Am. J. Trop. Med. Hyg., 60(2), 1999, pp. 300–306
1999 by The American Society of Tropical Medicine and Hygiene
SEROLOGIC EVIDENCE FOR AN EPIZOOTIC DENGUE VIRUS INFECTING TOQUE
MACAQUES (MACACA SINICA) AT POLONNARUWA, SRI LANKA
ARAVINDA M. DE SILVA, WOLFGANG P. J. DITTUS, PRIYANI H. AMERASINGHE,
FELIX P. AMERASINGHE
Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut;
Department of Zoological Research, National Zoological Park, Smithsonian Institution, Washington, District of Columbia;
Institute of Fundamental Studies, Kandy, Sri Lanka; Department of Zoology, Faculty of Science, University of Peradeniya,
Peradeniya, Sri Lanka
Abstract. Dengue is one of the most rapidly emerging diseases in the tropics. Humans are the principal reservoir
of dengue viruses. It is unclear if nonhuman primates also serve as a reservoir of human dengue viruses under certain
conditions. In this study, a cross-sectional serologic survey was carried out to characterize the pattern of transmission
of a recently identiﬁed dengue virus among toque macaques in Sri Lanka. The results indicated that an epizootic
dengue virus was active among the macaques. A single epizootic had taken place between October 1986 and February
1987 during which 94% of the macaques within the 3 km
study site were exposed to the virus. The epizootic was
highly focal in nature because macaques living 5 km from the study population were not exposed to the virus. The
transmission of dengue viruses among macaques in the wild may have important public health implications.
Dengue is the name given to four closely related but dis-
tinct arboviruses (serotypes DEN-1, DEN-2, DEN-3, and
DEN-4) belonging to the genus Flavivirus in the family Fla-
viviridae. Over the past four decades dengue viruses have
emerged as a major threat to the health of people living in
many tropical areas of the world.
Human dengue viruses
are mostly active in urban areas where the virus is main-
tained through a cycle in which humans are the principal
reservoir host and Aedes aegypti is the principal mosquito
Human dengue infections have also been documented in
rural areas in Asia, although the prevalence of infection is
lower than in the urban setting.
In rural areas with a low
prevalence of human infection, how does the virus maintain
itself? The virus may persist in the vector through direct
vertical and horizontal transmission between mosquitoes.
Dengue-infected female mosquitoes have genital tract infec-
tions that sometimes lead to the infection of eggs during
Furthermore, venereal transmission has also
been noted from infected males to females.
anism used by human arbovirus for maintaining themselves
is to have additional nonhuman reservoir hosts. Yellow fever
virus, a Flavivirus that shares many similarities to dengue,
is maintained by an urban cycle involving Ae. aegypti and
humans as well as in a sylvatic cycle with Aedes mosquitoes
and nonhuman primates.
It remains to be resolved if human
dengue viruses also use nonhuman primates as reservoir
hosts under certain conditions.
Dengue viruses have been isolated from monkeys and pri-
matophilic vectors. Sylvatic circulation is common in parts
of West Africa where both DEN-1 and DEN-2 viruses have
been isolated from monkeys and sylvatic Aedes species.
However, the sylvatic cycle in West Africa does not to ap-
pear to cross-over to humans because dengue infection was
rare or absent among people living close to the sylvatic cy-
In fact, genetic analysis has shown that viruses isolated
from human epidemics in Africa are distinct from the syl-
Rudnick and Lim carried out studies on the ecology of
dengue in Malaysia.
These studies led to the isolation of
DEN-1, DEN-2, and DEN-4 viruses from sentinel monkeys
(Presbytis obscura and Macaca fascicularis). Dengue virus
was also isolated from Ae. albopictus, a rural vector found
at ground level, as well as from other primatophilic Aedes
species found at the canopy level. Based on these ﬁndings,
Rudnick has proposed that in Southeast Asia dengue viruses
are maintained in a primal cycle involving monkeys and vec-
tors of the Ae. niveus group that feed at the canopy level.
