RESEARCH

Macacine Herpesvirus 1 inLong-Tailed Macaques,
Malaysia, 2009–2011
Mei-Ho Lee, Melinda K. Rostal, Tom Hughes, Frankie Sitam, Chee-Yen Lee, Jeffrine Japning,
Mallory E. Harden, Anthony Griffiths, Misliah Basir, Nathan D. Wolfe, Jonathan H. Epstein, Peter Daszak

Macacine herpesvirus 1 (MaHV1; B virus) naturally infects
macaques (Macaca spp.) and can cause fatal encephalitis in humans. In Peninsular Malaysia, wild macaques areabundant, and translocation is used to mitigate human–
macaque conflict. Most adult macaques are infected with
MaHV1, although the risk for transmission to persons who
handle them during capture and translocation is unknown.
We investigated MaHV1 shedding among 392 long-tailed
macaques (M. fascicularis) after capture and translocationby the Department of Wildlife and National Parks in Peninsular Malaysia, during 2009–2011. For detection of MaHV1
DNA, PCR was performed on urogenital and oropharyngeal
swab samples. Overall, 39% of macaques were shedding
MaHV1 DNA; rates of DNA detection did not differ between
sample types. This study demonstrates that MaHV1 wasshed by a substantial proportion of macaques after capture and transport and suggests that persons handlingmacaques under these circumstances might be at risk for
exposure to MaHV1.

M

acacine herpesvirus 1 (MaHV1; also known as B virus) is a zoonotic pathogen that is enzootic among
macaque (Macaca spp.) populations throughout Asia (1,2).
MaHV1 is an α-herpesvirus related to human herpes simplex viruses (HSV) 1 and 2 (3,4) and to herpesviruses that
infect other nonhuman primates such as baboons (5). Like
HSV infection in humans, MaHV1 infection in macaques
can clinically appear as vesicular lesions on the mucous
membranes of the buccal cavity and genital area (6,7).
However, macaques without clinically apparent lesions canstill shed MaHV1 (6).
Transmission of MaHV1 can occur transcutaneously(via bites) or permucosally (via exposure to macaque body
Author affiliations: EcoHealth Alliance, New York, New York, USA(M-.H. Lee, M.K. Rostal, T. Hughes, C.-Y. Lee, J.H. Epstein,
P. Daszak); Department of Wildlife and National Parks Peninsular
Malaysia, Kuala Lumpur, Malaysia (F. Sitam, J. Japning, M. Basir);Texas Biomedical Research Institute, San Antonio, Texas, USA(M.E. Harden, A. Griffiths); Metabiota, San Francisco, California,
USA (N.D. Wolfe)
DOI: http://dx.doi.org/10.3201/eid2107.140162

fluids) (8,9). Among humans, ≈40 cases of MaHV1 encephalitis have been reported; all patients were laboratory
workers who had come in contact with rhesus macaques(M. mulatta) only or with rhesus macaques and long-tailed
macaques (M. fascicularis) or their tissues in the research
environment (2,3). For these patients, signs and symptomsof MaHV1 infection included skin ulcers and lesions at thesite of injury, influenza-like illness, and infection of the peripheral and central nervous systems (which can develop
into brainstem encephalomyelitis and death) (7,9). The
mortality rate for humans with untreated MaHV1 infectionis >70% (7). This high case-fatality rate has led to strict
regulations for handling macaques and macaque clinicalsamples in laboratories and resulted in the designation of
MaHV1 as a Biosafety Level 4 (BSL-4) pathogen and, until
recently, a select agent (2,7,9).
In macaques, MaHV1 frequently remains latent in thetrigeminal and lumbosacral ganglia; however, in responseto stress, it can be asymptomatically reactivated and shed insaliva and urogenital excretions (10). Macaques typicallyacquire MaHV1 at sexual maturity (11); previous studies
have found IgG against MaHV1 in up to 100% of sexually mature wild or laboratory long-tailed and rhesus macaques (11,12). As with other viral infections, the presenceof IgG indicates previous exposure or infection but does
not indicate active virus shedding. During active infection, MaHV1 DNA can be detected in saliva or urogenital
samples by use of PCR. Virus culture is also possible but is
not routinely performed because doing so safely requires a
BSL-4 laboratory (11). Using PCR as a diagnostic method
has advantages over culture in that it can be performed under BSL-2 conditions, it produces results more rapidly, andits sensitivity and specificity are higher (13,14).
Little is known about the shedding rate of MaHV1 inmacaques outside the laboratory setting, the frequency oftransmission to humans, or the incidence of MaHV1 encephalitis among humans (particularly those with frequentcontact with macaques). In Asia, at least 50% of cases of
encephalitis are never diagnosed to the point of causative
agent identification (15). Understanding the ecology of
MaHV1 among macaques is essential for understanding

