Park, Côte d'Ivoire. J. Virol. 84(15):7427-7436.
) in Taï National Piliocolobus badius badiusMonkeys (
Diversity of Retroviruses in Wild Red Colobus
2010. High Prevalence, Coinfection Rate, and Genetic
Hedemann, et al.
Siv Aina J. Leendertz, Sandra Junglen, Claudia
) in Taï National Park, Côtebadius
Piliocolobus badius Monkeys (
Retroviruses in Wild Red Colobus
and Genetic Diversity of
High Prevalence, Coinfection Rate,
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JOURNAL OF VIROLOGY, Aug. 2010, p. 7427–7436
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 15
High Prevalence, Coinfection Rate, and Genetic Diversity
of Retroviruses in Wild Red Colobus Monkeys
(Piliocolobus badius badius) in Taï
National Park, Co ˆte d’Ivoire?
Siv Aina J. Leendertz,1,2,3Sandra Junglen,1† Claudia Hedemann,1Adeelia Goffe,1
Sebastien Calvignac,1Christophe Boesch,2Fabian H. Leendertz1,2*
Research Group Emerging Zoonoses, Robert Koch Institut, Nordufer 20, 13353 Berlin, Germany1; Department of Primatology,
Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, 04103 Leipzig, Germany2; Center for Epidemiology and
Biostatistics, Norwegian School of Veterinary Science, P.O. Box 8146 Dep, N-0033 Oslo, Norway3
Received 31 March 2010/Accepted 7 May 2010
Simian retroviruses are precursors of all human retroviral pathogens. However, little is known about the
prevalence and coinfection rates or the genetic diversity of major retroviruses—simian immunodeficiency virus
(SIV), simian T-cell lymphotropic virus type 1 (STLV-1), and simian foamy virus (SFV)—in wild populations
of nonhuman primates. Such information would contribute to the understanding of the natural history of
retroviruses in various host species. Here, we estimate these parameters for wild West African red colobus
monkeys (Piliocolobus badius badius) in the Taï National Park, Co ˆte d’Ivoire. We collected samples from a total
of 54 red colobus monkeys; samples consisted of blood and/or internal organs from 22 monkeys and addition-
ally muscle and other tissue samples from another 32 monkeys. PCR analyses revealed a high prevalence of
SIV, STLV-1, and SFV in this population, with rates of 82%, 50%, and 86%, respectively. Forty-five percent of
the monkeys were coinfected with all three viruses while another 32% were coinfected with SIV in combination
with either STLV or SFV. As expected, phylogenetic analyses showed a host-specific pattern for SIV and SFV
strains. In contrast, STLV-1 strains appeared to be distributed in genetically distinct and distant clades, which
are unique to the Taï forest and include strains previously described from wild chimpanzees in the same area.
The high prevalence of all three retroviral infections in P. b. badius represents a source of infection to
chimpanzees and possibly to humans, who hunt them.
Lentiviruses and deltaretroviruses that infect African non-
human primates have received considerable attention as they
are the precursors of all pathogenic human retroviruses: hu-
man immunodeficiency virus types 1 and 2 (HIV-1/HIV-2) and
human T-cell lymphotropic virus type 1 (HTLV-1). These hu-
man infections are the results of past zoonotic transfers of
simian immunodeficiency virus (SIV) and simian T-cell lym-
photropic viruses type 1 (STLV-1) from wild monkeys and
apes into local human populations, presumably through pri-
mate hunting and handling of primate bushmeat (13, 19, 43, 46,
55, 58, 59). Via the same route, zoonotic transmission of simian
foamy virus (SFV), a spumaretrovirus whose exact pathogenic-
ity in human hosts is still unknown, has also been shown (64).
The increasing contact between humans and wild primates
implies that further zoonotic transmission of retroviruses is
likely to happen (42, 63). Studying the occurrence and circu-
lation of simian retroviruses such as SIV, STLV-1, and SFV in
wild primate populations enables us to better understand ret-
rovirus evolution in primates and also provides tools for mon-
itoring possible future retroviral zoonotic events.
