ORIGINAL ARTICLE

Prevalence of antibody reaction with cercopithecineherpesvirus 1 antigen in Macaca cyclopis, Macacafascicularis, and Papio anubis in TaiwanF. Lee, Y-J. Lin, M-C. Deng, T-Y. Lee & C-C. Huang

Animal Health Research Institute, Tamsui, Taipei County, Taiwan

Introduction

Cercopithecine herpesvirus 1 (CeHV-1), also known as

Herpesvirus simiae, herpes B virus, or monkey B virus,

is a member of the genus Simplexvirus within the sub-

family Alphaherpesvirinae, family Herpesviridae [12].

Serological cross-reactivity between CeHV-1 and other

herpesviruses, such as herpes simplex virus 1 (human

herpesvirus 1), herpesvirus papio 2 (HVP-2, cercopithe-

cine herpesvirus 16) and simian agent 8 (SA8, cercopi-

thecine herpesvirus 2), has been described previously

[11, 15–17].

In non-human primates, CeHV-1 infection is usually

a subclinical or mild infection characterized by oral

vesicles, ulcers, and conjunctivitis. Following initial

infection, the virus remains latent in the dorsal root or

trigeminal ganglia of the infected primates. In a small

proportion of infected primates with stressed or

immunocompromised status, the virus may be reactiva-

ted and shed via oral, conjunctival, and mucosal

pathways. In human, CeHV-1 is zoonotic and able to

cause severe meningoencephalitis and high mortality

[6–8]. Human CeHV-1 infection usually involved direct

contact with macaques, their body fluid or tissue, or

indirect contact with CeHV-1 contaminated fomites.

Only one case of human-to-human CeHV-1 transmis-

sion has been documented. With an incubation period

of a few days to a week, the disease often starts with

influenza-like symptoms and neurological signs develop

when CeHV-1 spreads to the central nervous system.

Deaths are often attributed to respiratory failure

associated with ascending paralysis [12].

Keywords

Macaca cyclopis – Macaca fascicularis –

Papio anubis

Correspondence

Fan Lee, Division of Hog Cholera Research,

Animal Health Research Institute, 376

Chung Cheng Road, Tamsui, Taipei County

(25158), Taiwan.

Tel.: 886 2 26212111 (ext. 306);

fax: 886 2 26225345;

e-mail: fanlee@mail.nvri.gov.tw

Accepted January 3, 2007.

Abstract

Background and Methods A total of 284 non-human primate sera were

collected between December 2004 and September 2005 and tested by a

commercially available dot immunobinding assay for the antibodies to

cercopithecine herpesvirus 1, an alphaherpesvirus with high mortality for

infected humans.

Results Seropositive rates were 58% among non-human primates from

animal shelters and 38% among those from zoos and academic institutes.

Positive reactors were found in three species, the Formosan macaque

(Macaca cyclopis; 57%), the cynomolgus macaque (Macaca fascicularis;

11%) and the olive baboon (Papio anubis; 68%).

Conclusions Our results showed that natural infection by cercopithecine

herpesvirus 1 in Formosan macaques was highly prevalent, and to a certain

extent reflected the situation of the wild populations in Taiwan. The find-

ings raised the issues of zoonotic public health and the occupational

health of primate workers. High positive rate in olive baboons was also

found, although, it cannot be ruled out that the positivity was due to cross-

reactivity between cercopithecine herpesvirus 1 and other herpesviruses.

J Med Primatol doi:10.1111/j.1600-0684.2007.00211.x

J Med Primatol 36 (2007) 343–347 ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard 343

Cercopithecine herpesvirus 1 infection is prevalent in

many species of macaques (genus Macaca) [7, 17].

Seroreactivity indicative of CeHV-1 infection has been

reported in wild primate populations in Indonesia [6],

Nepal [14] and Puerto Rico [13], and in macaque col-

onies for research purpose in Brazil [1], Germany [4],

Indonesia [20], Japan [19], and the USA [21, 22]. Infec-

tion in rhesus macaque (Macaca mulatta) [1, 13, 14],

cynomolgus macaque (M. fascicularis) [1, 6, 20],

Japanese monkey (M. fuscata), Formosan macaque

(M. cyclopis), pig-tailed macaque (M. nemastrina),

moor macaque (M. maurus), stump-tailed macaque

(M. arctoides) have been reported [19]. CeHV-1-react-

ive antibodies have also been described in African Old

World, and New World species, including green mon-

key (Cercopithecus sabaeus), Hamadryas baboon

(Papio hamadryas), olive baboon (P. anubis) [19], and

capuchin monkey (Cebus apella) [4]. Prevalence of

CeHV-1 in macaque population is usually high after a

long-term circulation of CeHV-1 within the popula-

tion. However, through rigorous surveillance and

removal of positive animals from the population, it is

possible to establish CeHV-1-negative breeding colony

for research purpose [21].

