c) showed evidence
of interspecies recombination between HBV infecting chimpan-
zees and the human HBV-C genotype strain [6,18]. Phylogenetic
trees of the recombinant region and equivalent fragments either
Figure 1. Phylogenetic analysis based on the HBV genome and identified recombinant region 1560–2120 bp. Phylograms displaying
phylogenetic trees based on (a) complete HBV genome; (c) HBV recombinant region 1560–2120 bp and equivalent fragments immediately
preceding; (b) 999–1559 bp and succeeding; (d) 2121–2681 bp this region; with HBV reference sequences from human genotypes (A–H). Relative
species and sub-species HBV variants are identified as follows Pan troglodytes troglodytesm, Pan troglodytes ellioti., Pan troglodytes verus&, Pan
troglodytes schweinfurthii¤, Gorilla gorilla# and Hylobates spp. %, and the host specific cluster is identified by ]. The trees were rooted with the
woolly monkey HBV sequence, NC_001896,. Sequences from this study are in bold and underlined and while recombinant HBV variants have bold
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side, inclusive of all reference and study sequences, confirm the
sub-species association of HBV in NHPs (Fig. 1b to 1d). The
phylograms also support the recombinant data of the Tree order
and Grouping scan analysis, with respect to the location and
confidence level for the recombinant region and sequence (Fig. 1b).
The correlation of HBV sequences with the different subspecies of
chimpanzees indicates either that the HBV strains and their hosts
have co-evolved or alternatively have diverged through allopatric
The co-divergence hypothesis for the distribution of non-human
HBV genotypes in Africa and South East Asia presupposes that
the distinct variants of HBV found in different ape species and
subspecies arose during the period of their evolution, over the past
5-7 million years. However, such a hypothesis implies an
extraordinarily slow maintained substitution rate of HBV; the
5% divergence between gorilla and chimpanzee variants requires a
minimum substitution rate of 561029substitutions per site per
year (SSY), slower even than mammalian coding region
substitution rates and quite distinct from the 1024–1025SSY
rates estimated for HBV over shorter periods . The co-
divergence hypothesis would additionally predict that variants
infecting gorillas should be more divergent from and take an
outlier phylogenetic position to the subspecies-associated variants
of chimpanzees that would have diverged from each other
between 0.8–1.5 million years ago . This is clearly not the
case in the phylogenetic analysis shown in Fig. 1, where the gorilla
clade adopts an internal branching position among chimpanzee
sub-species associated variants. Similarly, the co-divergence
hypothesis cannot explain the inlier position of orangutan derived
HBV variants within the gibbon clade , nor the substantial
sequence diversity in HBV variants infecting humans and their
outlier position to NHP-derived HBV sequences. On the other
hand, co-divergence of virus and host and the implied extremely
low long-term viral substitution rates have been observed within
other primate associated viruses, including simian immunodefi-
ciency virus (SIV) and simian foamy virus [15,24]. In the case of
HBV, substitutions may accumulate slowly as a result of the
extreme constraints on sequence change in the HBV genome
imposed by the extensive use of overlapping reading fames for
protein coding, as well as RNA secondary structures required for
genome transcription and translation [22,24]. However, co-
divergence does not explain how HBV-like viruses infecting
rodents, squirrels and birds could have become so divergent from
human and primate variants over period perhaps only 10–20 times
as long as the period of ape divergence.
The alternative hypothesis which could account for the pattern
of sequence diversity of HBV in NHPs is divergence through
allopatric separation but ongoing transmission of HBV between
ape species and subspecies. This alternative hypothesis would also
account for the internal branching position of the gorilla clade
within the chimpanzee derived HBV sequences. As previously
discussed, it also accounts for the inlier position of orangutan-
derived sequences deep within the gibbon clade, and their close
genetic relationship with the sympatric Hylobates agilis gibbon
species. These two species occupy proximal or overlapping
habitats in Borneo, while HBV variants infecting gibbons from
elsewhere in Asia group separately [24,28]. The lack of sequence
diversity between infected orangutans implies a more recent
introduction of HBV into this species.