Systematic studies of SIV, STLV-1, and SFV in wild pri-
mates are relatively rare. Many use bushmeat samples, which
can vary in their quality and are prone to cross-contamination
from butchering and storage with other carcasses. Confiscated
primates are also not representative of the situation in the wild
since the animals are caught at a young age when the occur-
rence of different retroviruses may be extremely low (24). The
technical possibilities for the detection of various pathogens in
noninvasive samples such as urine and feces have greatly im-
proved and are frequently used; however, in general, the sen-
sitivity of detection methods is higher when blood and tissue
samples are used (25, 32, 47). Such samples can be collected if
fresh carcasses are found, or they can be collected by anesthe-
tizing live primates for sampling purpose, animal translocation,
or medical intervention, such as snare removal. The practical
and ethical issues of each of the sampling methods have been
discussed elsewhere (12, 14).
Red colobus monkeys [Procolobus (Piliocolobus)] are inter-
esting subjects for retroviral infection studies for a number of
reasons. First, they are widely distributed (yet in a fragmented
manner) from East to West Africa, which suggests that red
colobus species and subspecies, or more likely ancestor(s) of
these, could have been key hosts in transmitting retroviruses
across tropical Africa (4, 54). Second, as they are herbivore
primates, the hunting of other primates can be excluded as a
route of infection. Finally, these monkeys are frequently
hunted by humans and chimpanzees and represent a possibly
* Corresponding author. Mailing address: Robert Koch Institut,
Research Group Emerging Zoonoses, Nordufer 20, 13353 Berlin,
Germany. Phone: 49 30187542592. Fax: 49 30187542605. E-mail:
† Present address: Institute of Virology, University of Bonn Medical
Centre, Sigmund Freud Strasse 25, 53105 Bonn, Germany.
?Published ahead of print on 19 May 2010.
at ROBERT KOCH-INSTITUT July 8, 2010
large reservoir for retroviruses and other pathogens that ought
to be investigated further (2, 45).
Very little information is available about the prevalence and
coinfection of SIV, STLV-1, and SFV in wild red colobus
monkeys across Africa. In other colobine monkeys only SIV
has been documented: in olive colobus (Procolobus verus) in
Co ˆte d’Ivoire and in black and white colobus (Colobus guereza)
in Cameroon (7, 8). Based on fecal samples from habituated
adult individuals, the prevalence of SIV in West African red
colobus monkeys (SIVwrc; local subspecies, Piliocolobus ba-
dius badius) has been estimated to a minimum of 26% in the
Taï National Park, Co ˆte d’Ivoire, but the authors recognized
the low sensitivity of viral RNA detection in fecal samples (34).
Another study conducted on the same population revealed that
5 out of 10 blood samples were SIV positive (7). These results
highlight that the most reliable prevalence data are based on
analyses of blood/tissue samples although such sampling is not
always feasible for reasons discussed above. Published preva-
lence information concerning STLV-1 and SFV in wild red
colobus monkeys (STLV-1wrc and SFVwrc) in the same area is
restricted to results obtained from analyses of a limited num-
ber of blood and necropsy samples collected as a part of studies
whose focus was on cross-species transmission of these two
viruses to chimpanzees (27, 28). However, these samples indi-
cated a high prevalence of STLV-1wrc and SFVwrc in the red
colobus monkey population (56% and 90%, respectively). A
recent study from Uganda, East Africa, estimated the preva-
lence of SIV, STLV-1, and SFV in another red colobus species
(Piliocolobus rufomitratus tephrosceles) to be 22.6%, 6.4%, and
97%, respectively (15). The study was performed using blood
samples collected from anesthetized wild red colobus monkeys
living in their natural habitat, which allowed reliable assess-
ment of the prevalence and genetic diversity of these three
The preliminary data from the Taï National Park indicate
that there might be great variation in the prevalence of retro-
viruses across the African continent, even in closely related
species of wild primates. Here, we aimed at generating reliable
prevalence and coinfection data for SIVwrc, STLV-1wrc, and
SFVwrc based on the analysis of blood and tissue samples from
wild Western red colobus monkeys. We expected that this
would allow for proper comparison of retroviral prevalence in
the allied species P. b. badius and P. r. tephrosceles.
