PREVALENCE AND PHYLOGENETIC ANALYSIS OF HEPATITIS B

VIRUS AMONG NONHUMAN PRIMATES IN TAIWAN

Cho-Chih Huang, D.V.M., M.S., Yu-Chung Chiang, Ph.D., Ching-Dong Chang, D.V.M., Ph.D., and

Yung-Huey Wu, D.V.M, Ph.D.

Abstract: Hepatitis B virus (HBV) is a public health problem worldwide, and apart from infecting humans,

HBV has been found in nonhuman primates. This study investigated the prevalence and phylogenetic analysis of

hepatitis B virus (HBV) and hepatitis D virus (HDV) among nonhuman primates in Taiwan, an area where human

HBV remains endemic. Serum samples from 286 captive nonhuman primates (i.e., 32 great apes [Pan troglodytes

and Pongo pygmaeus], 42 gibbons [Hylobates sp. and Nomascus sp.], and 212 Cercopithecidae monkeys) were

collected and tested for the presence of HBV- and HDV-specific serologic markers. None of the Cercopithecidae

monkeys were reactive against serologic markers of HBV. In contrast, 21.9% (7/32) of great apes and 40.5% (17/42)

of gibbons tested positive for at least one serologic marker of HBV. Of these, five gibbons were chronic HBV

carriers, characterized by presence of HBV DNA and hepatitis B surface antigen in the serum. HBV DNA was also

detected in the saliva of three of the chronic carries. None of these HBV carrier gibbons exhibited symptoms or

significant change in serum clinical chemistry related to HBV infection. Phylogenetic analysis of the complete HBV

genome revealed that gibbon viruses clustered with other HBV isolates of great apes and gibbons from Southeast

Asia and separately from human-specific HBV. None of the HBV-infected animals were reactive against HDV.

These findings indicate that HBV found in these animals is indigenous to their respective hosts and might have been

introduced into Taiwan via the direct import of infected animals from Southeast Asia. To reduce the horizontal and

vertical transmission of HBV in captive animals, the HBV carriers should be kept apart from uninfected animals.

Key words: Hepatitis B virus, nonhuman primates, phylogenetic analysis, prevalence, Taiwan.

INTRODUCTION

Hepatitis B virus (HBV) is the prototypic

member of the Hepadnaviridae family of viruses.

The Hepadnaviridae family is classified into two

genera, corresponding to viruses that specifically

infect mammals (i.e., Orthohepadnaviruses) and

those that infect birds (i.e., Avihepadnaviruses).

Human-specific HBV, which has been reported in

various geographical regions, has been classified

into eight genotypes that range from A to H,

based on an 8% intergroup divergence threshold

in the complete nucleotide sequence.2,15,23,31 In

addition to human-specific HBV, members of the

Orthohepadnavirus genus have been isolated

from rodents such as ground squirrels (GSHV)27

and woodchucks (WHV),5 as well as nonhuman

primates such as chimpanzees (ChHBV),35 low-

land gorillas (GoHBV),6 gibbons (GiHBV),21

orangutans (OuHV),38 and woolly monkeys

(WMHBV).12 However, hepatitis B viruses iso-

lated from gorillas are only 5% divergent from

those isolated from chimpanzees and thus are

considered members of the chimpanzee-specific

HBV genotype.

Hepatitis D virus (HDV) is a defective RNA

virus in which the nucleocapsid is composed of a

delta antigen (HDAg). This virus requires the

hepatitis B surface antigen (HBsAg) for packag-

ing and transmission11 and thus must manifest as

a dual infection with HBV (i.e., a coinfection) or

as a secondary infection to chronic HBV (i.e.,

superinfection). To date, chimpanzees are the

only appropriate animal model for studying

HDV infection37 because they are easily infected

with human HDV and develop acute or chronic

hepatitis. However, naturally occurring coinfec-

tion or superinfection of HBV-infected apes with

HDV has never been documented.

Although it is widely accepted that primate

hepadnaviruses are indigenous to their hosts,

several studies have shown that mammalian

strains of hepadnavirus can be transmitted

between species, such as gibbons and chimpan-

zees,21 chimpanzees and gorillas,6 and chimpan-

zees and humans.8,32 Cross-species transmission

of primate-associated HBV increases the likeli-

hood of interspecies recombination. For example,

HBV isolates from two Vietnamese patients were

putatively identified as gibbon/genotype C re-

combinants, and both sequences clearly fell

From the Department of Veterinary Medicine,

National Pingtung University of Science and Technol-

ogy, Hseuh Fu Road, Neipu Hsiang, Pingtung 91201,

Taiwan (Huang, Chang, Wu); and the Department of

Life Science, National Pingtung University of Science

and Technology, Hseuh Fu Road, Neipu Hsiang,

Pingtung 91201, Taiwan (Chiang). Correspondence

should be directed to Dr. Wu (yhwu@mail.npust.edu.

tw).

