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Journal of Virology, June 2000, p. 5377-5381, Vol. 74, No. 11
Department of Medical Microbiology,
University of Göttingen, D-37075
Göttingen,1 Zoological and
Botanical Garden Wilhelma, D-70342 Stuttgart,2
and Department of Virology, Institute for Medical
Microbiology and Hygiene, University of Freiburg, D-79104
Freiburg,3 Germany
Received 21 October 1999/Accepted 28 February 2000
We characterized hepatitis B virus (HBV) isolates from sera of 21 hepatitis B virus surface antigen-positive apes, members of the
families Pongidae and Hylobatidae (19 gibbon
spp., 1 chimpanzee, and 1 gorilla). Sera originate
from German, French, Thai, and Vietnamese primate-keeping institutions.
To estimate the phylogenetic relationships, we sequenced two genomic
regions, one located within the pre-S1/pre-S2 region and one including
parts of the polymerase and the X protein open reading frames. By
comparison with published human and ape HBV isolates, the sequences
could be classified into six genomic groups. Four of these represented
new genomic groups of gibbon HBV variants. The gorilla HBV isolate was
distantly related to the chimpanzee isolate described previously. To
confirm these findings, the complete HBV genome from representatives of each genomic group was sequenced. The HBV isolates from gibbons living
in different regions of Thailand and Vietnam could be
classified into four different phylogenetically distinct genomic
groups. The same genomic groups were found in animals from European
zoos. Therefore, the HBV infections of these apes might have been
introduced into European primate-keeping facilities by direct import of
already infected animals from different regions in Thailand. Taken
together, our data suggest that HBV infections are indigenous in the
different apes. One event involving transmission between human and
nonhuman primates in the Old World of a common ancestor of human HBV
genotypes A to E and the ape HBV variants might have occurred.
Human hepatitis B virus
(HBV), the prototype member of the genus Hepadnaviridae, can
infect different primates. In nature, HBV infections occur mainly in
humans, although they have been also documented in chimpanzees
(16), gibbons (8, 10), and orangutans (17,
18). Other members of the family Orthohepadnaviridae infect rodents, e.g., woodchucks (3) and ground squirrels
(14, 15). A new hepadnavirus was recently isolated from a
woolly monkey, a New World primate (woolly monkey HBV [WMHBV])
(7). This virus is distantly related to the human HBV group
and is therefore considered to represent a progenitor of the human viruses.
HBV shows a remarkable genetic variability, which is reflected in the
occurrence of at least six human HBV genotypes and two nonhuman primate
HBV genotypes, consisting of a chimpanzee HBV isolate and a gibbon HBV
isolate (9, 10). All these primate HBV genotypes are defined
by an intergenotype variation of 8% based on complete viral genomes
(12). The different human HBV genotypes show characteristic
geographical distributions (11).
There are only a few reports about the occurrence of HBV infections in
ape populations (6, 17). It is still a matter of debate
whether these infections are of human origin or indigenous to the
different apes (7, 17). At present, the complete HBV sequence from ape isolates has been analyzed only for one chimpanzee and one gibbon (10, 16). These viruses might as well
represent human viruses introduced "artificially" into these
animals (7). In order to answer this question, we analyzed
in this study HBV isolates from HBV surface antigen (HBsAg)-positive
apes kept in different primate-keeping facilities in Germany, France,
Thailand, and Vietnam. A correlation between the geographic origins of
the apes and their HBV sequences was investigated by analyzing parts of
the viral genome. In addition, we tried to find chains of HBV transmission within the different primate-keeping institutions. The
sequences of two short HBV genomic regions were analyzed to characterize phylogenetically closely related viral isolates. One
region (A) encompasses the pre-S1 genomic region, and the second region (B) includes an area that codes for the viral polymerase and a part of the X protein. However, conclusions concerning possible new genotypes can only be drawn on the basis of complete genomes as
defined by Okamoto et al. (12). Thus, the complete genomes of representatives of each genomic group were then fully
sequenced in order to analyze the intergenotype variation.