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

1107

RESEARCH

the potential for human infection. Macaques have adapted
to urbanized human environments, and contact between
humans and macaques can occur in a variety of contexts(e.g., feeding in public recreational areas, capture of wild
macaques for the pet trade or biomedical research colonies, consumption, or population management by wildlife
authorities). Human–macaque contact can result in bites,
scratches, and indirect exposure to macaque body fluids(16,17). Simian foamy virus, a nonpathogenic retrovirus
found in nonhuman primates including macaques, has beentransmitted during occupational exposure to macaques via
bites and scratches in many of the aforementioned contexts
and in agricultural, suburban, and urban environments (18–20). Zoonotic transmission of simian foamy virus to humans has been demonstrated in Indonesia (18) and Bangladesh (19). Because exposure to MaHV1 can occur throughsimilar routes (8), its transmission under circumstances
similar to those of transmission of simian foamy virus in
Asia is plausible.
In Peninsular Malaysia, conflict between humans andmacaques in residential and public areas results from lossof macaque habitat, successful macaque adaptation to human environments, and subsequent macaque overpopulation. As a result, the Department of Wildlife and National
Parks (DWNP) in Peninsular Malaysia implemented a macaque population management program, which includes
the removal or translocation of macaques from a conflictarea. The possibility of exposure to MaHV1 during macaque capture and transport presents a potential occupational hazard to wildlife personnel.
Our aim with this study was to describe the prevalence of MaHV1 shedding among wild-caught long-tailed
macaques after capture and transport in Peninsular Malaysia. This study represents a step toward understanding
the potential for zoonotic transmission of MaHV1 outside
the laboratory.
Materials and Methods
Capture and Sample Collection

Independently of this study, DWNP, as part of their macaque management program throughout Peninsular Malaysia, captured and transported macaques from 6 states(Johor, Perak, Pahang, Pulau Pinang, Selangor, and Negeri
Sembilan) to DWNP holding facilities. Capture and opportunistic blood sampling was performed by DWNP and
EcoHealth Alliance during September–November 2009,
July–October 2010, and July 2011. The macaques captured
had been free ranging and lived in the peripheral vegetation of rural, suburban, and urban communities in several
states of Peninsular Malaysia. Trapped animals were transferred into transport cages and taken to the nearest local
DWNP facility, where they were held up to 72 h before
1108

being transported to the DWNP headquarters in Kuala
Lumpur or relocated to a new area. Macaques were kept ingroups in cages and provided with food and water throughout the holding period. Animals were captured in accordance with the protocols and guidelines of the Manual for
Human–Macaque Conflict Management in Peninsular Malaysia (21). This study was conducted under Institutional
Animal Care and Use Committee approval no. 18048 fromthe University of California (Davis, CA, USA). When handling and sampling macaques, personnel involved with thisstudy wore personal protective equipment (PPE; e.g., eyeprotection, double-layered nitrile gloves, Tyvek coveralls,
and P100 respirators) (22). Blood and swab samples werecollected from each animal at its arrival at the headquarters
or at the local DWNP facility before relocation. Macaques
were immobilized with an intramuscular injection of a
combination of 5 mg/kg ketamine and 5 mg/kg xylazine(21). After immobilization, oropharyngeal swab and urogenital swab samples were collected (when possible, urine
was also collected by cystocentesis). The samples wereplaced in 2 mL cryovials (Nalgene Nunc International,
Rochester, NY, USA) with 500 μL NucliSens lysis buffer (bioMérieux, Marcy l’Étoile, France) and immediately
stored at -80°C. Macaque weight, body condition, sex, andapproximate age were recorded. The age of the animals
was determined by assessing their weight, body size, andthe development of their incisors and genitals (23). Macaques were categorized as adult (>7 years), subadult (3–6
years), or juvenile (1–3 years). The sex and age of 2 animals and the sex of 1 adult animal were not recorded.
Molecular Testing

The samples were vigorously mixed; 100 μL of the sample
was used for mechanical nucleic acid extraction by use of
the NucliSENS miniMAG system (bioMérieux). The extracted nucleic acid was eluted with 60 μL of buffer. PCR
was performed as previously described and validated by
Scinicariello et al. (13). Briefly, MaHV1 primers (B virus
1, 5′-ACCTCACGTACGACTCCGACT-3′; and B virus
2, 5′-CTGCAGGACCGAGTAGAGGAT-3′; 2.5 μmol/L)
were each added to the extraction product and HotStarTaq
Plus Master Mix (QIAGEN, Hilden, Germany). The product was placed in a thermocyler at 94°C for 5 min and thenunderwent 30 cycles as follows: 94°C for 1 min, 56°C for
1 min, and 72°C for 1 min. The products (10 μL) were thenanalyzed by electrophoresis on 1% agarose gels. Of the≈10% of samples that were positive by PCR, 14 PCR products were randomly selected and purified with a PCR purification kit (QIAGEN) and sequenced by using the sameprimers to confirm identity. Sequences were 128 bp andwere analyzed by using a BLAST search of GenBank (24).
Because PCR is reported to be highly specific (13) and all 14
PCR products showed 93%–100% nucleotide homology to

