JOURNAL OF VIROLOGY, May 2004, p. 4700–4709
0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.9.4700–4709.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 9
Simian T-Cell Leukemia Virus (STLV) Infection in Wild Primate
Populations in Cameroon: Evidence for Dual STLV Type 1
and Type 3 Infection in Agile Mangabeys
(Cercocebus agilis)
Valerie Courgnaud,1 Sonia Van Dooren,2 Florian Liegeois,1 Xavier Pourrut,1
Bernadette Abela,1,3 Severin Loul,3 Eitel Mpoudi-Ngole,3
Annemieke Vandamme,2 Eric Delaporte,1
and Martine Peeters1*
UR36, IRD, and University of Montpellier I, Montpellier, France1; Department of Clinical and Epidemiological
Virology, Rega Institute for Medical Research, Leuven, Belgium2; and PRESICA, Yaounde, Cameroon3
Received 29 September 2003/Accepted 17 December 2003
Three types of human T-cell leukemia virus (HTLV)-simian T-cell leukemia virus (STLV) (collectively called
primate T-cell leukemia viruses [PTLVs]) have been characterized, with evidence for zoonotic origin from
primates for HTLV type 1 (HTLV-1) and HTLV-2 in Africa. To assess human exposure to STLVs in western
Central Africa, we screened for STLV infection in primates hunted in the rain forests of Cameroon. Blood was
obtained from 524 animals representing 18 different species. All the animals were wild caught between 1999
and 2002; 328 animals were sampled as bush meat and 196 were pets. Overall, 59 (11.2%) of the primates had
antibodies cross-reacting with HTLV-1 and/or HTLV-2 antigens; HTLV-1 infection was confirmed in 37
animals, HTLV-2 infection was confirmed in 9, dual HTLV-1 and HTLV-2 infection was confirmed in 10, and
results for 3 animals were indeterminate. Prevalences of infection were significantly lower in pets than in bush
meat, 1.5 versus 17.0%, respectively. Discriminatory PCRs identified STLV-1, STLV-3, and STLV-1 and
STLV-3 in HTLV-1-, HTLV-2-, and HTLV-1- and HTLV-2-cross-reactive samples, respectively. We identified
for the first time STLV-1 sequences in mustached monkeys (Cercopithecus cephus), talapoins (Miopithecus
ogouensis), and gorillas (Gorilla gorilla) and confirmed STLV-1 infection in mandrills, African green monkeys,
agile mangabeys, and crested mona and greater spot-nosed monkeys. STLV-1 long terminal repeat (LTR) and
env sequences revealed that the strains belonged to different PTLV-1 subtypes. A high prevalence of PTLV
infection was observed among agile mangabeys (Cercocebus agilis); 89% of bush meat was infected with STLV.
Cocirculation of STLV-1 and STLV-3 and STLV-1-STLV-3 coinfections were identified among the agile
mangabeys. Phylogenetic analyses of partial LTR sequences indicated that the agile mangabey STLV-3 strains
were more related to the STLV-3 CTO604 strain isolated from a red-capped mangabey (Cercocebus torquatus)
from Cameroon than to the STLV-3 PH969 strain from an Eritrean baboon or the PPA-F3 strain from a
baboon in Senegal. Our study documents for the first time that (i) a substantial proportion of wild-living
monkeys in Cameroon is STLV infected, (ii) STLV-1 and STLV-3 cocirculate in the same primate species, (iii)
coinfection with STLV-1 and STLV-3 occurs in agile mangabeys, and (iv) humans are exposed to different
STLV-1 and STLV-3 subtypes through handling primates as bush meat.
baboons, African green monkeys, guenons, mangabeys, orangutans, and chimpanzees, whereas STLV-2 has been identified
only in captive bonobos (Pan paniscus) from the Democratic
Republic of Congo (12, 13, 15, 24, 29, 40). The close relationship between HTLV-1 and STLV-1 suggests a simian origin for
HTLV-1. Moreover, phylogenetic analyses of African HTLV-1
and STLV-1 strains revealed that some HTLV-1 strains are
more closely related to STLV-1, suggesting the occurrence of
multiple cross-species transmissions between primates and humans and also between different primate species (18).