Sera from 19 gibbon spp., 1 chimpanzee, and 1 gorilla were part of the
primate serum bank stored at the Zoological and Botanical Garden
Wilhelma, Stuttgart, Germany. Sera were collected during routine
veterinary procedures in different zoos and primate-keeping facilities
in Europe, Thailand, and Vietnam (Table 2). All sera were positive for
at least one of the HBV serologic parameters HBsAg, HBeAg, anti-HBs,
and anti-HBc, as determined by microparticle enzyme immunoassays using
commercial test kits (IMx-HBsAg, IMs-HBe2, IMx-AUSAB, and IMx-CORE;
Abbott Laboratories). HBV DNA, detected by PCR, was present in all samples.
HBV DNA was extracted from serum as described previously
(5). Primers used for amplification of overlapping fragments
spanning the HBV genome are given in Table
1. Nested PCR was carried out (5) for 35 cycles, with denaturation at 95°C for 15 s, annealing at 57°C for 15 s, and extension at 72°C for
70 s, as described previously. Fragments 2 and 4 were
amplified from all samples to determine genomic groups of
regions A and B. We were able to amplify these target sequences from
almost all ape sera with the exception of that from gibbon 174. Here,
we could amplify fragment 2 only. Fragments 1 and 3 were amplified
additionally from one representative of each genomic group to
sequence the complete genome.
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Molecular Epidemiology of Hepatitis B Virus
Variants in Nonhuman Primates
ABSTRACT
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TABLE 1.
Primers used for amplification
and sequencinga
To determine the phylogenetic relationships, we sequenced two genomic regions, one located within the pre-S1/pre-S2 region (region A) and one including parts of the polymerase and the X protein open reading frame (region B). Nucleotide sequences were determined for both strands with the PRISM Ready Reaction DyeDeoxy Terminator Cycle sequencing kit on an ABI 373A DNA sequencer (Applied Biosystems, Weiterstadt, Germany) using PCR primers and internal oligonucleotides (Table 1).
Sequence analysis was performed using the program package GCG
(Wisconsin sequence analysis package, version 8.1, University of
Wisconsin, Madison). Some phylogenetic analyses were done with programs
from the PHYLIP package, version 3.57c (J. Felsenstein, Department of
Genetics, University of Washington, Seattle, 1993). The sequences
obtained in our study (Table 2) were
compared to published human, chimpanzee, and gibbon sequences.
|
Sequence analysis of the pre-S1/pre-S2 region (region A).
As
described previously for the classification of human HBV isolates
(4), the sequence analysis of region A allowed a rough estimate of the phylogenetic relationships among HBV isolates. A
dendrogram of the sequences of region A including human and gibbon HBV
isolates is given in Fig. 1. Here, human,
chimpanzee, and gibbon HBV isolates clustered in different groups. All
isolates obtained from gibbons were related to the gibbon HBV sequence (hbu46935; EMBL accession no. U46935) published by Norder et al.
(10). Furthermore, the isolate from the gorilla (gor97a) was
found to be distantly related to the chimpanzee HBV isolate (hpbvcg;
EMBL accession no. D00220) described by Vaudin et al.
(16). Surprisingly, the HBV isolate from the chimpanzee analyzed here (chimp82) was found within a gibbon HBV genomic group (gibI). The robustness of this tree was
confirmed by bootstrap resampling with 100 data sets. Here, the gibbon
HBV sequences were found within five distinct genomic groups
(gibI to gibV) with intergroup distances of 7 to
11 changes per 100 nucleotides, compared to intergenotype distances of
8 to 22 changes per 100 nucleotides for human HBV genotypes.
|
Sequence analysis of the complete HBV genome from representatives
of gibbon HBV genomic groups I to V.
The complete genome
of at least one representative of each group was sequenced. The results
of phylogenetic analysis of complete viral genomes were similar to the
ones obtained by analyzing region A (Fig.
2). There is one branch leading to the
nonhuman primate HBV isolates, including the isolates from a naturally
infected chimpanzee (hpbvcg) (16) and from a gorilla (whose
HBV isolate was analyzed in this work). The other branch leads to the
human HBV genotypes A to E. A third one leads to human HBV genotype F
and to WMHBV (af046996). All gibbon HBV sequences are found within one
monophyletic group, and within this gibbon HBV group, five
genomic groups can be distinguished. Distances among these groups are between 6.3 and 7.9 changes per 100 nucleotides, thus slightly lower than distances among genotypes, which are defined by a
distance of at least 8% (12). Intragroup distances were between 2.5 and 4.6%, comparable to intragroup distances within human
genotypes. A summary of the intra- and intergroup distances of human
HBV, chimpanzee HBV, WMHBV, and gibbon HBV variants is given in Table
3.