MATERIALS AND METHODS
Study site and animals. Field work was conducted in the evergreen rainforest
of the Taï National Park in Co ˆte d’Ivoire, West Africa (5°15? to 6°07?N, 7°25? to
7°54?W). The West African red colobus monkey (P. b. badius) is the most
abundant primate species in this forest and shares its habitat with eight other
diurnal primates: chimpanzee (Pan troglodytes verus), sooty mangabey (Cercoce-
bus atys), black-and-white colobus (Colobus polykomos), olive colobus (P. verus),
Diana monkey (Cercopithecus diana), lesser spot-nosed monkey (Cercopithecus
petaurista), Campbell’s monkey (Cercopithecus campbelli), and greater spot-
nosed monkey (Cercopithecus nictitans) (36). Samples were collected from wild
nonhabituated West African red colobus monkeys within an area of approxi-
mately 100 km2.
Sample collection. Blood samples were collected from 10 adult red colobus
monkeys under general anesthesia (28). At the same time other biological sam-
ples and anatomical measurements were collected. The samples were centrifuged
shortly after collection, and the cell-rich layer (buffy coat) was frozen immedi-
ately in liquid nitrogen. As part of a chimpanzee health monitoring project,
veterinarians have been performing necropsies on all carcasses of any species
found in the forest. Since the year 2001 necropsy samples have been collected
from 12 adult red colobus monkey carcasses, and these were transported on ice
directly to the camp. Chimpanzees in the area regularly hunt red colobus (2) and
sometimes leave behind pieces of muscle or other tissues from their prey. The
veterinary project collected such tissue samples from observed hunts of 32 indi-
vidual red colobus monkeys as soon as the chimpanzees had left the site. These
samples were collected using single-use gloves, transported at ambient temper-
ature, and preserved at camp a maximum of 12 h after the death of the monkey.
For the necropsies and chimpanzee meal remains, multiple samples were col-
lected if possible, and up to three samples per individual were analyzed. All
samples were stored in liquid nitrogen at the field site and later transported on
dry ice to Robert Koch Institute, Berlin, Germany, where samples were stored at
?80°C until analysis. All parts of the study were performed under permission of
the Ministry of Research and the National Park authorities of Co ˆte d’Ivoire.
DNA extraction, PCR, and sequencing. DNA was extracted using either a
DNA tissue kit or DNA blood kit (Qiagen, Hilden, Germany).
Samples were tested for SIV with a seminested PCR with primers specifically
designed for the detection of pol regions of SIV from the Western red colobus/
olive colobus (SIVwrc/SIVolc) group (SIVwrc S1 [CAT GGC AAA TGG ATT
GTA CTC A], SIVwrc R2 [GTG CCA TTG CTA ATG CTG TTT C], SIVwrc
S3 [CCA AAT TCT TGT TCT ATC CCT AAC C], and SIVwrc R3 [AGC AAA
AAT CAT ATC AGC AGA AGA T]). These primers were based on SIVwrc and
SIVolc sequences published by Courgnaud and colleagues (7). We used the
primers SIVwrc S1 and SIVwrc R2 in the first round PCR, and the primer pair
SIVwrc S1 and SIVwrc R3 (expected amplicon size approximately 250 bp) and
the pair SIVwrc S3 and SIVwrc R2 (expected amplicon size approximately 300
bp) were used in two parallel seminested PCRs. The cycler conditions were 94°C
for 5 min and 30 cycles of 94°C for 15 s, 55°C for 30 s, 72°C for 30 s, with a final
step of 72°C for 10 min and then cooling to 4°C. SIVwrc-negative samples and
one sample from each SIVwrc-positive individual were also tested with a generic
SIV PCR known to detect most primate lentiviruses to determine if the monkeys
also carried other types of SIV (6). We used the primers DR1 (TRC AYA CAG
GRG CWG AYG A) and DR2 (AIA DRT CAT CCA TRT AYT G) in the first
round PCR and primers DR4 (GGI ATW CCI CAY CCD GCA GG) and DR5
(GGI GAY CCY TTC CAY CCY TGH GG) in a nested PCR. The cycler
conditions were 94°C for 2 min and 30 cycles of 94°C for 15 s, 50°C decreasing
by 0.5°C each cycle to 35°C for 30 s, and 72°C for 1 min; this was followed by 15
cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 1 min, with a final step at 72°C
for 5 min and then cooling to 4°C. The expected amplicon size was 194 bp.