Journal of Zoo and Wildlife Medicine 40(3): 519–528, 2009

Copyright 2009 by American Association of Zoo Veterinarians

519

within the gibbon sequence clade.28 Similarly,

HBV isolates from wild Pan troglodytes schwein-

furthii from East Africa were identified as

recombinants of chimpanzee and human geno-

type C viruses.14 Thus, HBV has potential

implications for the conservation of endangered

animals and also for the bidirectional transmis-

sion of HBV between animals and humans, as

well as possibly hindering future efforts to

eradicate HBV from the human population.

Although Taiwan is not a native habitat for

apes, high numbers of baby gibbons and orang-

utans were imported into the country by illegal

pet traders in the 1980s. Fortunately, the wildlife

Conservation Act of 1989 drastically reduced the

ape trade, and many full-grown gibbons and

orangutans have since been donated to zoos and

animal shelters. A previous study indicated that

approximately 111 gibbons reside in Taiwan.3

Human HBV is hyperendemic in Taiwan, where

gibbons and orangutans in private homes have

long been in close contact with their owners.

However, there is still a marked lack of

information on the epidemiology, genome, and

pathogenicity of nonhuman primate HBV in

Taiwan. Thus, in this study, we examined the

epidemiology, pathogenicity, and potential reser-

voirs of HBV infection, as well as the possibility

of HDV infection, in nonhuman primates.

Furthermore, a phylogenetic analysis of HBV

isolates to investigate their genetic relationship

with the previously identified human and nonhu-

man primate strains of HBV was performed.

MATERIALS AND METHODS

Study population

Blood samples were collected from 286 captive

nonhuman primates, including 32 great apes (i.e.,

28 Pongo pygmaeus and four Pan troglodytes), 42

gibbons (i.e., 33 Hylobates sp. and nine Nomascus

sp.), and 212 Cercopithecidae monkeys (i.e., 156

Macaca cyclopis and 56 of other species) from

four zoos and two animal shelters in Taiwan

between December 2005 and October 2007

(Table 1). This project was approved by the

Animal Care and Use Committee of the National

Pingtung University of Science and Technology

(Taiwan). Animals were briefly sedated with

10 mg/kg ketamine hydrochloride (Imalgene

1000H, Merial Laboratoire Ltd., Lyon 69002,

France), and blood samples were obtained by

venipuncture during the animals’ routine annual

examinations. Sera were separated within 6 hr by

centrifugation at 3,000 rpm for 10 min, followed

by storage at 225uC. Saliva samples were

collected using swabs and stored at 225uC.

Serologic methods

Sera were screened for antibodies to hepatitis B

core antigen (anti-HBc) and hepatitis B surface

antigen (HBsAg) using the commercially available

AxSYM CORE 2.0 and ARCHITECT HBsAg kit

(Abbott Laboratories, Wiesbaden 65205, Ger-

many), respectively. Samples that tested positive

for anti-HBc or HBsAg were further assayed for

the presence of hepatitis B e antigens and

antibodies (HBeAg/anti-HBe), as well as antibod-

ies to hepatitis B surface antigen (anti-HBs) using

the commercially available ARCHITECT

HBeAg, AXSTM Anti-HBe 2.0 and AXSTM

AUSAB HBsAb kit, respectively (Abbott Labo-

ratories). Samples that tested positive for HBV

markers were further assayed for the presence of

anti-Delta antibodies using Murex anti-Delta

(total) kit (Abbott Laboratories).

To determine whether liver pathology was

associated with HBV infection, serum samples

from gibbon carriers of HBV, as well as HBsAg-

negative gibbons, were analyzed for the enzyme

levels of alanine aminotransferase (ALT) and

aspartate aminotransferase (AST) with a Johnson

& Johnson 950 biochemical analyzer (Johnson &

Johnson, San Diego, California 92121, USA).

Data were expressed as mean 6 standard

deviation (SD). Student’s t-tests were used to

calculate statistical differences between groups.