Similar to HTLV, other simian retroviruses such as human
immunodeficiency virus type 1 (HIV-1) and HIV-2 are of zoonotic origin, with their closest simian relatives in the common
chimpanzee (Pan troglodytes) and the sooty mangabey (Cercocebus atys), respectively (6, 11). First recognized in the early
1980s, HIV-1 has spread to most parts of the world, and today
it is estimated that more than 40 million individuals live with
HIV infection or AIDS (34). HTLV is less pathogenic than
Simian T-cell leukemia viruses (STLVs) are the simian
counterparts of human T-cell leukemia viruses (HTLV), and
these viruses are collectively called primate T-cell leukemia
viruses (PTLVs). HTLVs are separated into two serologically
and genetically distinct types, HTLV type 1 (HTLV-1) and
HTLV-2, and both types have simian counterparts, STLV-1
and STLV-2 (8, 36, 37). A third type, STLV-3, was isolated
from several African nonhuman primates such as hamadryas
baboons (Papio hamadryas) from east and west Africa and
red-capped mangabeys (Cercocebus torquatus) and greater
spot-nosed monkeys (Cercopithecus nictitans) from Cameroon
(22, 23, 35, 39). STLV-1 has been isolated from a wide variety
of Old World monkeys in Asia and Africa, including macaques,
* Corresponding author. Mailing address: Laboratoire Retrovirus,
UR036, IRD, 911 Avenue Agropolis, BP 64 501, 34394 Montpellier
Cdx 1, France. Phone: 33-4 67 41 62 97. Fax: 33-4 67 41 61 46. E-mail:
martine.peeters@mpl.ird.fr.
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VOL. 78, 2004
STLV INFECTION IN WILD PRIMATES
HIV, but HTLV-1 is known to be associated with lymphoma,
leukemia (adult T-cell leukemia), and some neurological disorders such as tropical spastic paraparesis (9, 17, 33).
Given that humans come in frequent contact with primates
in many parts of sub-Saharan Africa, the possibility of additional zoonotic transfers of retroviruses from primates has to
be considered. Prevalences of HTLV infection are high in
Africa, with the highest values in equatorial Africa and more
precisely in the tropical forest region (5). In central Africa,
prevalences of HTLV-1 infection increase with age, and in
rural areas women and pygmies are more frequently infected
(4, 21). All these epidemiological observations, together with
the phylogenetic relationships between HTLV and STLV, are
in favor of zoonotic transmissions. The risk for acquiring such
infections is expected to be the highest in individuals who hunt
primates and who prepare their meat for consumption, as well
as in people who keep primates as pet animals. Therefore, it is
important to study the prevalence, diversity, and geographic
distribution of these infections in wild primate populations. In
a similar way, it was previously shown that humans are exposed
to a plethora of simian immunodeficiency viruses in Cameroon
(26).
In the present study, we tested wild-caught primates from
Cameroon for STLV infection. Cameroon is known to harbor
a diverse set of primate species which are extensively hunted
for food and trade at various levels (3). Our study indicates
that in addition to frequent contamination with simian immunodeficiency virus, a considerable proportion of primate meat
sold for consumption is contaminated with STLV-1 and
STLV-3. These data also provide an approximation of the
magnitude of exposure and the variety of STLVs to which
humans are exposed and permit estimation of the prevalences
of STLV infection in wild primate populations in Cameroon.
In addition, our study further documents coinfection with
STLV-1 and STLV-3 in wild primate populations.
MATERIALS AND METHODS
Collection of primate tissue and blood samples. Blood was obtained from 524
monkeys all wild caught in Cameroon between January 1999 and July 2002.
Species were determined by visual inspection according to The Kingdon Field
Guide to African Mammals (14) and by use of the taxonomy described by Colin
Groves (10). Three hundred twenty-eight animals were sampled as bush meat
upon arrival at markets in Yaounde, the capital city, in surrounding villages, or
at logging concessions in southeastern Cameroon; the other 196 animals sampled
were pets from these same areas. Table 1 summarizes the numbers of each
primate species collected. All primate samples were obtained with government
approval from the Cameroonian Ministry of Environment and Forestry. The
bush meat samples were obtained by employing a strategy specifically designed
not to increase the demand for bush meat, i.e., women preparing and preserving
the meat for subsequent sale and hunters already involved in the trade were
asked for permission to sample blood and tissues from carcasses which were then
returned to their owners; animals and bush meat confiscated by the national
program against poaching were also sampled. For the bush meat animals, blood
was collected by cardiac puncture. Information provided by the owners indicated
that most of the animals had died 12 to 72 h prior to sampling. For the pet
monkeys, blood was drawn by peripheral venipuncture after the animals were
tranquilized with ketamine (10 mg/kg). Plasma and cells were separated on site
by Ficoll gradient centrifugation. All samples, including peripheral blood mononuclear cells, plasma, whole blood, and other tissues, were stored at ⫺20°C.