Rudnick also proposed that the rural dengue vector (Ae. al-
bopictus) may introduce the sylvatic virus into the human
Much of the work in Malaysia was done in the
1960s and the sylvatic and human dengue viruses were not
sequenced to establish the relationship between human and
sylvatic dengue viruses in Asia.
Dengue is an important cause of human morbidity and
mortality in Sri Lanka.
While dengue viruses are primar-
ily active in the urban areas in the southwestern part of the
island, serologic surveys indicate low-level human exposure
in other parts of the country.
Four species of nonhuman
primates are present in Sri Lanka, and they often live in
proximity to people in rural villages.
of a population of toque macaques (Macaca sinica)atPo-
lonnaruwa has been intensely studied by Dittus and others
Evidence for a sylvatic dengue cycle among
these macaques was recently reported by Peiris and others,
who found that 94% of the monkeys tested had antibodies
that neutralized DEN-2 virus.
The objective of the current
study was to further characterize the pattern of infection of
this virus among macaques at Polonnaruwa. We report here
that the virus causes epizootics among the macaques, rather
than being enzootic as was previously thought.
MATERIALS AND METHODS
Study site and macaque population. The population of
toque macaques used in the current study inhabit the natural
dry evergreen forest within the Nature and Archaeological
Reserve at Polonnaruwa (Figure 1).
The behavior, ecology,
demography, and genetics of these wild monkeys have been
intensively studied since 1968 by Dittus and others.
1995, the population comprised nearly 1,000 monkeys dis-
tributed among some 28 social groups. All the macaques in
the population have been individually identiﬁed and the
dates of birth and life histories of nearly all the animals are
DENGUE EPIZOOTIC AMONG MACAQUES IN SRI LANKA
1. Location and map of study site (adapted from Hoelzer
). The position of the study site and camp site at Polon-
naruwa are indicated.
known. The ages of animals whose births were not observed,
such as immigrants into the study population, were estimated
based on known relationships between morphologic devel-
opment and age.
Macaque serum samples. Blood was collected for ge-
netic and epidemiologic analyses by trapping the macaques
in cages and tranquilizing them with ketamine hydrochloride
as previously described.
Blood was collected by venipunc-
ture and separated into serum the same day and stored in
liquid nitrogen. Sera collected in 1995, 1987, and 1986 were
used in the current study. Control sera samples from labo-
ratory rhesus macaques (M. mullata) infected with dengue
or yellow fever viruses were provided by the Yale Arbovirus
Research Unit (YARU) (New Haven, CT).
Serologic tests. An ELISA was used to screen macaque
sera for dengue antibody. Each plate was coated for 2 hr at
C with the 4G2 anti-ﬂavivirus monoclonal antibody (pro-
vided by YARU).
The plate was washed and incubated
with a blocking solution (5% horse serum in phosphate-buf-
fered saline [PBS]) for 15 min at 37
C. Next, the plates were
incubated overnight at 4
C with the virus antigen. Flavivirus
antigens (DEN-1, DEN-2, DEN-3, DEN-4, yellow fever,
Japanese encephalitis, and Zika viruses) were prepared and
provided by YARU. Since the antigens were prepared in
mouse brain, uninfected mouse brain antigen was used as a
negative control. The following day the unbound antigen
was washed away with PBS containing 0.5% Tween 20
(PBS/Tween) and the macaque sera were added at a 1:100
dilution and incubated for 1 hr at 37
C. The plates were
washed with PBS/Tween and incubated for 30 min at 37
with horseradish peroxidase–conjugated goat anti-monkey
immunoglobulin (Sigma Biosciences, St. Louis, MO). Fi-
nally, the plates were developed by adding commercially
prepared peroxidase substrate (Zymed Laboratories, San
Francisco, CA). Each serum sample was tested in duplicate
against each of the test antigens. Sera that gave an optical
density (OD) that was at least four times greater than the
normal mouse brain negative control were designated as pos-
Enzyme-linked immunosorbent assay for detecting
dengue antibody in monkeys. Flavivirus antigens are no-
torious for generating antibodies that cross-react between
Flaviviruses. Thus, it was important to determine if the ELI-
SA used to screen macaque sera was capable of distinguish-
ing between Flaviviruses. For this purpose sera from labo-
ratory monkeys infected with dengue or yellow fever were
used. As expected, the sera cross-reacted with Flavivirus an-
tigens other than the virus used in the original infection (Fig-
ure 2). However, for each of the sera the highest signal was
observed in the presence of antigen from the infecting virus
(Figure 2). Thus, although cross-reactivity was observed, it
was possible to clearly distinguish the infecting Flavivirus
from other Flaviviruses. These results validate the use of this
ELISA as a means of screening macaques for Flavivirus
antibodies, as well as characterizing at a serologic level the
relationship of macaque Flaviviruses to known Flaviviruses.