Whole genome amplification

Serum and saliva samples that tested positive

for HBV markers were assayed for the presence of

HBV DNA via nested PCR analysis. DNA was

extracted from 200 ml of serum with the QIAamp

MinElute viral DNA extraction kit (Qiagen Inc.,

Hilden 40724, Germany) according to the manu-

facturer’s instructions. DNA from saliva samples

was extracted with the QIAamp blood DNA

extraction kit (Qiagen Inc.) according to the

manufacturer’s instructions. HBV sequences were

amplified by polymerase chain reaction (PCR)

with the use of previously described sets of nested

primers that amplify overlapping segments of

DNA and span the complete genome (Ta-

ble 2).13,24 PCR amplifications were performed in

a 50-ml reaction volume with 5 ng of template

DNA, 5 ml of 10 times reaction buffer (Promega

Inc., Madison, Wisconsin 53711, USA), 5 ml of

MgCl2 (25 mM), 5 ml of dNTP mix (8 mM),

10 pmol of each primer, and 2 U of DNA Taq

520 JOURNAL OF ZOO AND WILDLIFE MEDICINE

polymerase (Promega Inc.). The first and second

rounds of amplification represented a total of 35

cycles, each consisting of 94uC for 1 min, anneal-

ing (temperature ranging from 54–56.5uC accord-

ing to the primer sets) for 1 min and 72uC for

1.5 min. The initial start cycle was performed at

94uC for 3 min, and the final extension step was

performed at 72uC for 10 min. Amplification

products were analyzed by electrophoresis on

1.5% agarose gel and visualized by ethidium

bromide staining and ultraviolet transillumina-

tion to ensure the absence of nonspecific ampli-

fication products.

DNA purification and sequencing

The second round of amplified products were

purified with the QIAquick PCR purification kit

(Qiagen Inc.). The products were then ligated to

the pGEM-TEasy vector (Vector SystemII kit,

Promega Inc.) and transferred into JM109

competent cells (Promega Inc.). At least two

plasmid DNA clones (i.e., resulting from two

independent PCR reactions) were randomly

selected and purified with the Qiagen plasmid

minikit (Qiagen Inc.). Purified plasmid DNA was

sequenced in both directions with the BigDye

Terminator v3.1 Cycle Sequencing Kit (Applied

Biosystems, Foster City, California 94404, USA)

and the ABI 3130 automated sequencer (Applied

Biosystems). Sequencing was performed with the

T7 and SP6 universal primers, which are specific

to a region within the pGEM-TEasy Vector

termination site.

Nucleotide comparison and phylogenetic analysis

The complete genome sequences were aligned

with the previously published HBV sequence by

the BioEdit program (Ibis Biosciences, Carlsbad,

California 92008, USA), version 7.0, and the

CLUSTAL-W multiple alignment method. Phy-

logenetic analyses were conducted using MEGA

software, version 4.33 A phylogenetic tree was

constructed using a neighbor-joining algorithm

with the settings specified by Kimura’s two-

parameter model10 and pairwise deletion treat-

ment of gaps. Data were resampled 1,000 times

and clade confidence was demonstrated by the

bootstrap method.4 As an outgroup to root the

Table 1. Taxonomy and viral hepatitis status of nonhuman primate serum samples.

Primate family SpeciesTotal no.of tests

Viral hepatitis

HBV markers

HDVtotaldeltaAb+

Anti-HBc2 Anti-HBc+

Anti-HBc+

Anti-HBc+

HBsAg+

Anti-HBs+ HBeAg+

Anti-HBs2 Anti-HBs+ Anti-HBe+ HBV-DNA+

Pongidae Pan troglodytes 4 4 0 0 0 NDa

Pongo pygmaeus 28 21 5 2 0 0

Total no. of apes 5 32

Hylobatidae Hylobates agilis 19 10 4 2 3 0

H. lar 10 7 3 0 0 0

H. muelleri 3 2 1 0 0 0

H. concolor 1 1 0 0 0 ND

Nomascus

gabriellae 2 1 0 0 1 0

N. leucogenys 7 4 1 1 1 0

Total no. of gibbons 5 42

Cercopithecidae Macaca cyclopis 156 156 0 0 0 ND

M. fascicularis 20 20 0 0 0 ND

M. nemestrina 10 10 0 0 0 ND

M. fuscata 7 7 0 0 0 ND

M. nigra 5 5 0 0 0 ND

Papio hamadryas 14 14 0 0 0 ND

Total no. of monkeys 5 212

a ND, not done.