Serology. Plasma or whole blood samples were tested for the presence of
HTLV-cross-reactive antibodies by using a commercially available enzymelinked immunosorbent assay (ELISA), the MUREX HTLV-I⫹II test (Abbott
Laboratories, Wiesbaden, Germany), using as antigens synthetic peptides and
recombinant proteins representing immunodominant regions of the envelope
4701
and transmembrane regions of HTLV-1 and HTLV-2. Samples reactive in the
ELISA were retested with a commercially available line immunoassay, INNOLIA HTLV I/II (Innogenetics, Ghent, Belgium), which discriminates between
HTLV-1- and HTLV-2-cross-reactive antibodies. This test configuration includes
HTLV-1 and HTLV-2 recombinant proteins and synthetic peptides that are
coated as discrete lines on a nylon strip. The antigenicity exhibited by these
proteins and peptides is either common to HTLV-1 and HTLV-2 or specific to
one of the two viruses to allow confirmation and discrimination in a single assay.
Two Gag (p19-I or p19-II and p24-I or p24-II) and two Env (gp46-I or gp46-II
and gp21-I or gp21-II) bands are applied as non-type-specific antigens, which are
used to confirm the presence of antibodies against HTLV-1 and HTLV-2. The
type-specific antigens for HTLV-1 (Gag p19-I and Env gp46-I) and HTLV-2
(Env gp46-II) are applied to differentiate between HTLV-1 and HTLV-2 infections. In addition to these HTLV antigens, control lines are present on each strip:
one sample addition line (3⫹) containing anti-human immunoglobulin (Ig) and
two test performance lines (1⫹ and ⫹/⫺) containing human IgG. Values represent reaction intensity. All assays were performed and interpreted according to
the manufacturer’s instructions.
PCR. DNA was isolated from whole blood or peripheral blood mononuclear
cells using Qiagen DNA extraction kits (Qiagen, Courtaboeuf, France). To
confirm the presence of PTLVs in samples with HTLV-cross-reactive antibodies,
a previously described diagnostic tax-rex PCR allowing generic as well as typespecific detection of PTLVs was done (38). The generic PCR proved to be highly
sensitive in detecting PTLV strains, and the discriminatory PCRs had high
sensitivities and specificities.
For a subset of STLV-1- and STLV-3-positive samples, we also sequenced part
of env and/or the long terminal repeat (LTR). For STLV-1, the complete LTR
(755 bp) was amplified with a combination of previously described primers (20).
A 522-bp region of the env gene, coding for most of gp21 and part of the
carboxyl-terminal region of gp46, was amplified and sequenced with previously
described primers (20). For STLV-3, a 540-bp fragment in the LTR region was
amplified with a combination of previously described and newly designed primers: AV51 (38) and pX-LTRAS (5⬘-TTTATAGGACCCAGGGTTCTT-3⬘ [positions 8450 to 8470 in PH969]) for the first round and pX-LTRS (5⬘-CRGGC
ACACRGGYCTACTCCC-3⬘ [positions 7932 to 7952 in PH969]) and pXLTRAS for the second round. R represents A or G; Y represents C or T. PCRs
for both rounds were performed using the Expand High Fidelity PCR kit (Roche
Molecular Biochemicals, Mannheim, Germany) and included a hot start (94°C
for 2 min) with the following cycle conditions: 10 cycles of denaturation at 94°C
for 15 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min followed by
25 cycles with extension at 72°C for 1 min in the first cycle and for times
increasing by an increment of 5 s per cycle thereafter. Amplification was completed by a final extension at 72°C for 7 min. PCR products were sequenced using
cycle sequencing and dye terminator methodologies (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq FS DNA polymerase [PE Biosystems, Warrington, England]) on an automated sequencer
(ABI 373, stretch model; Applied Biosystems) either directly or following cloning
into the pGEM-T vector (Promega, Lyon, France).
To test for DNA degradation, a 1,151-bp region of the glucose-6-phosphate
dehydrogenase gene was amplified using the primers GPD-F1 (5⬘-CATTACCA
GCTCCATGACCAGGAC-3⬘) and GPD-R1 (5⬘-GTGTTCCCAGGTGACCCT
CTGGC-3⬘) in a single-round PCR with the following conditions: 94°C for 2 min
and then 35 cycles at 94°C for 20 s, 58°C for 30 s, and 72°C for 1 min (26).