Analysis of 1995 sera. Two hundred forty-four serum
samples collected between July and October 1995 were
screened for the presence of antibodies against dengue-2 vi-
rus. Twenty-one percent (52 of 244) of the animals tested
positive for dengue virus antibody. The macaque sera were
also tested using antigens from yellow fever, Japanese en-
cephalitis, and Zika viruses. Most of the dengue-positive
sera also cross-reacted with these antigens, although the sig-
nal was consistently less than the signal for the dengue-2
virus antigen (Figure 3). The mean ODs for the dengue virus
antigen were 1.7, 3, and 3.3 times greater than the mean
ODs for the yellow fever, Japanese encephalitis, and Zika
virus antigens, respectively (Figure 3). None of the dengue
virus–negative sera bound to the other Flavivirus antigens.
These results indicate that the Flavivirus infecting the ma-
caques was antigenically most closely related to dengue vi-
rus and was distinct from the antigenic complexes that in-
clude Japanese encephalitis and Zika viruses.
The ELISAs were also performed to determine the dengue
virus serotype circulating among the macaques. Plates were
coated with antigen prepared with the different dengue virus
serotypes. Five representative positive sera were selected
and serial dilutions of each sera was tested with the different
dengue viral antigens. The positive sera cross-reacted with
the four antigens and, in spite of the serial dilutions, it was
not possible to single out any particular antigen as belonging
to the serotype responsible for the original infection.
Analysis by sex and social group (1995 sera). The ratio
of males to females in the study sample (1:1.13) was similar
to the ratio of males to females among the seropositive an-
imals (1:1.38), indicating that both sexes were equally sus-
ceptible to the virus. The 244 animals tested in this study
belonged to 16 of the 28 different social groups living within
the study area. The data were analyzed to determine the
prevalence of infection in the different social groups. Ani-
mals from all the groups tested had at least one infected
individual and the prevalence of infection in the different
groups ranged from 6% to 50% (Table 1), depending on the
number of group members nine years of age or older. Thus,
DE SILVA AND OTHERS
2. The ELISA for detecting Flavivirus antibodies in macaque sera. The ELISA plates were coated with normal mouse brain (NMB)
and dengue-2 (D-2), Japanese encephalitis (JE), yellow fever (YF), and Zika virus antigens. Each plate was incubated with sera from a monkey
infected with yellow fever or DEN-2 viruses. Each serum and antigen combination was tested in duplicate. OD
Prevalence of dengue antibody among macaques from different so-
cial groups in Polonnaruwa, Sri Lanka
Group ID No. tested No. positive (%)
Total 244 52 (21)
3. Analysis of sera showing that ﬂavivirus antibody–pos-
itive macaque sera bound best to the dengue virus antigen. The 52
ﬂavivirus antibody-positive macaque sera were tested against normal
mouse brain (NMB), Dengue-2, Japanese encephalitis (JE), yellow
fever (YF), and Zika virus antigens. The mean value obtained from
all 52 positive sera against each of the antigens is displayed. Bars
show the mean
the virus was active in an area encompassing the entire study
site, if not a larger area.