HUANG ET AL.—HEPATITIS B VIRUS INFECTION IN APES 521

phylogenetic trees, the full-length HBV sequence

included representative data from human geno-

types A–H and nonhuman primate sequences

from chimpanzees, gibbons, orangutans, and the

woolly monkey. Further comparisons were made

with the use of gibbon and orangutan sequences

derived from the intact region of the S gene

(including per-S1, pre-S2, and S genes).

RESULTS

Serology

Serum samples from 21.9% (7/32) of the great

apes and 40.5% (17/42) of the gibbons in our

study tested positive for at least one serologic

marker of HBV, suggesting a past or current

HBV infection. In contrast, HBV infection was

not detected in any of the Cercopithecidae

monkeys tested (Table 1). The most common

serologic profile (i.e., anti-HBc/anti-HBs) was

observed in 12 (seven gibbons and five orangu-

tans; 50%) of the 24 HBV-positive animals,

suggesting that these animals developed immuni-

ty after a previous infection. Seven (five gibbons

and two orangutans; 29.1%) of the 24 HBV-

positive animals tested positive for anti-HBc,

anti-HBs, and anti-HBe, suggesting that these

animals had resolved HBV infections. The

remaining five gibbons (20.8%) were defined as

chronic carriers on the basis of positive results for

HBV DNA and HBsAg, and negative results for

anti-HBs.20 All of the HBV carriers were mem-

bers of Hylobates agilis (i.e., G15, G18, and G50),

Nomascus garbriellae (i.e., G51), and Nomascus

leucogenys (i.e., G56). Interestingly, these HBV

carriers also tested positive for HBeAg, indicating

that the virus had entered a highly replicative

phase. Furthermore, HBV DNA was also detect-

ed in saliva samples from three of the chronic

carrier gibbons (i.e., G15, G18, and G56).

However, samples from the 24 apes with current

or past HBV infections did not cross-react with

anti-HDV antibodies.

To assess the relationship between HBV

infection and liver damage, ALT and AST

enzyme levels were measured in serum samples

from 25 gibbons (five chronic carriers and 20

seronegative animals). Significant differences

between the two groups with regard to levels of

ALT (chronic carriers 5 24.2 6 7.25 mU/ml;

seronegative animals 5 24.5 6 9.7 mU/ml) and

AST (chronic carriers 5 15.6 6 5.77 mU/ml;

seronegative animals 5 18.8 6 7.2 mU/ml) (Wu,

unpubl. data) were not observed.

Table

2.

Seq

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T-1

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24

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G-6

84

514

54.5

Inte

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196-C

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16

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47

54.5

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15

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-267

809

54.5

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T-1

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40

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54

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b2700-C

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54

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55

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34

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316

938

56.5

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-772

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24

522 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Gibbon HBV nucleotide sequences

Hepatitis B viral DNA samples from five

chronic carrier gibbons were used as targets for

DNA sequencing. Each HBV sequence was 3,182

base pairs (bp) long and aligned with previously

published gibbon HBV sequences. Each sequence

contained intact reading frames (i.e., for the core,

pol, X, and S genes) in similar locations as the

corresponding gibbon HBV sequences. Com-

pared with human HBV genotypes B and C,

which are endemic in Southeast Asia and Taiwan,

the gibbon HBV isolates have a 33-bp deletion at

the 59 end of the pre-S1 gene. However, the

deletion does not result in a frame shift. The

nucleotide sequences obtained in this study have

been submitted to GenBank and bear the

accession numbers xxxxxxxx – xxxxxxxx.

Within this intact region of the gibbon S gene,

variability in the major hydrophilic region that

contains the a, d/y, and r/w antigenic determi-

nants was observed. The HBV serotypes could be

differentiated on the basis of the presence of a

glycine (G) residue at position 145 of the a

determinant and the presence of lysine (K) and

arginine (R) residues at positions 122 and 160,

respectively, of the small S protein.22 Thus, HBV

variants from the G15, G18, and G50 samples

were predicted to have an adw serotype, whereas

variants from the G51 and G56 samples

were expected to have ayw and adr serotypes,

respectively.