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

Macacine Herpesvirus 1 in Macaques, Malaysia

MaHV1, we considered the other PCR products with identical amplicon size to also be positive for MaHV1. PCR is
more sensitive than culture for detecting HSV (25,26), andwe considered the detection of MaHV1 DNA in a sample
as an indication of virus shedding, although viral load was
not obtained through culture or quantitative PCR.
The positive control was produced in a BSL-4 facility and removed from containment by use of inactivation
procedures approved by the Texas Biomedical ResearchInstitute Biohazard Committee. Briefly, macacine herpesvirus 1 strain E2490 virion “mini-prep” DNA was generated as previously described (27). With the same B virus
1 and 2 primers, the region between nt 54886 and 54993
was amplified by PCR from the viral genome (13) by using
the FailSafe PCR Enzyme Mix (Epicenter, Madison, WI,
USA). This region corresponded to a region in the UL28
open reading frame. The resulting 128-nt fragment wascloned into pCR2.1-TOPO by using the TOPO TA kit (Invitrogen, Carlsbad, CA, USA) to generate the pMHUL28
gene. The insert was sequenced and confirmed.
PCR sensitivity (limit of detection) was determined byusing DNA from pMHUL28 and 2 confirmed-positive samples by diluting the DNA to copy numbers of 2.71 × 104 for
pMHUL28 and 1.59 × 1011 for the samples. The sensitivity
limit for pMHUL28 by PCR was 9.13 × 102 molecules, andfor the swab samples it was ≈1 × 104 molecules. The PCRsensitivity was previously determined to be ≈100 gene copies by using purified viral DNA, and specificity was determined by SacII restriction enzyme analysis and Southernblot hybridization by using an MaHV1-specific internal
probe (5′-GGAGAAGACGTCGCGGTCGTAC-3′) that
discriminates MaHV1 from HSV (13).
Immunoassay

A subset of 149 animals, randomly chosen to represent eachage group, were tested by the MaHV1 ELISA as describedby Ohsawa et al. (28). Although the exact specificity andsensitivity of the original MaHV1 ELISA was not determined, it had been validated by testing of known MaHV1positive (n = 14) and negative (n = 6) serum, and the assay
correctly detected 100% of the positive samples and provided negative results for 100% of the negative samples(R. Eberle, pers. comm., 2013). In brief, MaHV1-infected
and noninfected cell antigens were added to 96-well roundbottom plates and prepared as previously described (28).
Wells were blocked with phosphate-buffered saline containing 5% bovine serum albumin and 0.05% Tween 20(PBS-BSA-Tw) and were incubated at 37°C for 1 h, thenrinsed with PBS-Tw. Serum samples were diluted to 1:100
with PBS-BSA-Tw, added to the plate (50 μL/well), andincubated at room temperature for 2 h. The wells were
washed 5 times with PBS-Tw. Biotinylated anti-human
IgG (Vector Laboratories, Burlingame, CA, USA) was

diluted 1:5,000 with PBS-BSA-Tw (50 μL/well) and incubated at room temperature for 1 h. The wells were washed 5times with PBS-Tw. A complex of avidin and biotinylated
peroxidase was prepared according to the manufacturer’s
instructions, diluted to 1:32, added to washed wells (50μL/well), and incubated at room temperature for 1 h. The
wells were washed 5 times with PBS-Tw. A 3,3′,5,5′-tetramethylbenzidine substrate solution was added (100 μL/well), and the plates were incubated without light for 8–12
min. The reaction was stopped with 2 mol/L sulfuric acid
(50 μL/well), and the optical density at 450 nm was measured by using a microplate absorbance reader (Bio-Rad
Laboratories, Hercules, CA, USA). An ELISA result wasconsidered positive when the optical density was >0.1 (28).
Statistical Analyses