Phylogenetic analyses. Newly derived STLV nucleotide sequences were
aligned with reference sequences from GenBank using CLUSTAL W (32) with
minor manual adjustments. Gaps in the alignment were omitted from further
analyses. The STLV-1 LTR and env phylogenetic trees were constructed using
the neighbor-joining (NJ) method and/or the maximum likelihood (ML) method
with the Tamura Nei substitution model using PAUP*4.0b10 software (30). The
reliability of branching orders was tested using the bootstrap approach (1,000
replicates) for the NJ tree, whereas P values were obtained for the ML tree with
the zero branch length test. The STLV-3 LTR (463 nucleotides) and PTLV tax
(180 nucleotides) phylogenies were investigated with the software package
PAUP* version 4.0b10 (30). NJ and ML trees were constructed under the most
appropriate evolutionary model tested with MODELTEST 3-06 (27). For the
LTR and tax sequences, the transitional model and transversional model, respectively, each allowing five different substitution rate categories including
gamma distribution rate heterogeneity, provided the best fit to the data. The NJ
trees were constructed by iterative optimization of the model parameters, followed by a bootstrap analysis of 1,000 replicates. The ML trees were constructed
by starting from the NJ tree with optimized parameters by using a heuristic
search with the nearest-neighbor interchange and the subtree-pruning-regrafting
3 (0.6)
10 (1.9)
9 (1.7)
37 (7.1)
524
3 (0.9)
10 (3.0)
9 (2.7)
34 (10.3)
328
0
0
0
0
1
0
0
0
0
0
0
2
0
0
0
0
0
0
2
196
Total (%)
3 (1.5)
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
2
0
1
2
0
0
0
2
40
1
16
14
16
19
31
5
0
0
0
2
35
0
0
11
0
18
6
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
5
1
16
3
16
1
25
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1
0
0
2
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
7
0
0
0
0
0
0
2
0
0
0
0
0
17
1
5
2
15
17
21
6
5
38
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
48
1
12
0
1
0
104
1
8
80
24
0
0
10
0
0
2
0
0
65
2
17
2
16
17
125
7
13
118
24
0
0
0
3
0
1
0
0
2
7
0
0
0
0
0
0
0
0
2
10
0
0
0
0
0
0
0
0
0
J. VIROL.
Agile managabey
Red-capped mangabey
Grey-cheeked mangabey
Drill
Mandrill
Olive baboon
Mustached monkey
Mona monkey
de Brazza’s monkey
Greater spot-nosed
monkey
Cercopithecus pogonias Crested mona
Cercopithecus preussi
Preuss’s monkey
Chlorocebus tantalus
Tantalus monkey
Miopithecus ogouensis Gabon talapoin
Erytrocebus patas
Patas monkey
Colobus guereza
Mantled guereza
Pan troglodytes
Chimpanzee
Gorilla gorilla
Gorilla
Common name
Species
Cercocebus agilis
Cercocebus torquatus
Lophocebus albigena
Mandrillus leucophaeus
Mandrillas sphinx
Papio anubis
Cercopithecus cephus
Cercopithecus mona
Cercopithecus neglectus
Cercopithecus nictitans
HTLV-1
No. of bush meat animals positive for:
Total no. of animals positive for:
Total
no. of
HTLV-1 Indeterminate animals
HTLV-1 Indeterminate
HTLV-2
and
and
tested HTLV-1 HTLV-2
HTLV
HTLV
HTLV-2
HTLV-2
COURGNAUD ET AL.
No. of pet animals positive for:
No. of
No. of
pet
bush meat
HTLV-1 Indeterminate
animals
animals
and
tested HTLV-1 HTLV-2
tested
HTLV
HTLV-2
TABLE 1. Detection of HTLV-1- and HTLV-2-cross-reactive antibodies in primate species in Cameroon
4702
branch-swapping algorithm (28). Additionally, the P values were estimated for
the branches of the ML trees with the branch length confidence test.
Nucleotide sequence accession numbers. The new sequences have been deposited in GenBank under the following accession numbers: AY496626 to
AY496638 (LTR from STLV-1), AY496596 to AY496606 (env from STLV-1),
AY496607 to AY496618 (tax from STLV-1), AY496588 to AY496595 (tax from
STLV-3), and AY496619 to AY496625(LTR from STLV-3).
RESULTS
Estimates of prevalence of STLV infection in bush meat and
pet monkey samples. Blood specimens were obtained from a
total of 524 nonhuman primates representing 18 different species. All the animals were wild caught in Cameroon. Whole
blood was collected from 328 animals that were sold as bush
meat at markets in the capital city of Yaounde, in nearby
villages, and at logging concessions in southeastern Cameroon.
The great majority (86.4%) of these animals were adults. We
also collected blood from 196 pet primates, most of which
(74.4%) were still infants or juveniles at the time of sampling.
Most primates originated from the southern part of the country.