Detection of recombinants efforts might therefore focus on
geographical regions where different non-human primate species
and sub-species come into contact, for example the upper reaches
of the Sanaga River in Cameroon P.t.ellioti and P.t.troglodytes may
occasionally mix  and southern Cameroon where P.t.troglodytes
and G.gorilla distributions overlap. The later hypothesis may
potentially explain why no recombinant variants have been
detected in P.t.verus, a subspecies found in West Africa that is
geographically isolated from other non-human ape species. The
timescale for the proposed geographical isolation of HBV variants
infecting different NHP species and sub-species is not known. If we
take the substitution rate for HBV measured over short periods
, the introduction and geographical differentiation of HBV in
African apes may have occurred relatively recently indeed,
approximately 5,000 years using the previously described
substitution rate of 1025substitutions per site per year [26,30].
Recombination affecting a short region between either end of
the polymerase gene (partial X gene and Pre-core/core) in one
Gorilla gorilla isolate is the first recorded occurrence of recombi-
nation between chimpanzee- and gorilla-derived HBV variants.
The recombination event between the gorilla and P.t.ellioti variants
likely occurred in the wild as gorillas and chimpanzees are never
co-housed in captivity. The position of the breakpoint region is
close to several documented previously in human genotypes 
(Fig. 2a). A recent study of duck HBV  recorded a similar
breakpoint event between position 1010–2304 bp, incorporating
the region of the X gene, which is believed to promote cell growth
and inactivate growth regulating molecules [32,33].
Complex epidemiological factors such as transmission routes
affect the pool of circulating HBV variants; however their spread
may be enhanced by the evolution of recombinant variants,
allowing the virus to transmit more efficiently between species.
Phylogenetic studies have previously indicated that recombination
events in HBV are quite common [12,22] and recombinant strains
have been shown to possess distinct biological features and
produce different clinical outcomes compared to their parental
strains . Further work is required to investigate the distribution
of HBV recombinants in Cameroon, their potential impact on
host species and the evolution of HBV in NHPs in the wild.
The evidence of animal reservoirs, cross-species transmission
and recombination between human and ape HBV variants have
Figure 2. Tree Order Scan of HBV sequences. Figure 2(a). TreeOrder Scan of HBV sequences, indicating positions of individual sequences (y
axis) in Phylogenetic trees generated from sequential 250-base sequence fragments, incrementing by 50 bases. Changes in sequence order as a result
of changes in phylogeny at the 70% bootstrap level are shown. Sequences are colour coded by genotype and host species, as indicated by the labels
in left and right margin: genotype A, purple; B, light blue; C, wine; D, emerald; E, royal blue; F, orange; G, pale green; H, navy; Gorilla, blue (Gor);
Chimpanzee, green (Pan); and Woolly monkey (WM-out-group on line 1), red. For comparison the Tree Order Scan has been aligned with scale
genome of HBV (top panel). Recombinant sequences are highlighted as by dashed lines; black gorilla/P.t.e ECO50003LIP3, green FJ798099 P.t.e/P.t.t,
pink FJ798098 P.t.e/P.t.t, orange AB046525 P.t.t and purple AF498266 P.t.s 2(b). Tree Order Scan of HBV sequences, indicating positions of individual
sequences (y axis) in phylogenetic trees generated from sequential 250-base sequence fragments, incrementing by 50 bases. Changes in sequence
order as a result of changes in phylogeny at the 70% bootstrap level are shown. Sequences are colour coded by host species and sub-species of
chimpanzee, as indicated by the labels in left and right margin: Gorilla gorilla, blue (Gor); Pan troglodytes troglodytes, yellow (Ptt); Pan troglodytes
ellioti, green (Pte); Pan troglodytes verus, purple (Ptv); Pan troglodytes schweinfurthii, violet (Pts); and Hylobates pileatus (Hyl) (out-group-line 1-GII), red.
For comparison the Tree Order Scan has been aligned with scale genome of HBV (top panel). Recombinant sequences are highlighted as by dashed
lines; black gorilla/P.t.e ECO50003LIP3, green FJ798099 P.t.e/P.t.t, brown FJ798098 P.t.e/P.t.t, orange AB046525 P.t.t and blue AF498266 P.t.s.
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important implications for the eradication of HBV worldwide.