We calculated standard prevalence rates and 95% CIs (29)for differences in shedding prevalence based on macaquesample type, sex, and age. For pairwise analysis (2 parameters) of proportions, we conducted a z-test. A general linear model was used to investigate the effect of geographic
location by state, sex, and age, and the Akaike Information Criterion was used to select the best-fit model (with nosignificant interactions between the variables). An analysis of variance of the general linear model with a post hoc
Tukey HSD (honest significant difference) test was used toassess the significance of each factor. Statistical analyses
were conducted by using the R Statistical package (R Core
Team, Vienna, Austria). A p value of <0.05 was considered
statistically significant.
Results
Samples from 392 long-tailed macaques from 6 states
within Peninsular Malaysia (Figure) were screened by
PCR; 149 of these were also screened by ELISA (Table1). The overall detection of MaHV1 DNA in macaques, inurogenital and/or oropharyngeal samples (n = 392 tested),
was 39.3% (95% CI 34.5%–44.1%). All 14 sequenced
DNA samples displayed 93%–100% homology withthose of MaHV1. Shedding status did not differ significantly among age groups: 37.6% (95% CI 31.2%–43.9%)
among 221 adults, 38.8% (95% CI 28.1%–49.4%) among
80 subadults, and 43.8% (95% CI 33.5%–54.1%) among
89 juveniles (for 2 animals, age was not recorded). Malemacaques were more likely than females to be shedding
the virus at the time of sampling; prevalence was 44.1%(95% CI 37.5%–50.7%) among 220 males and 33.1%(95% CI 26.0%–40.2%) among 169 females; the sex of 3
animals was not recorded (z-statistic = 2.192466, degrees
of freedom [df] = 1, p = 0.0001) (Table 1). Males werealso significantly more likely than females to shed virus
in saliva; prevalence was 26.4% (95% CI 20.5%–32.2%)
among 220 males and 16.0% (95% CI 10.5%–21.5%)

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

1109

RESEARCH

seroprevalence differed significantly among age groups:
IgG was found in 70.0% of 50 adults, 46.0% of 50 subadults, and 30.6% of 49 juveniles (χ2 = 15.6333, df = 2, p
= 0.0004) (Table 3). Among macaques tested by ELISA,
24.2% (95% CI 17.3%–31.0%) were positive according
to PCR but negative according to ELISA results, althoughthese animals did not differ significantly by age, sex, or
shedding site (oropharyngeal vs. urogenital) (Table 4).
The geographic origin of macaques that were MaHV1
positive by PCR was as follows: 4 (75.0%; 95% CI 32.6%–
100%) were from Johor, 44 (54.5%; 95% CI 39.8%–69.3%)
from Perak, 24 (54.2%; 95% CI 34.2%–74.1%) from Pahang, 8 (37.5%; 95% CI 4.0%–71.0%) from Pulau Pinang,
251 (37.5%; 95% CI 31.5%–43.4%) from Selangor, and61 (27.9%; 95% CI 16.6%–39.1%) from Negeri Sembilan(Figure). An analysis of variance of the general linear model indicated that both sex and geographic location (state)
were significantly associated with detection by PCR, but
only the effect of sex (F-statistic = 3.97, df = 1, p = 0.047)
had enough power to remain significant after a post hoc
Tukey HSD (for categorical data) was applied (p = 0.049).

Figure. State of origin and prevalence of macacine herpesvirus 1
shedding within sampled groups of macaques (no. positive/total
tested) from Peninsular Malaysia, September 2009–July 2011.

among 169 females (z-statistic = 2.457458, df = 1, p =0.007). Overall, the proportion of urogenital and oropharyngeal samples positive for MaHV1 DNA did not differ
significantly: 24.7% (95% CI 20.5%–29.0%) of urogenital samples and 21.9% (95% CI 17.8%–26.0%) of oropharyngeal samples were positive. We detected viral DNA in
oropharyngeal and in urogenital swabs for 18.8% (95%
CI 12.7%–25.0%) of the 154 macaques with positive results by PCR (Table 2).
Overall, IgG against MaHV1 was found in 73 (49.0%;
95% CI 38.5%–57.0%) of 149 macaques. We found that

Discussion
We examined MaHV1 shedding among free-ranging macaques after capture and transport, a scenario under which
occupational exposure could occur. In addition to the riskthat capture and transport poses for handlers, macaques areprobably under increased physiologic stress during capture
and transport, which might result in increased virus activation and shedding.
Despite little published data for shedding prevalencein free-ranging or recently captured wild macaques withwhich to compare our findings, serologic evidence fromwild-caught macaques transported from India to the UnitedStates in the 1950s for polio vaccine testing indicates thatthe stress of transport probably led to increased MaHV1seroprevalence from 10% before transit to 70% after transit (30). These animals were young (1.0–1.5 years of age)
and were kept in groups of 60. PCR studies of laboratory
macaques (rhesus and long-tail) have reported a shedding
prevalence range of 0%–71% (11,14,31). Sample sizes inthese studies were generally very small, and most reported

Table 1. PCR results for macacine herpesvirus 1 in macaques, by age and sex. Malaysia, 2009–2011*
Male
FemaleUnspecified†
No. (%; 95% CI)
No. (%; 95% CI)
No. (%)
Age group, yNo.
positiveNo.
positiveNo.
positive
Adult, >6
120 55 (45.8; 36.9–54.7)
100 28 (28.0; 19.2–36.8)
10Subadult, 3–6
48
19 (39.6;25.7–53.4)
32
12 (37.5; 20.7–54.3)
00Juvenile, <352
23 (44.2; 30.7–57.7)
37
16 (43.2; 27.3–59.2)
00
Unspecified†
0
0
0
0
2
1 (50.0)
Total
220 97 (44.1; 37.5–50.7)‡
169 56 (33.1; 26.0–40.2)‡
3
1 (33.3)*NA, not applicable.
†2 macaques of unspecified age and sex and 1 adult macaque of unspecified sex were included in the study.
‡Indicates a significant difference (p<0.05) between the 2 groups marked.