Analysis by age (1995 sera). The age of the animals test-
ed for dengue virus antibody ranged from less than one-
month-old infants to 33-year-old adults. The prevalence of
infection in the different age groups of macaques was ana-
lyzed. Figure 4 shows the number of animals tested in each
age cohort, as well as the number of animals exposed to the
virus. Very few (2.6%) animals in the eight year of age and
younger cohorts were exposed to the virus whereas 88% of
the animals in the nine year of age and older cohorts were
exposed to the virus. The pattern of exposure in the different
age cohorts is highly suggestive of an epizootic virus that
was active 8–9 years (between 1986 and 1987) prior to the
time when the blood was collected in 1995. Thus, all the
animals that were born after the epizootic in 1986–1987,
which includes 80% of the animals in the eight-year-old co-
DENGUE EPIZOOTIC AMONG MACAQUES IN SRI LANKA
4. Prevalence of dengue antibody among macaques in different age groups (1995 sera).
Dengue antibodies among macaques in the eight-year-old cohort
(1995 sera) in Polonnaruwa, Sri Lanka
Animal ID number Date of birth ELISA result
Aug 16, 1986
Dec 28, 1986
Jan 5, 1987
Jan 6, 1987
Feb 4, 1987
Feb 11, 1987
Feb 21, 1987
Feb 26, 1987
Mar 10, 1987
Apr 7, 1987
Apr 8, 1987
Apr 8, 1987
Apr 14, 1987
Apr 27, 1987
May 1, 1987
May 3, 1987
May 9, 1987
May 10, 1987
May 19, 1987
May 31, 1987
hort and all of the animals in the seven-year-old and younger
cohorts, were not exposed to the virus. The three positive
animals in the 0-year-old cohort are almost certainly due to
maternal antibody. In fact, two of the mothers of these in-
fants were tested and found to be positive animals more than
nine years of age. The mother of the third positive infant
was not tested in this study. Prior to the point at which trans-
mission stopped in 1986–1987, there might have been a sin-
gle epizootic that exposed almost all the individuals in the
population or the virus may have been enzootic among the
Detailed analysis of eight-year-old animals (1995 sera).
To determine more accurately when this virus stopped cir-
culating among the macaques, all the eight-year-old animals
bled in 1995 (this sample included 10 animals from the orig-
inal 246 animal samples, as well as 10 animals not included
in the original sample, but also trapped in 1995) were tested
for dengue-2 virus antibody (Table 2). Three of the 20 ani-
mals in the eight-year-old cohort were positive for dengue
virus antibody. The dates of birth of the eight-year-old ani-
mals and their serologic status is presented in Table 2. It is
clear from these data that only the oldest animals in this
cohort (i.e., those born before January 6, 1987) were ex-
posed to this virus, while none of the animals born after
February 4, 1987 were exposed. These results suggest that
transmission abruptly ceased among the macaques starting
sometime between January and February 1987.
Analysis of 1986 and 1987 sera. The serologic analysis
of macaque samples collected in 1995 indicate that the an-
imals were exposed to a dengue virus whose transmission
abruptly ceased before February 1987. Prior to February
1987, the virus may have been responsible for a single large
epizootic that occurred towards the end of 1986 or beginning
of 1987 or the virus may have been enzootic among the
macaques. To further characterize the pattern of transmission
during the critical period between 1986 and 1987, blood
samples collected in September 1986 (pre-epizootic?) and
blood samples collected between June and August 1987
(post-epizootic) were tested for dengue virus antibody. These
results, which are presented in Table 3, conﬁrm that 81% of
the macaques were infected due to a single epizootic that
DE SILVA AND OTHERS
Prevalence of dengue antibody among macaques bled in 1986 and
1987 in Polonnaruwa, Sri Lanka
1986 (September) 1987 (July–September)
No. of animals tested
No. positive (ELISA)
* The two positive animals were 15 and 20 years old at the time of bleeding in 1986.
Rainfall at Polonnaruwa, Sri Lanka before, during, and after the
northeast monsoon of 1986–1987
Month Rainfall (mm) No. of days of rain
took place at the end of 1986. Of the 16 animals sampled
in September 1986, only two older animals were seroposi-
tive. In contrast, when the same population was sampled in
1987 the seroprevalence was 94% (41 of 44), indicating that
a major epizootic took place between 1986 and 1987. The
two positive animals in the 1986 sample indicate that some-
time in the past there may have been another epizootic, a
possibility that is supported by the observation that the two
positive animals (15 and 20 years of age) were among the
oldest of the animals in the 1986 sample.