Phylogenetic analyses

Phylogenetic analyses of the complete HBV

genomes revealed that sequences from the five

gibbon isolates corresponded to data from

previous studies.1,6,8,12,20,24,32,34,36,38 In addition, a

clade with a 99% supporting bootstrap value that

revealed that the five gibbon isolates were

distinctly separate from the eight known human

HBV genotypes (i.e., clades A–H) and from the

HBV genotypes in African apes (i.e., chimpan-

zees; Fig. 1) was reconstructed. Phylogenetic

analyses of the gibbon samples revealed marked

clustering of HBV sequences according to host

species, such as Hylobates lar and Hylobates

pileatus. However, the HBV sequences from H.

agilis (i.e., samples G15, G18, and G50) were

closely related to the HBV sequences from two

Bornean orangutans (i.e., samples AF193863 and

AF193864) kept in Kalimantan, Indonesia, al-

lowing for the construction of a monoclade with

a 99% supporting bootstrap value. Similarly,

sequences from the Nomascus species grouped

together: G51 (Nomascus gabriellae) was closely

related to AY077736, and G56 (Nomascus

leucogenys) was closely related to AJ131573, with

bootstrap values of 99% and 100%, respectively.

Samples AY077736 and AJ131573 were obtained

from Nomascus concolor animals at the Krabok

Koo wildlife breeding center and the Dusit Zoo

in Thailand, respectively. An advanced phyloge-

netic comparison of the intact region of the S

gene sequence was performed to analyze partial

genomic data obtained from gibbons and orang-

utans (Fig. 2). The phylogenetic topology results

obtained for the intact region of the S gene

sequence resembled those obtained for the whole

genome.

DISCUSSION

The findings of this study reveal that approx-

imately 41% of gibbons and 22% of great apes in

Taiwan are currently, or were previously, infected

with HBV. Active infection, as defined by the

presence of HBV DNA and HBsAg, was only

observed in gibbons (i.e., at a rate of approxi-

mately 12%). These results indicate that captive

gibbons and apes in Taiwan experience a high

prevalence of HBV. On phylogenetic analysis,

genomic sequences from the five HBV-infected

gibbons grouped within the gibbon HBV clade

and were distinctly separate from the human

HBV clade, suggesting that these viruses are

indigenous to their hosts. Furthermore, the five

HBV variants clustered closely with those from

gibbons kept in Thailand and with Bornean

orangutans kept in Kalimantan, Indonesia. The

observation is analogous to a previous report that

HBV isolates from primates living in European

zoos could be integrated into the Thailand

group.6 Thus, these results suggest that the

HBV found in these gibbons is indigenous to

their respective hosts and not recent acquisitions

from humans. These HBV variants might have

been introduced into Taiwan via the direct

import of infected animals from Southeast Asia.

The serologic survey of 15 species of captive

nonhuman primates in Taiwan provides further

evidence that HBV infection is restricted to

gibbons and orangutans. This observation is

consistent with previous reports that HBV does

not appear to infect Old World monkeys.17–19,29 A

previous study screened 195 Old World monkeys

with the use of nested PCR and failed to detect

any additional cases of HBV infection, despite

the fact that the sets of HBV primers were

designed from a highly conserved region of the

surface gene that matched not only all human

HUANG ET AL.—HEPATITIS B VIRUS INFECTION IN APES 523

Figure 1. Phylogenetic analysis of the complete HBV genomic sequence from gibbons. The gibbon sequence was

compared with previously published HBV sequences from apes and with representative sequences from human

genotypes A–H, with the woolly monkey HBV sequence used as an outgroup. Phylogenetic trees were estimated by the

neighbor-joining method. Bootstrap values (i.e., a total of 1,000) greater than or equal to 50 are indicated on the

branches. Horizontal branch lengths are drawn to scale. All sequences were submitted to GenBank and bear the

accession numbers TIM209512 (G15), TIM209513 (G18), TIM209514 (G50), TIM209515 (G51), TIM209516 (G56).

524 JOURNAL OF ZOO AND WILDLIFE MEDICINE

and nonhuman primate HBV variants, but also

the sequences of HBV-like viruses recovered from

rodents, including ground and arctic squirrels

and woodchuck.29 It remains unclear why HBV

infections are generally restricted to members of

the Pongidae and Hylobatidae families. Similarly,

24 HBV-infected animals were screened with a

commercial human HDV serologic assay and

none of the samples cross-reacted with anti-

HDV. Whether HDV-like viruses circulate in

nonhuman primates remains unclear. It is hy-

pothesized that if the nonhuman counterparts of

human hepatitis viruses exist, they must be highly

divergent and have remained undetected using

the currently available diagnostic test.