1110

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

No.
221
80
89
2
392

Total
No. (%; 95% CI)
positive83 (37.6; 31.2–43.9)
31 (38.8; 28.1–49.4)
39 (43.8; 33.5–54.1)
1 (50.0; NA)
154 (39.3; 34.5–44.1)

Macacine Herpesvirus 1 in Macaques, Malaysia

Table 2. PCR results for macacine herpesvirus 1 in macaques, by sample type, Malaysia, 2009–2011*
Oropharyngeal
Urogenital
Oropharyngeal and urogenital
Sex
No.
No. (%; 95% CI) positiveNo.
No. (%; 95% CI) positiveNo.
No. (%; 95% CI) positiveM
220
58 (26.4; 20.5–32.2)†
220
56 (25.5; 19.7–31.2)
97
17 (17.5; 10.0–25.1)
F169
27 (16.0; 10.5–21.5)†
169
41 (24.3; 17.8–30.7)
56
12 (21.4; 10.7–32.2)
Unknown‡
3
1 (33.3; 0–86.7)
3
0
0
0
Total
392
86 (21.9; 17.8–26.0)
392
97 (24.7; 20.5–29.0)
29 (18.8)
29 (18.8; 12.7–25.0)*For animals with positive results for both oropharyngeal and urogenital samples, percentages are of the total number of positive animals.
†Indicates a significant difference (p<0.05) between the 2 groups marked for oropharyngeal swabs.
‡3 macaques of unspecified sex were included in the results.

prevalence rates were <10%. Shedding prevalence determined in our study certainly falls within these ranges.
We observed that shedding prevalence was significantly
higher among male than female macaques, although seroprevalence did not differ. This difference could be related tosociobiological dominance behavior by which females typically remain at the dominance level of their mother, whereas
males lose that dominance rank when they leave the group at
the time of dispersal. Thus, males continually must earn theirranking as they change groups (32). This behavior might
predispose males to greater social stress during capture andtransport. Unfortunately, we were unable to separate sexbased differences in physiologic stress from the potential effects of the stress of capture and transport, which might haveaffected male macaques differently than females.
We observed a significant difference in seroprevalence among macaques in different age groups; seroprevalence was highest among adults. This finding was consistent with findings of previous studies (11,12); however,
we did not observe an age-based difference in shedding
prevalence. One potential bias in our sampling strategy
was that the age groups, which were composed of randomly selected individuals, did not reflect the age ratioof the overall group, which might have contributed to thelack of difference among age groups in shedding prevalence. We had expected to see a lower rate of shedding
among juvenile than among adult animals because seroconversion is evident at sexual maturity for most laboratory and free-ranging macaques (33). It could be thatyounger animals were experiencing primary infection
from exposure during capture and transport, which could
explain the higher than expected shedding prevalence forthis age group and in the overall study.
Our primary aim with this study was detection of
MaHV1 DNA in macaques. However, we included serologic test results from a subset of animals to identify

antibody seroprevalence among macaques in different age
groups and to determine whether shedding occurs in theabsence of detectable antibodies. We detected viral DNA
in 36 seronegative macaques. This finding may have been
the result of a recent primary infection before detectable
IgG response. Some animals could have been infected byconspecifics during transport or just before capture. It isalso possible that these animals were experiencing acutevirus reactivation resulting from the stress of capture andtransportation during the 6–72 h before sampling because
previously infected animals would probably be seropositive. Unfortunately, data for the duration of time between
capture and sampling were not available. The incubation
period for HSV-1 or HSV-2 in humans is 2–12 days (34).
Although the time between onset of stress and MaHV1
reactivation has not been determined for macaques, inmice, HSV can reactivate in as little as 14 h after exposure to a stressor (35). Approximately 80% of the animals
shedding MaHV1 in the absence of detectable IgG were
subadults or juveniles, suggesting that this infection was
their first.
The low frequency of simultaneous MaHV1 detectionin oropharyngeal and urogenital swab samples suggests avariable shedding pattern among individuals, which was
not unexpected given the fact that the virus can sequester
itself in the ganglia of the trigeminal nerve, sacral nerve(36,37), or both, which would probably affect the route ofvirus excretion. Other MaHV1 studies have also reported
inconsistent detection of virus in oral and genital secretions
from infected laboratory macaques sampled repeatedly
over time (11,31). Sensitivity of the PCR assay we used
was lower than that of the one used by Scinicariello et al.
(13), which might have resulted in underdetection of viral
DNA in macaque clinical samples. Use of real-time PCR(not available for this study), such as that developed by
Huff et al. (38), which has a sensitivity of 10 viral particles,