Samples were tested for proviral DNA of STLV-1 by a tax-specific real-time
PCR (23). We used the primers SK43 (CGG ATA CCC AGT CTA CGT GT)
and SK44 (GAG CCG ATA ACG CGT CCA TCG) and the probe HTLV TAX
TM (6FAM-CGC CCT ATG GCC ACC TGT CCA GA XT P; 6FAM is 6-car-
boxyfluorescein), and the cycler conditions were 95°C for 10 min and 45 cycles of
95°C for 15 s and 60°C for 35 s. The expected amplicon size was approximately
190 bp. A fragment of the long terminal repeat (LTR) region was then sequenced
from positive samples as this region of the primate T-cell lymphotropic virus type
1 (PTLV-1) genome evolves more rapidly and is frequently used for phylogenetic
analyses. We used the primers S10, H, and X (26, 27) derived from nucleotides
7929 to 7948, 8756 to 8735, and 8296 to 8316, respectively, from the prototype
HTLV-1 sequence ATK (accession number J02029) (48). We used primer S10
(GGC CCT AAT AAT TCT ACC CG) and primer H (AGT TCA GGA GGC
ACC ACA GGC G) for the first round and primer X (GAG CTC GAG CAG
ATG ACA ATG ACC ATG AG) and primer H in a seminested PCR. The cycler
conditions were 94°C for 5 min and 35 cycles of 94°C for 30 s, 58°C for 30 s, and
72°C for 1 min (30 s for seminested PCR), with a final step at 72°C for 10 min and
then cooling to 4°C. The expected amplicon size for the seminested PCR was
Samples were tested for SFV with a PCR specifically designed to amplify a
fragment of SFVwrc pol (28). We used the primers SFVwrc 1s (CAT ACA ATT
ACC ACT CCA AGC CT), SFVwrc 2as (CAG ACA AAT CCA GTC ATA
CCA TC), SFVwrc 3s (CTC AGT ACT GGT GGC CAA ATC TTA GA), and
SFVwrc 4as (CCA GTC ATA CCA TCG ACT ACT ACA AGG). In the
first-round PCR we used primers SFVwrc 1s and SFVwrc 2as, and then for two
parallel seminested PCRs we used primers SFVwrc 1s and SFVwrc 4as (expected
amplicon size of approximately 430 bp) and primers SFVwrc 3s and SFVwrc 2as
(expected amplicon size of approximately 270 bp); the cycler conditions were
96°C for 5 min and 40 cycles of 96°C for 1 min, 56°C for 30 s, and 72°C for 1 min,
with a final step at 72°C for 10 min and then cooling to 4°C. One sample from
each individual was also tested with a generic SFV PCR (15) to check for the
presence of other non-red colobus strains of SFV. We used the primers SIF2
(TAG CWG AYA ARC TTG CCA CCC AAG G) and SIR1 (GTC GTT TWA
TIT CAC TAT TTT TCC TTT CCA C) in the first round and the primers SIF3
7428LEENDERTZ ET AL.J. VIROL.
at ROBERT KOCH-INSTITUT July 8, 2010
(CCA ARC CTG GAT GCA GAG YTG GAT CA) and SIR3 (ACT TTG GGG
RTG RTA AGG AGT ACT G) in a nested PCR. The cycler conditions were
95°C for 5 min and 40 cycles of 95°C for 30 s, 45°C for 45 s, and 72°C for 1 min,
with a final step at 72°C for 10 min and then cooling to 4°C. The expected
amplicon size for was approximately 630 bp.