In order to detect PTLV infection in nonhuman primates,
we used commercially available HTLV-1–HTLV-2 assays since
all previously reported STLV infections were also identified
through cross-reactivity with HTLV antigens. A total of 59
(11.2%) of 524 samples tested reacted strongly in the HTLV1-HTLV-2 ELISA (Table 1). All ELISA reactive samples were
retested with the INNO-LIA HTLV I/II confirmatory assay,
and the results are summarized in Table 1. Among the 59
samples, 37 were confirmed as HTLV-1 positive, 9 were confirmed as HTLV-2 positive, 10 were confirmed as HTLV-1 and
HTLV-2 positive, and results for 3 were indeterminate. Figure
1 illustrates the kind of INNO-LIA reactivities that were typically observed. Overall, HTLV-cross-reactive antibodies were
detected in 9 of the 18 primate species tested, and the prevalences of seroreactivity (positive or indeterminate results) in
the different species ranged from 0.8 to 66%. Moreover, we
identified for the first time STLV sequences in talapoins, mustached monkeys, and gorillas. As expected, prevalences were
significantly lower in pet animals, which were mainly infants or
juveniles, than in bush meat primates, which were predominantly adult animals (1.5 versus 16.9%, respectively; data not
shown). Surprisingly, corresponding to differences in species,
extreme differences in prevalences of HTLV-cross-reactive antibodies were observed: up to 89% of agile mangabey bush
meat was infected with STLV, whereas only 0.96% of mustached monkeys were infected. For the majority of the 9 out of
18 primate species without HTLV antibodies, the numbers of
adult animals tested were low, which may explain the lack of
reactivity. For example, we did not observe a positive reaction
in samples from red-capped mangabeys (Cercocebus torquatus)
and mona monkeys (Cercopithecus mona), although STLV-3
and STLV-1 infections, respectively, were previously documented in these species (23, 24). Interestingly, we observed 10
agile mangabeys with antibodies that cross-reacted with
HTLV-1- and HTLV-2-specific antigens.
Confirmation of STLV infection by confirmatory and discriminatory PCR analysis of the tax gene. In order to confirm
whether animals with HTLV-cross-reactive antibodies were
infected with a PTLV and to determine with which type of
VOL. 78, 2004
STLV INFECTION IN WILD PRIMATES
4703
FIG. 1. Detection of HTLV-1- and HTLV-2-cross-reactive antibodies in sera from agile mangabeys (Cercocebus agilis) by using a line
immunoassay (INNO-LIA HTLV confirmation; Innogenetics). The HTLV antigens include recombinant proteins and synthetic peptides which are
either common to HTLV-1 and HTLV-2 or specific to one of the two viruses. The first three control lines contain human IgG in different
concentrations and are followed by four confirmation lines (two gag and two env HTLV-1 and HTLV-2 antigen lines) and three discriminatory lines
(two gag HTLV-1 peptides and one env HTLV-2 peptide) at the bottom of the strip. Plasma samples from HTLV-1- and HTLV-2-negative and
-positive individuals are shown as controls on the left. Lanes labeled 01-CM1040 and 01-CM1129 represent STLV-1-seropostive Cercocebus agilis;
lane 01-CM1053 represents STLV-3-seropositive Cercocebus agilis, and lanes 01-CM1038 and 01-CM1106 represent STLV-1- and STLV-3seropositive Cercocebus agilis. Lane 01-CM1003 represents an example of plasma with indeterminate serology.
PTLV, we performed PCR using highly cross-reactive tax
primer pairs previously shown to amplify sequences from a
variety of divergent HTLV and STLV strains and known to
have a high specificity in characterizing the PTLV type. Among
the 59 samples with a positive or indeterminate serology, 41
samples for which sufficient additional material was available
were tested by generic PCR followed by type-specific PCR to
discriminate between STLV-1, STLV-2, and STLV-3. Among
the 41 samples, 24 were positive for HTLV-1, 9 were positive
for HTLV-2, 5 were positive for HTLV-1 and HTLV-2, and 3
had an indeterminate serology in the INNO-LIA HTLV I/II
assay. The two HTLV-2-positive samples from greater spotnosed monkeys have been previously described and were identified as being infected with a new SLTV-3 variant (39). The tax
PCR results are summarized in Table 2. Among the 24 HTLV1-positive samples, 5 were negative by PCR, 18 were positive
for STLV-1, and in 1 sample (from animal 01CM-1135)
STLV-1 and STLV-3 were detected. This dual infection was
confirmed by sequence analysis of the tax fragments. All seven
HTLV-2-seropositive samples were reactive with the STLV-3specific tax primers only, which was confirmed by sequence
analysis of four samples (Fig. 2). Among the five samples
reactive with HTLV-1- and HTLV-2-specific antigens in the
line immunoassay, three were determined to carry both
STLV-1 and STLV-3, and the remaining two were found to
carry only HTLV-1. In two of the three samples with indeterminate HTLV serology, no viruses could be amplified with the
generic primers, and in the remaining sample, STLV-3 was
present and confirmed by sequence analysis of the tax fragment.
Overall, we confirmed the presence of STLVs in eight of the
nine primate species in which we observed HTLV-cross-reactive antibodies. Only among chimpanzees, where we observed
one animal with indeterminate HTLV serology, could no
STLV infection be demonstrated by PCR. All samples which
were identified as HTLV-2 positive by serology were in fact
infected with an STLV-3 strain. More interestingly, we observed that one primate species can be infected with two different STLV types; more precisely, STLV-1 and STLV-3 infections were observed in agile mangabeys and in greater spotnosed monkeys. In addition, we showed that the same animal
can be infected with both viruses at the same time. We identified four agile mangabeys (animals 01CM-1038, 01CM-1122,
01CM-1135, and 01CM-1272) that were coinfected with
STLV-1 and STLV-3. Figure 2 shows the phylogenetic tree
analysis of the tax sequences and encompasses results of the
diagnostic and discriminatory tax PCRs for a subset of samples.