Table 3. ELISA results for macacine herpesvirus 1 antibodies in , by age and sex, Malaysia, 2009–2011
Male
FemaleUnspecified
No. (%; 95% CI)
No. (%; 95% CI)
No. (%; 95%
Age group, yNo.
positiveNo.
positive
No. CI) positive
Adult, >6*32
21(65.6; 49.2–82.1)
17 13 (76.5; 60.0–96.6)
1
1 (100.0; NA)
Subadult, 3–6*
29
12 (41.4; 23.5–59.3)
21 11 (52.4; 34.5–73.7)
00 (NA)
Juvenile, < 3*26
8 (30.8; 13.0–48.5)23
7 (30.4; 12.7–49.2)00 (NA)Total
87
41 (47.1; 36.6–57.6)
61 31 (50.8; 40.3–63.4)
1
1 (100.0; NA)*Indicates a significant difference (p<0.05) between the age groups marked.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

No.
50
50
49
149

Total
No. (%; 95% CI)
positive
35 (70.0; 53.5–82.7)
23 (46.0; 28.1–59.8)
15 (30.6; 12.9–43.5)
73 (49.0; 38.5–57.0)

1111

RESEARCH

Table 4. ELISA and PCR results for macacine herpesvirus 1 in macaques, by age, Malaysia, 2009–2011
No. (%) animals
PCR positive,
PCR positive,
PCR negative,
Age group, y
No. animals
ELISA positive
ELISA negative
ELISA positive
Adult, >650
10 (20.0)
8 (16.0)25 (50.0)
Subadult, 3–6
50
9 (18.0)
13 (26.0)
14 (28.0)
Juvenile, <349
4 (8.2)15 (30.6)
11 (22.4)
Total
149
23 (15.4)
36 (24.2)
50 (33.6)

would substantially improve sensitivity of future studies
that screen macaques for MaHV1.
The observed MaHV1 shedding patterns suggest that asubstantial proportion of animals shed virus after, and potentially during, transport and that the risks for exposure
to MaHV1 by wildlife personnel or others handling macaques under these circumstances should be seriously considered. Appropriate PPE, including coveralls, gloves, N95
or P100 respirators, and eye protection, are recommended
for wildlife personnel when handling macaques (and anyother nonhuman primate) under conditions in which stressand prolonged confinement with other macaques may contribute to increased shedding of MaHV1 and potentiallyother pathogens. Indeed, as a result of this study, DWNP
is strengthening its existing policies requiring personnel
handling macaques to wear PPE and use proper work area
biosafety and disinfection techniques to reduce the risk fortransmission of MaHV1 and other zoonotic pathogens, inaccordance with established safety protocols (22). Personnel working with macaques have received additional training to increase their awareness of the potential risks for
exposure to MaHV1.
Future studies should determine whether zoonotic transmission has occurred among those who have occupational
contact with macaques during procedures such as capture,sample collection, treatment, and translocation (e.g., wildlife personnel) and should determine the incidence rate for
infection among high-risk populations. Questions remain
about the etiology of viral encephalitides throughout Asia
and what proportion of these may be caused by MaHV1.
Studies that examine the shedding prevalence of MaHV1
in free-ranging macaques will improve our understandingof shedding in the absence of anthropogenic stressors and,coupled with human surveillance, will enable further assessment of the potential risk for zoonotic transmission.
These results will be of particular relevance to professionals who are occupationally exposed to macaques.

This work was supported by funding from the US Agency
for International Development’s Emerging Pandemic Threats:
PREDICT program, the Skoll Foundation, and Google.org.
Ms. Lee is a research scientist at EcoHealth Alliance, currently
working in Kuala Lumpur, Malaysia. Her research interest is theapplication of molecular and microbiology to public health andconservation.
References
1.
2.

3.

4.

5.
6.
7.
8.

9.

10.

11.

Acknowledgments
We express our gratitude to the wildlife officers and veterinarians of the DWNP in Peninsular Malaysia who provided access to
macaques for this study. We thank Richard Eberle for generously
providing ELISA plates and technical guidance, Nona Yeoh forfacilitating the ELISA, and Parviez Hosseini and Andrew Huff
for consultation on statistical methods.
1112

PCR negative,
ELISA negative
7 (14.0)
14 (28.0)
19 (38.8)40 (26.8)

12.

13.