PCR products were visualized with gel electrophoresis before being purified.
Gel extraction was performed when necessary. Sequencing was performed in
both directions using the Sanger method, with all PCR products being sequenced
on both strands. Comparison to the public database using NCBI BLAST (1)
always confirmed that the expected proviral sequences had been amplified.
Prevalence and coinfection of SIV, STLV-1, and SFV and correlation between
infections. The prevalence of the three retroviruses was calculated in Stata
(Stata/SE, version 10.0, for Windows; Stata Corp., College Station TX), as well
as the corresponding 95% confidence interval (CI) for proportions (normal
approximation). We calculated the percentage of monkeys with single infections
of the individual viruses, and in order to investigate if infections were linked to
each other, we also calculated the percentage of individual monkeys with dual
(for all possible viral combinations) or triple infections. Kendall tau-b test,
including Fisher’s exact test, was used to determine the degree of correlation
between the infections. Prevalence and coinfection of SIV, STLV-1, and SFV
and the correlation between infections were calculated on basis of the results
obtained from buffy coat samples and necropsy samples only. The samples
collected after chimpanzee meals were considered not to be of sufficient quality
to be included in these analyses. These samples, however, were used to obtain
additional nucleotide sequences for the phylogenetic analyses.
Sequence analyses. Newly generated sequences were added to data sets con-
sidered to encompass the overall genetic diversity of SIV, STLV-1, and SFV.
Alignments were edited manually using SeaView, version 4 (16). To allow de-
tection of saturation, the number of transitions and transversions versus diver-
gence was first plotted in DAMBE (65). If distances calculated under the global
time reversible (GTR) model are chosen as a measure of divergence, then both
transitions (ts) and transversions (tv) should increase in a linear manner with
GTR distance. In general, ts should also accumulate faster than tv. However,
with GTR distance increasing, multiple substitutions are expected to occur at the
same sites, ultimately leading to the loss of the initial correlation. Saturation
should thus translate into plateauing plots, with tv outnumbering ts (30). While
the PTLV-1 data set was apparently exempt from saturation, the SIV and SFV
data sets exhibited clear patterns of ts saturation at the third positions of codons.
Accordingly, we performed all following analyses on SIV and SFV data sets
stripped of this position (a conservative approach). All three data sets were
haplotyped (reduced to unique sequences) using FaBox (60). The overall process
resulted in our starting data sets being composed as follows: (i) SIV pol, 75 taxa
and 152 bp; (ii) PTLV-1 LTR, 47 taxa and 422 bp; and (iii) SFV pol, 51 taxa and
Nucleotide substitution models to obtain the best fit for the data were then
selected using jModeltest, version 0.1.1 (17, 44). According to the Akaike infor-
mation criterion (AIC), comparisons of model likelihoods were most favorable
to GTR?I?G (GTR with a proportion of invariant sites [I] and gamma-distrib-
uted [G] rate heterogeneity) (SIV), Hasegawa Kishino and Yano (HKY)?I?G
(PTLV-1), and GTR?G (SFV). Phylogenetic analyses were performed in both
maximum-likelihood (ML) and Bayesian frameworks, under the appropriate
model of nucleotide substitution. ML analyses were performed on the PhyML
webserver (http://www.atgc-montpellier.fr/phyml/) (17, 18). Equilibrium frequen-
cies, topology, and branch lengths were optimized; the starting tree was deter-
mined using BioNJ and both nearest-neighbor interchange (NNI) and subtree
pruning and regrafting (SPR) algorithms of the tree search were used (keeping
the best outcome). Branch robustness was assessed by performing nonparametric
bootstrapping (500 replicates). Bayesian analyses were performed using BEAST,
version 1.5.3 (11). Besides allowing modeling nucleotide substitution processes,
BEAST also allows for modeling rate variation among tree branches and tree
shape. All analyses were run under the assumption of a relaxed, uncorrelated
log-normal clock. For SIV and SFV data sets, analyses were performed assuming
two different tree shape speciation models (Yule and birth-death processes).