It is clear from this figure that the previously reported tax
STLV-3 sequences from greater spot-nosed monkeys formed a
distinct, well-supported (90% bootstrap support for NJ; P of
⬍0.05 for ML) cluster within the STLV-3 group. All other
STLV-3 strains clustered together but separately from the
greater spot-nosed monkey STLV-3 strains, with a reasonable
bootstrap support for NJ (86%) and statistical support for ML
(P ⬍ 0.05). The further clustering pattern among these eastern,
western, and central African STLV-3 strains was more or less
according to geographic origins of the STLV host species. Due
to the low genetic diversity in tax and the shortness of the
fragment, however, the topology among these STLV-3 strains
was not well supported. Based on this 180-bp fragment, five
4704
COURGNAUD ET AL.
J. VIROL.
TABLE 2. PTLV confirmation and discrimination by generic and type-specific tax PCR for HTLV-1 and HTLV-2 antibody cross-reactive
samples
Species
Cercocebus agilis
Cercopithecus nictitans
Cercopithecus pogonias
Chlorocebus tantalus
Miopithecus ogouensis
Mandrillus sphinx
Cercopithecus cephus
Pan troglodytes
Gorilla gorilla
Subtotal
No. of
samples
tested
STLV-1
STLV-3
STLV-1 and
STLV-3
HTLV-1
HTLV-2
HTLV-1 and HTLV-2
Indeterminate HTLV
HTLV-1
HTLV-2
HTLV-1
HTLV-1
HTLV-1
HTLV-1
HTLV-1
Indeterminate HTLV
HTLV-1
14
7
5
2
2
2
2
1
1
1
1
1
2
8
0
2
0
2
0
2
1
1
1
1
0
2
0
7
0
1
0
2a
0
0
0
0
0
0
0
1
0
3
0
0
0
0
0
0
0
0
0
0
5
0
0
1
0
0
0
0
0
0
0
1
0
HTLV-1
HTLV-2
HTLV-1 and HTLV-2
Indeterminate HTLV
24
9
5
3
16
0
2
0
0
9
0
1
1
0
3
0
5
0
0
2
41
20
10
4
7
Total
a
No. of samples positive by tax PCR for:
Virus identification by
INNO-LIA HTLVI/II
No. of samples negative
by tax PCR
Previously reported to be infected with STLV-3.
sequences from agile mangabey virus strains were even identical to sequences from the previously described strains from
red-capped mangabeys (CTO602 and CTO604) (23). The
STLV-1 strains clustered with other African HTLV-1 and
STLV-1 strains, but the support here was also rather low.
Moreover, we identified for the first time STLV sequences in
talapoins, mustached monkeys, and gorillas. Therefore, we further investigated longer fragments from these STLV-1 and
STLV-3 strains in more divergent gene regions such as the
LTR and/or env.
env and LTR sequence analysis of STLV-1 strains obtained
from different primate species. The complete STLV-1 LTR
was sequenced for 10 STLV-1-infected and 3 STLV-1- and
STLV-3-coinfected animals. The 10 STLV-1-infected animals
were representatives of the following primate species: agile
mangabeys (four), mustached monkeys (one), crested monas
(one), mandrills (two), and talapoins (two). The three coinfected animals (01CM-1038, 01CM-1122, and 01CM-1272)
were all agile mangabeys. Figure 3 shows the phylogenetic tree
analyses of the LTRs and env. Phylogenetic tree analyses of the
new sequences together with previously published STLV and
HTLV sequences representing the different HTLV-1-STLV-1
subtypes using both NJ and ML revealed that all sequences
from agile mangabeys were closely related to one another (98.9
to 100% identity) and to a previously published STLV-1 sequence obtained from an agile mangabey (25) (99.3 to 99.7%
identity) captured in southeast Cameroon (NJ, bootstrap values of 54 and 100%; ML, P of ⬍0.001 and 0.008). The STLV-1
LTR sequence from the only mustached monkey also clustered
with the STLV-1 sequences from agile mangabeys (99.1 to
99.3% identity) from the same area in Cameroon (25) (NJ,
bootstrap value of 61%; ML, P of ⬍0.001). The LTR sequences from STLVs obtained from agile mangabeys and mustached monkeys clustered with the sequence from subtype F
identified in an individual from Gabon (96 to 96.6% identity
with the Lib2 sequence) with 86% bootstrap support for NJ
and a P value of ⬍0.001 for ML. The STLV-1 sequences from
the mandrills from our study and from the crested mona clustered with high support values with sequences from the central
African subtype D in both trees (96% identity between sequences from strains 1228 and H23 and 99.5% identity between sequences from strains ML4 and H23). The sequences
obtained from talapoins clustered with those of STLVs from
western Africa and western central Africa (NJ, bootstrap value
of 60%; ML, P of ⬍0.001). The STLV-1 strain obtained from
a gorilla clustered with HTLV-1 subtype B (98.2% identity
with H24; NJ, bootstrap value of 82%; ML, P of ⬍0.001).