Palmer AE. B virus, herpesvirus simiae: historical perspective.
J Med Primatol. 1987;16:99–130.
Holmes GP, Chapman LE, Stewart JA, Straus SE, Hilliard JK,
Davenport DS. Guidelines for the prevention and treatmentof B-virus infections in exposed persons. Clin Infect Dis.
1995;20:421–39.
Holmes GP, Hilliard J, Klontz K, Rupert A, Schindler CM,
Parrish E, et al. B virus (Herpesvirus simiae) infection in humans:
epidemiologic investigation of a cluster. Ann Intern Med.
1990;112:833–9. http://dx.doi.org/10.7326/0003-4819-112-11-833
Killeen AM, Harrington L, Wall L, Kelly D. Nucleotide
sequence analysis of a homolog of herpes simplex virus type 1
gene US9 found in the genome of simian herpes B virus.
J Gen Virol. 1992;73:195–9. http://dx.doi.org/10.1099/
0022-1317-73-1-195
Eberle R, Black DH, Lipper S, Hilliard J. Herpesvirus papio 2, an
SA8-like alpha-herpesvirus of baboons. Arch Virol; 1995;140:529–45.
Weigler BJ. Biology of B virus in macaque and human hosts: areview. Clin Infect Dis. 1992;14:555–67. http://dx.doi.org/10.1093/
clinids/14.2.555
Elmore D, Eberle R. Monkey B virus (Cercopithecine herpesvirus
1). Comp Med. 2008;58:11–21.
Centers for Disease Control and Prevention. Fatal cercopithecine
herpesvirus 1 (B virus) infection following a mucocutaneous
exposure and interim recommendations for worker protection.
MMWR Morb Mortal Wkly Rep. 1998;47:1073–6.
Cohen JI, Davenport D, Stewart J, Deitchman S, Hilliard J,
Chapman L, et al. Recommendations for prevention of and therapy
for exposure to B virus (Cercopithecine herpesvirus 1). Clin InfectDis. 2002;35:1191–203. http://dx.doi.org/10.1086/344754
Lees DN, Baskerville A, Cropper L, Brown D. Herpesvirus simiae(B virus) antibody response and virus shedding in experimental
primary infection of cynomolgous macaques. Lab Anim Sci.
1991;41:360–4.
Huff JL, Barry P. B-virus (Cercopithecine herpesvirus 1) infection inhumans and macaques: potential for zoonotic disease. Emerg InfectDis. 2003;9:246–50. http://dx.doi.org/10.3201/eid0902.020272
Engel GA, Jones-Engel L, Schillaci MA, Komang GS, Putra A,
Fuentes A, et al. Human exposure to herpesvirus B–seropositive
macaques, Bali, Indonesia. Emerg Infect Dis. 2002;8:789–95.
http://dx.doi.org/10.3201/eid0808.010467
Scinicariello F, Eberle R, Hilliard J. Rapid detection of B virus(Herpesvirus simiae) DNA by polymerase chain reaction. J InfectDis. 1993;168:747–50. http://dx.doi.org/10.1093/infdis/168.3.747

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

Macacine Herpesvirus 1 in Macaques, Malaysia
14.
15.

16.
17.

18.

19.

20.

21.

22.
23.
24.
25.

26.

Black DH, Eberle R. Detection and differentiation of primate
α-herpesviruses by PCR. J Vet Diagn Invest. 1997;9:225–31.
http://dx.doi.org/10.1177/104063879700900301
Granerod J, Tam CC, Crowcrof NS, Davies NWS, Borchert M,
Thomas SL. Challenge of the unknown: a systematic review
of acute encephalitis in non-outbreak situations.
Neurology. 2010;75:924–32. http://dx.doi.org/10.1212/
WNL.0b013e3181f11d65
Eudey A. The crab-eating macaque (Macaca fascicularis)widespread and rapidly declining. Primate Conservation.
2008;23:129–32. http://dx.doi.org/10.1896/052.023.0115
Engel G, Hungerford LL, Jones-Engel L, Travis D, Eberle R,
Fuentes A, et al. Risk assessment: a model for predicting crossspecies transmission of simian foamy virus from macaques(M. fascicularis) to humans at a monkey temple in Bali, Indonesia.
Am J Primatol. 2006;68:934–48. http://dx.doi.org/10.1002/ajp.20299
Jones-Engel L, Engel GA, Schillaci MA, Rompis A, Putra A,
Suaryana KG, et al. Primate-to-human retroviral transmissionin Asia. Emerg Infect Dis. 2005;11:1028–35. http://dx.doi.org/
10.3201/eid1107.040957
Jones-Engel L, May CC, Engel GA, Steinkraus KA, Schillaci MA,
Fuentes A, et al. Diverse contexts of zoonotic transmission ofsimian foamy viruses in Asia. Emerg Infect Dis. 2008;14:1200–8.
http://dx.doi.org/10.3201/eid1408.071430
Fuentes A. Human culture and monkey behavior: assessing thecontexts of potential pathogen transmission between macaquesand humans. Am J Primatol. 2006;68:880–96. http://dx.doi.org/
10.1002/ajp.20295
Haji Saaban S, Sulai P, Rasdi I, Zanudin M, Yatim H,
Samsudin AR. Manual for the management of human–macaqueconflict in Peninsular Malaysia. Kuala Lumpur (Malaysia): Department of Wildlife and National Parks of Peninsular Malaysia; 2006.
PREDICT One Health Consortium 2013. Protocol for primate
sampling methods [cited 2015 Apr 20]. http://www.vetmed.ucdavis.
edu/ohi/predict/PREDICT_Publications.cfm#Protocols
Seethamchai S, Putaporntip C, Malaivijitnond S, Cui L,
Jongwutiwes S. Malaria and Hepatocystis species in wild macaques,
southern Thailand. Am J Trop Med Hyg. 2008;78:646–53.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic
local alignment search tool. J Mol Biol. 1990;215:403–10
http://dx.doi.org/10.1016/S0022-2836(05)80360-2.
Wald A, Huang M-L, Carrell D, Selke S, Corey L. Polymerase
chain reaction for detection of herpes simplex virus (HSV) DNA on
mucosal surfaces: comparison with HSV isolation in cell culture.
J Infect Dis. 2003;188:1345–51.
Nahass GT, Goldstein BA, Zhu WY, Serfling U, Penneys NS,
Leonardi CL. Comparison of Tzanck smear, viral culture, and DNA