No article about CeHV-1 seroreactivity in captive or

free-ranged non-human primates living in Taiwan has

been published. In this article, the results demonstrate

a high prevalence of CeHV-1 infection among Formo-

san macaques in Taiwan.

Materials and Methods

A total of 266 sera was collected from non-human pri-

mates in two zoos, five animal shelters administered by

county governments, and two academic institutes

(Table 1) between December 2004 and September

2005. The 113 macaques from animal shelters were

molecular characterized and 104 of which were identi-

fied as Formosan macaques. Of the 113 macaques,

seven with phenotypes similar to that of Formosan

macaque cannot be identified as Formosan macaques

by molecular characterization as based on mitochon-

drial DNA sequence (C.C. Huang, unpublished data).

Eleven male, 3-year-old cynomolgus macaques impor-

ted from Mauritius in 2005 were sampled. The impor-

ted cynomolgus macaques are wild-captured in

Mauritius and have been reared there for more than

6 months for quarantine. Whole blood was sampled

from the femoral vein, and serum and clot were separ-

ated by centrifugation. Serum samples were stored at

)20�C until testing.

The kit used for the detection of specific antibodies

to CeHV-1 was Herpes B Virus DIAdot (Esoterix Inc.,

San Antonio, TX, USA), a dot immunobinding assay

on the basis of inactivated CeHV-1 antigen dotted on

nitrocellulose sheet [9]. Use of the kit was according to

its instruction manual. The nitrocellulose sheet on the

filter paper pad, supplied by the kit, was pre-dotted

with inactivated CeHV-1 antigen and Vero cell culture

control by the manufacturer. After the nitrocellulose

sheet was saturated with phosphate-buffered saline

(PBS)-Tween 20, excess PBS-Tween 20 was removed

from the nitrocellulose sheet. The filter paper was cut

into strips and each strip was dipped into individual

diluted serum sample (five-fold diluted with PBS), and

then placed directly onto the antigen-dotted nitrocellu-

lose sheet. Kit-supplied negative and positive sera were

also treated as samples. Following an incubation of

30 minutes at room temperature (25–30�C), the strip

was removed and the nitrocellulose sheet was washed

Table 1 Seroprevalence of cercopithecine

herpesvirus 1 among non-human primates

in TaiwanOrigins of samples/species

No. of

samples

No. of positive

1+ 2+ 3+ 4+ Total (%)

Animal shelters

Formosan macaque (Macaca cyclopis) 104 10 10 22 18 60 (58)

Cynomolgus macaque (Macaca fascicularis) 2 0 (0)

Unidentified macaque species 7 2 1 2 0 5 (71)

Zoos and research institutes

Formosan macaque (Macaca cyclopis) 61 6 8 15 5 34 (56)

Cynomolgus macaque (Macaca fascicularis) 58 3 2 2 1 8 (14)

Pig-tailed macaque (Macaca nemastrina) 6 0 (0)

Patas monkey (Erythrocebus patas) 5 0 (0)

Olive baboon (Papio anubis) 28 4 4 9 2 19 (68)

De Brazza’s monkey (Cercopithecus neglectus) 1 0 (0)

Mandrill (Mandrillus sphinx) 1 0 (0)

Importation

Cynomolgus macaque (Macaca fascicularis) 11 0 (0)

Cercopithecine herpesvirus 1 infection in Taiwan Lee et al.

J Med Primatol 36 (2007) 343–347 ª 2007 The Authors

344 Journal compilation ª 2007 Blackwell Munksgaard

with 50 ml of PBS-Tween 20. The kit-supplied conju-

gate, goat anti-human IgG conjugated with alkaline

phosphatase, was then added onto the nitrocellulose

sheet and the sheet was incubated at room temperature

with gentle agitation for 30 more minutes. Finally, the

conjugate solution was poured and the nitrocellulose

sheet was washed three times with 20 ml of PBS-

Tween 20 for 5 minutes in each wash followed by the

addition of BCIP/NBT chromogen for color develop-

ment. The chromogen-dipped nitrocellulose sheet was

agitated gently until the positive control developed a

three to four plus staining intensity. Color develop-

ment was stopped by the addition of EDTA solution.

The test results were interpreted by comparing the

staining intensity of the samples to the color chart pro-

vided with the kit. The sample that developed a two to

four plus intensity was interpreted as a positive reac-

tor. The sample that developed a one plus intensity

was considered equivocal and was retested. If the

staining intensity of the re-tested results was the same

or stronger, it was interpreted as positive.