The HBV genome replicates via reverse tran-

scription of an RNA intermediate. Thus, the

entire genome (i.e., particularly the pre-S1/S2

region) is prone to mutation.7,21 Two of the HBV-

infected gibbons (i.e., G15 and G18) originated

from different monogamous families and were

cohoused in separate cages but share a common

exhibition area in a zoo. Nucleotide sequences

from the most divergent part of the HBV genome

(i.e., the 465-bp pre-S1/S2 region) were 99.4%

similar in G15 and G18 (Wu, unpubl. data).

Assuming a hepadnaviral nucleotide substitution

rate of 1.75 3 1025 to 7.62 3 1025 substitutions

per site, per year,23 this level of sequence identity

indicates recent, possibly horizontal, transmission

between the two HBV-infected gibbons.

Hepatitis B infection in humans can lead to

acute and chronic hepatitis and even cirrhosis.

More than one million people die each year from

HBV-associated liver disease and hepatocellular

carcinoma.9 Although the previous study found

that HBsAg and HBcAg localization within

hepatocytes occurred in a fashion similar to

humans and HBV-infected gibbons,1 the hepatic

pathologic changes of human HBV infection

findings are not consistently observed in all

nonhuman primate HBV infections. The findings

of this study indicate that ALT and AST enzyme

levels are not significantly different in the chronic

HBV carrier gibbons and the seronegative

animals. This finding is consistent with the results

of a previous study in HbsAg-positive gibbons.25

However, an earlier study reported elevated ALT

concentrations in the serum of HBsAg-positive

compared with HBsAg-negative gibbons.20 It is

difficult to interpret those findings with respect to

HBV pathogenesis. Indeed, past studies of HBV

pathogenicity in nonhuman primates have been

limited by the number of animals studied and the

lack of long-term studies on the effects of chronic

infection. Longer term studies are needed to

determine whether such pathologic changes occur

in the indigenous host during the course of

chronic infection.

In this study, saliva samples from three HBV

carrier gibbons (i.e., G15, G18, and G56) tested

positive for HBV DNA, demonstrating a poten-

tial for infection through contact with body fluid.

HBV can be transmitted through human bites30

and experimental transmission to gibbons via the

intradermal administration of HBV-positive hu-

man saliva.26 Thus, it should be noted that HBV-

infected primates pose a risk for animal keepers

and veterinary personnel, especially in the event

of an accidental bite. Although the HBV vaccine

that is specific for nonhuman primates is not

available, most commercially available HBV

vaccines consist solely of a neutralizing epitope

on the a determinant of the small S protein that is

encoded by the S region, and antibodies to the

subtype determinants do not confer protection.16

Because all samples of gibbon HBV isolated from

this study contain the glycine residue at position

145 of the a determinant, it is expected that the

human HBV vaccine could provide cross-protec-

tion. These observations strongly suggest the need

for routine HBV serologic marker screenings and

vaccination of animal keepers and veterinary staff

who work with HBV-infected animals.

There is a high prevalence of HBV among

captive gibbons and apes in Taiwan. Phylogenetic

analyses indicate that the HBV found in these

gibbons is indigenous to their respective hosts,

and these HBV variants might have been

introduced into Taiwan via the direct import of

infected animals from Southeast Asia. Although

none of these HBV carrier gibbons exhibited

symptoms or significant change in serum clinical

chemistry related to HBV infection, for reduced

horizontal and vertical transmission in captive

gibbon populations, the HBV carriers should be

kept apart from uninfected animals to reduce the

rate of transmission. Furthermore, with regard to

the negativity of the Cercopithecidae monkeys

tested against HBV and the negativity to HDV-

related virus, the possibility of a nonhuman

counterpart of human hepatitis viruses in non-

human primates cannot be ruled out.

Acknowledgments: This work was supported

by a grant from the Bureau of Animal and Plant

Health Inspection and Quarantine, Council of

Agriculture, Executive Yuan, Taiwan (95AS-

6.1.2-BQ-B1).

HUANG ET AL.—HEPATITIS B VIRUS INFECTION IN APES 525

Figure 2. Molecular phylogenetic tree showing the intact region of the S gene (including per-S1, pre-S2, and S

genes) sequences from gibbons and orangutans. The tree was created by the neighbor-joining method, with

bootstrap values (i.e., a total of 1,000) greater than or equal to 50 indicated on the branches. Horizontal branch

lengths are drawn to scale. All sequences were submitted to GenBank and bear the accession numbers TIM209512

(G15), TIM209513 (G18), TIM209514 (G50), TIM209515 (G51), TIM209516 (G56).

526 JOURNAL OF ZOO AND WILDLIFE MEDICINE

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