The transmission of recombinant gorilla/chimpanzee HBV and
endemic infection among apes in the wild will hinder efforts to
eradicate HBV globally, particularly in regions where apes and
humans come into contact . Further sampling of isolated
populations of NHPs and in areas of sympatry is required to
further investigate the currently conflicting evolutionary hypoth-
eses for HBV diversity and to determine how and when HBV
spread between Africa and Asia. Understanding the relationship
between human and NHP HBV variants will aid in resolving this
question and explain the possible role humans have played in
disseminating HBV globally.
Materials and Methods
A total of 164 non-human primate plasma samples were
screened for the presence of HBV DNA. Samples were collected
from animals at three wildlife sanctuaries in Cameroon between
August 2004 and August 2009. Animals were brought to the
sanctuaries following confiscation by the authorities or abandon-
ment by owners. All chimpanzees and gorillas sampled were wild-
born, while other species included both wild and captive-born
individuals. Gorilla gorilla and Pan troglodytes were generally housed
separately in the sanctuaries, while some species of monkeys were
housed in mixed groups. However, the captive history of some
animals is unknown as some were held in captivity prior to their
arrival in the sanctuaries and the sanctuaries themselves were not
always under the current management.
Blood samples were collected via venepuncture from 73 apes
comprising 11 gorillas (Gorilla gorilla) and 62 chimpanzees (Pan
troglodytes troglodytes and Pan troglodytes ellioti), and from a variety of
Old World Monkey species: Cercocebus agilis (n=7), C. torquatus
(n=2), Cercopithecus cephus (n=3), C.erythrotis (n=4), C. l’hoesti preussi
(n=4), C. mona (n=9), C. nictitans (n=3), C. pogonias (n=1), C.
tantalus (n=3), Erythrocebus patas (n=3), Lophocebus albigena (n=5),
Mandrillus leucophaeus (n=20), M. sphinx (n=9), and Papio anubis
(n=20). Plasma was separated by centrifugation and frozen at
280uC until testing. Samples were shipped to the United
Kingdom from Cameroon in compliance with UK and Cameroon
laws and the Convention on International Trade in Endangered
Species of Wild Fauna and Flora (CITES) .
DNA extractions were performed with 50 ml of plasma using the
AllPrep DNA/RNA minikit (Qiagen) according to the manufac-
turer’s instructions with DNA eluted in a final volume of 50 ml.
Samples were screened by nested PCR using first round primers
S1, S5 and second round primers S3 and S6 as previously
published  .Three ml of extracted DNA was then amplified in a
PCR mixture containing Promega Access reagents (Promega,
Chilworth, Southampton, United Kingdom). First-round amplifi-
cation involved 30 cycles of 94uC for 18 s, 55uC for 21 s, and
72uC for 1.5 min; and 1 cycle of 72uC for 5 min. One ml of first
round PCR product was amplified further in a second-round PCR
using the internal primers and conditions previously described .
PCR positive samples with screening primers included samples
from 9 apes (n=2 gorillas, n=7 chimpanzees) and 2 Old World
Monkeys. The entire HBV genome was successfully sequenced for
2 gorillas (ECO50003LIP3 and ECO50065) and 6 chimpanzees
(ECO50083, ECO50210, ECO51109, ECO51212, ECO51394
AND ECO51377) in overlapping fragments using primers as
published  in addition to new primer sets: Set 1: Outer Sense 49
(CTGGATGTGTCTGCGGCGTT position 375) and Outer
Anti-Sense 51 (GCACAGACGGGGAGACCGCG position 1542)
followed by Inner Sense 48 (CCAATTTGTCCTGGYTATCG
position 395) and Inner Anti-Sense 50 (TAAAGAGAGGTG-
CGKCCCGT position 1522), Set 2: Outer Sense 52 (CWTTR-
TATGCATGTATACAAGC position 1082) and Outer Anti-Sense
55 (GGCTTCMCGGTACARAGCTGA position 2054) followed
by Inner Sense 53 (TCGCCAAYTTAYAAGGCCTT position
1121), and Inner Anti Sense 54 (GCGGTGTCRAGRAGAR-
CACG position 2033), and finally Set 3: Outer Sense 56 (TTG-
CCTKCTGAYTTCTTTCC position 2025) and Outer Anti-Sense
59 (CCCATGCTGTAGCTCTTGTTCC position2888), followed
by Inner Sense 57 (CGTGATCTYCTYGACACCGC position
2052) and Inner Anti Sense 58 (CAAGAATATGGTGACCCACA
position 2868). First round PCR involved 35 cycles of 94uC for 18 s,
55uC for 21 s, and 72uC for 1.5 min; and 1 cycle of 72uC for 5 min,
followed by second round PCR of 40 cycles at matching conditions.