Table ofsContent

27.
28.
29.
30.
31.
32.

33.

34.
35.
36.

37.

38.

diagnostic methods in detection of herpes simplex and varicellazoster infection. JAMA. 1992;268:2541–4.
http://dx.doi.org/10.1001/jama.1992.03490180073029
Coen DM, Weinheimer S, McKnight S. A genetic approach to promoter recognition during trans induction of viral gene expression.
Science. 1986;234:53–9. http://dx.doi.org/10.1126/science.3018926
Ohsawa K, Lehenbauer TW, Eberle R. Herpesvirus papio 2:
alternative antigen for use in monkey B virus diagnostic assays.
Lab Anim Sci. 1999;49:605–16.
Smith RD. Veterinary clinical epidemiology: a problem-oriented
approach. 2nd ed. Boca Raton (FL): CRC Press LLC; 1995.
Hull RN. The significance of simian viruses to the monkey colony
and the laboratory investigator. Ann N Y Acad Sci. 1969;
162:472–82. http://dx.doi.org/10.1111/j.1749-6632.1969.tb56398.x
Zwartouw HT, Boulter EA. Excretion of B virus in monkeys
and evidence of genital infection. Lab Anim. 1984;18:65–70.
http://dx.doi.org/10.1258/002367784780864929
Van Noordwijk MA, Van Schaik CP. Male migration and rankacquisition in wild long-tailed macaques (Macaca fascicularis).
Anim Behav. 1985;33:849–61. http://dx.doi.org/10.1016/S00033472(85)80019-1
Weigler BJ, Hird DW, Hilliard JK, Lerche NW, Roberts JA,
Scott LM. Epidemiology of Cercopithecine herpesvirus 1 (B virus)
infection and shedding in a large breeding cohort of rhesus
macaques. J Infect Dis. 1993;167:257–63. http://dx.doi.org/
10.1093/infdis/167.2.257
Whitley RJ, Roizman B. Herpes simplex virus infections.
Lancet. 2001;357:1513–8. http://dx.doi.org/10.1016/S0140-6736(00)04638-9
Sawtell NM, Thompson R. Rapid in vivo reactivation of herpes
simplex virus in latently infected murine ganglionic neurons after
transient hyperthermia. J Virol. 1992;66:2150–6.
Boulter EA. The isolation of monkey B virus (Herpesvirus simiae)from the trigeminal ganglia of a healthy seropositive rhesus
monkey. J Biol Stand. 1975;3:279–80. http://dx.doi.org/10.1016/
0092-1157(75)90031-1
Weigler BJ, Scinicariello F, Hilliard JK. Risk of venereal B virus(Cercopithecine herpesvirus 1) transmission in rhesus monkeys
using molecular epidemiology. J Infect Dis. 1995;171:1139–43.
http://dx.doi.org/10.1093/infdis/171.5.1139
Huff JL, Eberle R, Capitanio J, Zhou S, Barry PA. Differential
detection of B virus and rhesus cytomegalovirus in rhesus macaques.
J Gen Virol. 2003;84:83–92. http://dx.doi.org/10.1099/vir.0.18808-0

Address for correspondence: Jonathan H. Epstein, EcoHealth Alliance,
17th Fl, 460 W 34th St, New York, NY 10001, USA: email:epstein@ecohealthalliance.org

GovDelivery
Manage your email alerts so you only
receive content of interest to you.

Emailedto youSign up for an Online Subscription:
wwwnc.cdc.gov/eid/subscribe.htm

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 7, July 2015

1113

This content is in the Public Domain.