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5 and 7.62 × 105 substitutions per site per year (13),
this sequence identity indicates a recent transmission event among
these apes. A second viral transmission was found in a gibbon family
group living in a German zoo. The HBV isolates of all three individuals
(gibbons 644, 645, and 700) were nearly identical in regions A and B
and cluster within genomic group gibII. Low
distances were again caused by ambiguous nucleotide positions. The
transmission events are indicated in Table 2.
There are some reports about the occurrence of HBV infections in ape
populations (6, 17). The question of whether these infections were of human origin or indigenous to the different ape
populations is still a matter of debate (7, 17, 18). We
found strong evidence for the presumption that these HBV infections are
indigenous to the different nonhuman primate populations by analyzing a
large number of ape HBV sequences. All isolates analyzed in this work
cluster within one monophyletic group, including the previously
described chimpanzee and gibbon sequences. Phylogenetic analysis shows
that there is one branch leading to human HBV genotypes A to E, a
second leading to the ape HBV genomic groups, and a third one
leading to human HBV genotype F and to the New World WMHBV. All ape
isolates were from animals of different geographic origins and include
different species. These facts are consistent with the presumption that
there was a single transmission event between humans and apes in the
Old World involving a common ancestor of human HBV genotypes A to E and
the ape viruses. Prior to this, the branch leading to the New World HBV
isolates, WMHBV, and human HBV genotype F might have split off. Norder
et al. (11) found indications that group F represented the
genomic group of HBV among populations with origins in the New World.
With few exceptions, gibbon isolates from the different Thai and the
Vietnamese locations could be classified into four genomic groups (gibI, -III, -IV, and
-V, with genetic distances between 6.3 and 7.9%),
corresponding to the geographical origins of the apes. This finding is
analogous to the finding by Norder et al. (12) of different
geographical distributions of the different human HBV genotypes. Thus,
genotype definition (12) should be discussed for the gibbon
HBV variants.
Genomic gibbon HBV group gibII represents nearly identical
isolates from a family group living in a German zoo. The HBV isolates from the primates living in other European zoos could be integrated into the Thai genomic groups gibI,
gibIII, and gibIV. Therefore, the HBV infection
of these apes might be introduced by captured wild apes which were
already infected. Surprisingly, the sequence of the HBV isolate of the
chimpanzee analyzed here showed a very close phylogenetic relationship
to the gibbon isolates within group gibI. Most likely, this
reflects an infection of this chimpanzee by a gibbon HBV variant, as
this animal was kept with some gibbons in another zoo. Unfortunately,
we did not get serum samples from apes of this zoo. However, this is
the first probable "quasinatural" interspecies transmission of HBV.
The infectivity of a gibbon HBV variant to a chimpanzee was documented
by Mimms et al. (8). In addition, different experimental
studies revealed that human HBV isolates can infect gibbons and
chimpanzees (1, 2). In general, these results imply that
nonhuman primate HBV variants may be transmittable to humans. At least
they infect representatives of the families Pongidae and
Hylobatidae. However, at present it remains to be elucidated
if nonhuman primate HBV variants are transmittable to humans and
whether they cause disease in humans. We think that this is likely,
because the transmission of human HBV variants to nonhuman primates is
well documented. Thus, we strongly recommend routine screening for HBV
markers and HBV vaccination of zoo-kept apes, primate-handling zoo
keepers, and veterinarians.
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ACKNOWLEDGMENTS |
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S.G. and J.-O.H. contributed equally to this work.
We thank Rainer Thomssen for helpful discussion and personal support.
J.-O.H. was supported by the Grimminger Foundation for Zoonosis Research, Stuttgart, Germany.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Hermann-Herder-Str. 11, D-79104 Freiburg, Germany. Phone: 49-761-203.6591. Fax: 49-761-203.6603. E-mail: hufert{at}ukl.uni-freiburg.de.
Present address: Landeskriminalamt Rheinland-Pfalz, D-55118 Mainz, Germany.
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Journal of Virology, June 2000, p. 5377-5381, Vol. 74, No. 11
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