Timing of infection in relation to weather. Most rainfall
at Polonnaruwa normally occurs between October and Jan-
uary due to inter-monsoonal convectional rains in October
and the northeast monsoon from November to January (Ta-
ble 4). February is normally dry with clear skies. The onset
of the virus infection among the macaques at Polonnaruwa
after September 1986 and its cessation before February 1987
coincided with the rainy season. The fact that transmission
took place during the rainy season is consistent with the
virus being transmitted by a mosquito vector.
Analysis of camp animals. The tests performed with sera
from macaques living within the study site at Polonnaruwa
indicated that almost all the animals were exposed to a den-
gue virus towards the end of 1986. Was this a focal event
restricted to animals at the study site, or did the 1986 epi-
zootic represent an island-wide incident? To examine the
geographic extent of the epizootic, toque macaque sera col-
lected in 1986 and in 1995 from animals at a site 5 km from
the study site and separated from the study site by cultivation
(Figure 1, camp site) were tested for dengue virus antibody.
These animals have not been extensively studied and bio-
graphic data including the dates of birth were not available.
The ages of these animals was estimated using morphologic
criteria as previously described.
Two seropositive older an-
imals and two seronegative younger animals from the study
site were also tested in this experiment as controls. None of
the animals trapped at the camp site in 1986 were positive.
More importantly, none of the animals trapped in 1995,
many of whom were estimated to be more than nine years
of age, were positive for dengue virus antibody. This result
was in sharp contrast to the data obtained for animals at the
study site where nearly all the older animals were positive.
These data indicate that the 1986–1987 epizootic was focal
in nature and restricted to animals living in the immediate
proximity of the Polonnaruwa study site.
This study has provided serologic evidence for an epizo-
otic dengue virus among toque macaques in Sri Lanka. One
must be cautious when using serologic data to identify Fla-
viviruses because antibodies in response to Flavivirus infec-
tions are notorious for cross-reacting with other Flaviviruses.
However, the available data suggest that the macaque virus
is a dengue virus. First, while four Flavivirus antigens (den-
gue, Japanese encephalitis, Zika, and yellow fever viruses)
were tested in the current study, the highest signal was ob-
served consistently with the dengue antigen. Second, and
more importantly, in the previous study by Pieris and others,
they performed neutralization tests and observed that all the
positive sera neutralized dengue-2 virus (64 of 64), while
very few sera neutralized JE (3 of 64) or West Nile (1 of
Based on these results, it is reasonable to des-
ignate the macaque virus as a dengue virus. The unambig-
uous identiﬁcation of the agent is only possible once the
virus has been isolated from macaques. Unfortunately, at-
tempts to isolate the virus from macaque sera have so far
proven unsuccessful. As in humans, dengue is an acute in-
fection in monkeys and the virus does not persist in pri-
The failure to isolate virus from the sera is not sur-
prising since none of the samples were collected during the
actual epizootic between October 1986 and February 1987.
In the previous study by Pieris and others, a smaller sam-
ple of the same population of macaques was tested in 1987.
These investigators concluded that the macaques were grad-
ually exposed to the virus over time and that by six years
of age more than 90% of the animals had seroconverted.
These results were consistent with a highly active, enzootic
virus. The present study with blood samples collected from
the same population eight years later (1995), as well as blood
samples collected in 1986 and 1987, has revealed that this
is an epizootic virus characterized by outbreaks infecting
almost the entire population of macaques within at least a 3
area followed by prolonged (at least eight years for the
Polonnaruwa population) periods with no transmission. The
high seroprevalence observed by Pieris and others was due
to the fact that the population was sampled in 1987 imme-
diately after a major epizootic.