Results

The results of our testing for the presence of

CeHV-1-specific antibodies are shown in Table 1. Sero-

positive rates were 58% among non-human primate

sera sampled from animal shelters and 38% among

those sampled from zoos and academic institutes,

respectively. Positive reactors were found in the For-

mosan macaque, cynomolgus macaque (except those

imported), and olive baboon. The positive rates were

57% (94/165), 11% (8/71), and 68% (19/28), respect-

ively. The 25 positive reactors that scored one plus in

the initial testing were re-tested and all of them gave

similar color intensity and interpreted as positive. The

cynomolgus macaques imported from Mauritius in

2005 tested negative. No positive reactor was detected

in samples from the pig-tailed macaque, patas monkey,

De Brazza’s monkey, and mandrill.

Discussion

This study is the first to conduct a serological investiga-

tion of CeHV-1 infection among non-human primates

in Taiwan. It revealed that the positive rate demonstra-

ted in Formosan macaques in Taiwan was comparable

with the studies recorded earlier [1, 6, 13, 14, 19, 22].

Although the present investigation was not an epidemi-

ological sampling and therefore it was not possible to

estimate the prevalence in wild population of Formo-

san macaque, our findings suggested that CeHV-1

infection might reside naturally in Formosan macaques,

and reflected, to a certain extent, the situation of wild

populations. A broad survey of wild populations of

Formosan macaques therefore warrants further investi-

gation. A large proportion of the positive Formosan

macaques with three-plus or stronger seroreactivity to

CeHV-1 implied that humoral immune responses of

these strong-positive animals might be boosted occa-

sionally, resulting in high serum antibody level.

The kit employed in this study was a modified

enzyme immunoassay created to identify CeHV-1-spe-

cific antibodies on the basis of inactivated CeHV-1

antigens, and it is considered as a reliable assay for

detecting antibodies to CeHV-1 with limited cross-

reaction to other herpesviruses. The assay for CeHV-1

antibody detection in rhesus macaque sera shows a

specificity of 89% and is at least 10 times more sensi-

tive than serum neutralization test and fluorescent anti-

body assay [9]. Declared by the manufacturer, the

sensitivity and specificity of the kit are both higher

than 96%. Previous study also showed that the results

of the assay are highly correlated with those of serum

neutralization test (kappa statistics ¼ 0.849) [10].

Although the serological cross-reactivity between her-

pesviruses is known and tests such as ELISA have

been developed for differentiating various herpesvirus

infections [2, 11, 18], we could not confirm our results

by parallel testing as these tests were not commercially

available. As warned in the kit’s instruction, false-

positive results did occur; however, the reaction den-

sity for a false-positive reactor usually referred to a

weak positive reaction. Moreover, Herbling et al. [10]

stated that CeHV-1 seropositive sera, tested by dot

immunobinding assay, can present greater intensity

against CeHV-1 antigen as they can react with both

simian and human viral antigens. We believed that the

results obtained by the kit were fairly valid. In our

study, of the 126 positive sera detected, only 25 sam-

ples (20%) gave one plus results, supporting that most

positive reactors in our study might be true positive.

With regard to the high positive rate in olive

baboons, serological cross-reactivity between CeHV-1

and HVP-2 or SA8 could not be completely ruled out,

especially in that baboons (Papio spp.) in particular

are considered natural hosts for HVP-2 and HVP-2

infection which is prevalent in certain areas of Africa

[5]. The olive baboons sampled were imported from

Africa about 10 years ago and had no history of con-

tact with Formosan macaques. It seemed likely that

the positive reactions were caused by infection with

simplex-like viruses specific to the species.

To work safely with non-human primates should be

a serious consideration for veterinarians and animal

caretakers. However in Taiwan, many of them wore

Lee et al. Cercopithecine herpesvirus 1 infection in Taiwan

J Med Primatol 36 (2007) 343–347 ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard 345

no or improper protective equipment when handling

primates. Furthermore, they were supported by a weak

occupational health safety system. Fortunately, no

clinical human case of CeHV-1 infection has been

reported in Taiwan, possibly because no rhesus maca-

que has been imported and most documented CeHV-1

human cases are associated with exposure to rhesus

macaques or contaminants of their derivatives [3]. Our

findings also raised public health issues with regard to

human/wildlife interactions. The number of free-ran-

ging Formosan macaques is approximately 250,000

(statistics by the Council of Agriculture in 2001). With

the wide geographic distribution in Taiwan, their habi-

tats unavoidably overlap suburbs, farms and public

recreation sites. Damaging crops and fruit trees by

free-ranging macaques and their food-begging behav-

iors resulted in increased contact between humans and

macaques potentially infected with CeHV-1 and other

associated zoonotic pathogens. For the primate work-

ers, providing personal protection equipment and

establishing occupational health safety support are crit-

ically necessary. From a public health perspective, it is

important to educate the public on the subject of zoo-

noses, enforce a ban on feeding wildlife, and draw up

guidelines for emergency medical care.

Acknowledgment

The study was supported by the Project Establishment

of Animal Models for Evaluating the SARS Vaccines

(NSC93-2751-B062-001-Y) from the National Science

Council, Taiwan.

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J Med Primatol 36 (2007) 343–347 ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard 347