Partial env sequences were also obtained from the abovedescribed samples except from that from the mustached monkey. Phylogenetic tree analysis of the env sequences showed
clustering patterns similar to those determined by analysis of
the LTR region.
LTR sequence analysis of STLV-3 strains obtained from
STLV-3-infected and STLV-1- and STLV-3-coinfected agile
mangabeys. Fragments of 540 bp comprising the LTR regions
of STLV-3 strains were obtained from four STLV-3-infected
and three STLV-1- and STLV-3-coinfected animals. All the
new STLV-3 sequences were closely related to one another
(97.8 to 100% identity) and were also most closely related to an
STLV-3 sequence (97.8 to 99.6% identity) obtained from a
recently described red-capped mangabey from Cameroon (23).
Even with this limited number of STLV-3 LTR sequences
available in the GenBank database, we observed a tendency
toward STLV-3 clustering according to the geographic origins
of the viral host species. Similar to those of STLV-1, STLV-3
sequences from coinfected animals did not form a separate
subcluster. For the STLV-3-infected greater spot-nosed monkeys, for which the tax sequences were previously reported, we
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STLV INFECTION IN WILD PRIMATES
4705
FIG. 2. PAUP* NJ tree of a 219-bp tax-rex fragment including sequences from reference strains of each PTLV type and subtype with the
bootstrap values (in percentages) and P values (**, P ⬍ 0.001; *, P ⬍ 0.05) noted on the branches.
were not able to amplify the LTR fragment due to the degraded nature of the DNA (39). Figure 4 shows the phylogenetic tree analysis of the STLV-3 LTR sequences.
DISCUSSION
The majority of previous studies of STLV infection have
relied almost exclusively on surveys of captive monkeys or apes
that were either kept as pets or housed at zoos or primate
centers. The great majority of pet monkeys are acquired at a
very young age, often when their parents are killed by hunters.
STLV infection rates of captive monkeys may thus not accurately reflect STLV infection prevalence rates in the wild.
Phylogenetic tree analysis of HTLV and STLV strains
showed that zoonotic transfers of STLV to humans have occurred on several occasions (37), but no study has examined
the prevalences of STLV infection among African primates
that are frequently hunted or kept as pets. In this paper, we
collected blood from 524 monkeys representing 18 different
species. All of the animals were wild caught in the rain forests
of Cameroon and sampled as either bush meat or pet animals.
This approach allowed us simultaneously to identify STLV
infection prevalence rates in wild primate populations and to
determine to what extent humans are exposed to STLVs. We
detected cross-reactive antibodies suggesting PTLV infection
in 11% of all tested animals. STLV infection was confirmed by
PCR in 8 of the 18 species tested, and phylogenetic analyses
revealed the presence of STLVs clustering in different PTLV
types. We confirmed STLV-1 infection in three species previously identified as STLV carriers by serology only, namely,
mustached monkeys (Cercopithecus cephus), talapoins (Miopithecus ogouensis), and gorillas (Gorilla gorilla). We showed
also for the first time the presence of STLV-3 infection in agile
mangabeys (Cercocebus agilis), and even coinfection with
STLV-1 and STLV-3 was observed in this primate species.
Our data reveal for the first time that a considerable proportion of wild-living primates in Cameroon are infected with
STLV and that these primates may be a source of infection to
those who come in contact with them. Although new STLVinfected host species were identified and new STLV variants
were characterized, it is likely that our data represent only
minimal estimates concerning STLV prevalences and STLV
diversity in Cameroon because not all native primate species
were tested and many were undersampled because they were
either rare or absent in the regions of Cameroon where we
sampled for this study. For example, the absence of reactive
sera from mona monkeys (Cercopithecus mona) and redcapped mangabeys (Cercocebus torquatus), two species known
to harbor STLV, must be due to the low numbers of blood
samples analyzed (23, 24).
4706
COURGNAUD ET AL.