Although the virus was highly infectious to macaques, the
animals did not appear to have serious illness. These animals
have been intensively studied and no increased mortality or
obvious signs of morbidity were observed by ﬁeld assistants
during the period of the epizootic (Dittus WPJ, unpublished
data). These ﬁndings are consistent with the agent being a
dengue virus since previous ﬁeld and laboratory studies have
documented that infected monkeys are mainly asymptomatic
DENGUE EPIZOOTIC AMONG MACAQUES IN SRI LANKA
although they do develop viremia and can serve as a reser-
The current study has raised the following important ques-
tions. What is the origin of this virus and how is the agent
maintained in Sri Lanka? Over a large part of the Amazon,
yellow fever virus moves in waves through monkey popu-
lations leading to epizootics in particular regions with a pe-
riodicity of 8–10 years.
In Senegal, epizootics of dengue
among monkeys were observed in 1974 and again in 1981.
The toque macaque is found in many forested areas of the
island, excluding the climatic extremes of arid and cold. Un-
like the rhesus macaque (M. mulatta) of India, however, it
does not cohabit with people in towns. It is conceivable that
this virus may sweep through the island infecting a troop of
macaques and then moving on to the next troop as immunity
builds up in the original group. A troop that has been in-
fected may again become susceptible to an epizootic as the
number of naive individuals increase in the population. The
population at the study site at Polonnaruwa may be poised
for another epizootic since the majority (79%) of the animals
tested in 1995 did not show evidence of exposure to this
When considering the reservoirs of the macaque virus, it
is important to consider other vertebrates, especially pri-
mates living on the island. In addition to toque macaques,
two other species of monkeys (Semnopithecus entellus and
Trachypithecus vetellus) and one species of prosimian, the
slender loris (Loris tardigradus), are found at the study site.
In areas of sympatry, the langurs are more common than the
The virus may be enzootic among one of these
other primates and the toque macaques may simply serve as
an amplifying host. However, if the virus is enzootic in an-
other species at the study site, it is difﬁcult to understand
why there was no evidence of infection among any of the
susceptible macaques over the eight-year period between
1987 and 1995. When considering the origin of the macaque
virus, it is important not to forget humans, the most abundant
primate on the island and a known reservoir of dengue vi-
ruses. It is possible that the epizootic in 1986 was due to
cross-over of a human dengue virus to the macaque popu-
lation. Future efforts will be directed towards isolating the
virus from macaques for unambiguous identiﬁcation and to
determine the relationship of the macaque virus to dengue
viruses infecting humans in Sri Lanka.
Acknowledgments: During this study we received support from col-
leagues in Sri Lanka and the United States. Foremost, we thank Dr.
Rebeca Rico-Hesse for advice and support throughout the project.
We thank Shirley Tirrell-Peck of the Yale Arbovirus Research Unit
for guidance at various stages of the project. In Sri Lanka, we thank
Taya Dias, Wanaja Dittus, Sunil Goonatilake, Gayan Gunewardane,
Vajira Hettige, Don Melnick, Beatrice Sweeney-Perez, Anna Pethi-
yagoda, S. P. Ranasinghe, and Jennifer Wilcox for technical assis-
tance. Aravinda de Silva thanks Amy Weil for encouragement.
Financial support: Field research was supported by grants to Wolf-
gang P. J. Dittus from the National Science Foundation (BNS-
9104649, 9510894), the Harry and Frank Guggenheim Foundation,
Friends of the National Zoo, and Earthwatch. The laboratory studies
were supported by U.S. Army grant DAMD17-94-J-4004.
Authors’ addresses: Aravinda M. de Silva, Department of Microbi-
ology and Immunology, University of North Carolina, Campus Box
7290, Chapel Hill, NC 27599. Wolfgang P. J. Dittus, Institute of
Fundamental Studies, Hantana Road, Kandy, Sri Lanka. Priyani H.
Amerasinghe and Felix P. Amerasinghe, Department of Zoology,
Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka.
Reprint requests: Aravinda M. de Silva, Department of Microbiology
and Immunology, University of North Carolina, Campus Box 7290,
Chapel Hill, NC 27599.
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