Touch-down PCR between 65uC and 50uC with 0.5uC decline per
cycle was also applied.
Mitochondrial sequencing to confirm host species and sub-
species for the 9 complete genomes was carried out using primate
specific primers: Forward: PrCOI: CTATTYGGYGCATGAGC-
NGG Reverse: PrCOI: TARAAGAARGTRGTRTTRAGGT-
TRC, followed by Forward: PrCOI CAGCCCTAAGYCTYC-
TYATTCG and Reverse: PrCOI GAYDGATCAGACRAA-
YARGGG (Where R: A/G, Y: T/C, D: G/A/T and N: G/A/
T/C). First and second round PCR conditions involved 30 cycles
of 94uC for 22 s, 50uC for 24 s, 72uC for 1.5 min; and 1 cycle of
72uC for 5 min.
Sequencing of PCR products and sequence analysis
Positive second round PCR amplicons were sequenced in both
directions using the inner sense and inner antisense primers used
in the second round of amplification. Sequencing was executed
using Big Dye Terminator v3.1 (Applied Biosystems) according to
the manufacturer’s instructions. Sequences were read at the Gene
Pool facility (University of Edinburgh) and analyzed using SSE
v1.0 software. Sequences obtained in this study have been assigned
the GenBank accession numbers JQ664502–JQ664509.
Phylogenetic trees were constructed using a bootstrap neigh-
bour-joining method with 100 replications and incorporating the
Kimura-2-Parameter model of nucleotide substitution and a
uniform rate variation among sites using the MEGA 5.01 
software package with pairwise deletion for missing data. Tree
construction involved two datasets of 31 complete HBV genome
sequences from Pan t. troglodytes, Pan t. ellioti, Pan t. verus, Pan t.
schweinfurthii and Gorilla gorilla GenBank sequences and final
phylogenetic analysis that included representative human HBV
sequences of genotypes A–H.
Figure 3. Grouping Scan analysis. Sequence fragments of 250 bases incrementing by 100 bases with 100 bootstrap replicates, were used to
compare and analyse (a) P.t.troglodytes/P.t.ellioti recombinant FJ98098.1 (b) P.t.ellioti/P.t.troglodytes recombinant FJ98099.1 (c) P.t.schweinfurthii isolate
A498266; (d) P.t.troglodytes AM117396 (e) P.t.troglodytes recombinant AB046525 (f) study recombinant Gorilla gorilla HBV sequence (ECO50003); to
sequence groups from Gorilla gorilla (red), Pan troglodytes ellioti (blue), Pan troglodytes troglodytes (green), Pan troglodytes verus (yellow), Pan
troglodytes schweinfurthii (purple) and human genotype HBV/C (light blue) with respect to A498266. Values .0.5 indicate clustering within the
HBV Recombination between Non-Human Ape Variants
PLoS ONE | www.plosone.org8March 2012 | Volume 7 | Issue 3 | e33430
Recombination analysis was carried out using Tree Order Scan
package of SSE v1.0 [Manuscript in preparation] generating an
image of individual sequence positions in phylogenetic trees
generated from sequential 250-base sequence fragments, incre-
menting by 25 bases. Changes to the sequence order due to
changes in phylogeny at the 70% bootstrap level are reported.
The authors would like to thank the Cameroon Ministry of Forestry and
Fauna, which provided the necessary permits for this work, Ape Action
Africa, and the US Embassy in Cameroon for their support. N.D.W. is
supported by the NIH Director’s Pioneer Award (DP1-OD000370).
Conceived and designed the experiments: SL CS PS. Performed the
experiments: SL. Analyzed the data: SL CS. Contributed reagents/
materials/analysis tools: SL CS PS MLB CFD. Wrote the paper: SL.
Animal handling and sample collection: MLB CFD JAK FL TGB UT JF.
GVFI global site director: NDW.
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