J. VIROL.
FIG. 3. Phylogenetic relationships among new STLV-1 strains from Cercocebus agilis, Cercopithecus cephus, Gorilla gorilla, Mandrillus sphinx,
Miopithecus ogouensis, and Cercopithecus pogonias and known STLV-1 and HTLV-1 strains from the different subtypes. Phylogenetic relationships
were determined using LTR (A) and env (B) sequences as described in Materials and Methods. The numbers along the branches are the bootstrap
values (in percentages), and two asterisks indicate that the branch has a P value of ⬍0.001 in the ML analysis. Horizontal branch lengths are drawn
to scale.
Similar to that with HIV, human infection with HTLV-1 and
HTLV-2 most likely resulted from cutaneous or mucous membrane exposure to infected blood during the hunting and
butchering of STLV-infected primates for food or from bites
from STLV-infected pet animals. Although no HTLV-3 infection is yet described in humans, our study shows that humans
are exposed to STLV-3-infected primate bush meat from
greater spot-nosed monkeys and agile mangabeys and possibly
other, not-yet-identified STLV-3-harboring primate species.
Samples from STLV-3-infected animals either reacted with
HTLV-2 antigens in the INNO-LIA assay or had an indeterminate HTLV serology. It will thus be important to genetically
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STLV INFECTION IN WILD PRIMATES
4707
FIG. 3—Continued.
characterize human samples with HTLV-2 or indeterminate
HTLV serology to study whether STLV-3 cross-species transmission between primates and humans has already occurred
and, if so, whether this infection is associated with any disease
in humans. Indeterminate HTLV Western blot patterns are
frequently observed in central Africa, and although a majority
of such patterns may be due to other environmental (viral or
paretic) factors, the possibility for HTLV-3 infection has to be
further explored (19). Bush meat hunting, to provide animal
proteins for the family and a source of income, has a longstanding tradition throughout sub-Saharan Africa (1, 7). How-
ever, the bush meat trade has increased in the last decades.
Commercial logging, together with road construction into remote forest areas, led to human migration and the development of social and economic networks in previously inaccessible forest areas. Villages around logging concessions have
grown from a few hundred to several thousand inhabitants in
just a few years (2, 41). These socioeconomic changes suggest
that the magnitude of human exposure to primates infected
with STLV has increased, as have the social and environmental
conditions that would be expected to support the emergence of
new zoonotic infections with STLVs.
4708
COURGNAUD ET AL.
J. VIROL.
FIG. 4. Phylogenetic relationships among new STLV-3 strains from Cercocebus agilis and known STLV-3 strains from different primate species.
Phylogenetic relationships were determined using LTR sequences as described in Materials and Methods. The bootstrap values (in percentages)
and P values (**, P ⬍ 0.001; *, P ⬍ 0.05) are indicated on the branches.
Our study shows clearly that significantly more adult monkeys than juveniles are infected (1.5% prevalence in pets versus 16.9% in bush meat samples), thus suggesting a low vertical
transmission rate and confirming that estimates of the prevalence of STLV infection have to be made using adult animals.
We also observed extreme differences in prevalence rates
among different primate species. We tested large numbers of
greater spot-nosed and mustached monkeys, but only a few
animals were determined to be positive. In contrast, more than
80% of adult agile mangabeys were infected with a PTLV, and
even STLV-1 and STLV-3 infections and STLV-1-STLV-3
coinfections were observed among these animals in the wild.
Another study among wild primate populations in Ethiopia
also revealed discrepancies among STLV infection prevalences
among different baboon species (31). STLV-3 and STLV-1
infections were observed, and one hybrid baboon was positive
by STLV-1- and STLV-L-specific PCR, suggesting a dual infection (31). It is known for HTLV infection in humans that
geographic and/or intrafamilial clusters with high prevalences
of infection exist (16). It has to be further investigated whether
the high prevalences observed in certain monkeys are specific
for the species or whether, similar to those among humans,
geographic clusters also exist among nonhuman primates.
Therefore, additional prevalence studies of STLV infections
among wild primate populations have to be done in other
regions of Cameroon and Africa.
In conclusion, our study shows that humans are exposed to
a large variety of STLV-1 and STLV-3 strains. Further studies
are needed to determine whether zoonotic transmissions of
STLVs, especially STLV-3, from primates has occurred. In
order to understand the evolution of PTLVs, it will be important to identify and compare STLVs from primate species from
western, central, and eastern Africa, as well as to determine
STLV infection prevalences among wild primate populations.
These studies will allow us to understand the origin, evolution,
and spread of these viruses into different primate and human
populations.
ACKNOWLEDGMENTS
We thank the Cameroonian Ministries of Health and of Environment and Forestry for permission to perform this study and the staff
from project PRESICA for logistical support and assistance in the
field.
This work was supported in part by grants from the National Institutes of Health (RO1 AI 50529), the Agence National de Recherche
sur le SIDA (ANRS), and the Fonds voor Wetenschappelijk Onderzoek (grant 0288.01).
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