Given the expected relatively shallow depth of the PTLV-1 phylogenetic tree, a
speciation model (birth-death process) and a coalescent model (constant popu-
lation size) were employed. Two runs of 10,000,000 generations were run per
data set per tree shape model (i.e., four runs total for each data set). Trees and
numerical values taken were sampled every 1,000 generations. Tracer, version
1.5, was used to check that individual runs had reached convergence, that inde-
pendent runs converged, and that chain mixing was satisfactory (effective sample
size values of ?200) (11). Trees sampled in duplicate runs were then gathered
into a single file (after removal of a visually conservative 10% burn-in period)
using LogCombiner, version 1.5.3 (distributed with BEAST), and the informa-
tion of 18,000 trees per data set per tree shape model was summarized onto the
maximum clade credibility tree using TreeAnnotator, version 1.5.3 (distributed
with BEAST). Posterior probabilities (pps) were taken as a measure of branch
For Bayesian analyses, no major discrepancy in topology or branch support
was detectable using different tree shape models. ML and Bayesian methods
globally supported congruent topologies with consistent branch supports (even
though bootstrap and posterior probability are not directly comparable ). All
xml files (including sequence alignments) used for Bayesian analyses are avail-
able at http://sebastiencalvignac.fr/emergingzoonoses/index.html. Figures sum-
marizing phylogenetic analyses were drawn using FigTree, version 1.3.1 (http:
Nucleotide sequence accession numbers. All sequences generated in this study
were deposited in GenBank under the accession numbers FN825787 to
FN825803 and FN859997 to FN860025.
Prevalence of SIV, STLV-1, and SFV. All three viruses were
detected by specific PCR and confirmed with sequencing and
BLAST. Further, all newly sequenced SIV, STLV-1, and SFV
strains could be linked to strains previously found in red colo-
bus monkeys. Results are summarized in Table 1. SIV1wrc was
detected in 8 out of 10 anesthetized monkeys and in 10 out of
12 carcasses. The overall prevalence was 82% (95% CI, 66 to
98%). STLV-1wrc was detected in six anesthetized monkeys
and in five carcasses. The overall prevalence was 50% (95% CI,
29 to 71%). SFVwrc was detected in all anesthetized monkeys
and in nine carcasses. The overall prevalence was 86% (95%
CI, 72 to 100%). In the samples from chimpanzee meal re-
mains (attributed to 32 red colobus monkeys), SIV, STLV-1,
and SFV were detected in five, nine, and six individuals, re-
SIV, SFV, and STLV-1 coinfections and correlation between
infections. Forty-five percent (n ? 10) of the monkeys were
coinfected with all three viruses, 27% (n ? 6) were infected
with SIV in combination with SFV, and 5% (n ? 1) were
infected with SIV in combination with STLV-1. Fourteen per-
cent (n ? 3) were infected with SFV only, and 5% (n ? 1) were
infected with SIV only. Five percent (n ? 1) were negative for
all viruses. Of note, no monkey was infected with STLV-1 only
or with the virus combination STLV-1 and SFV. There was no
statistically significant correlation between any of the infec-
tions (Kendall tau-b coefficient and Fisher exact P value of 0.47
and 0.09 for SIV/STLV-1, 0.16 and 0.47 for SIV/SFV, and 0.13
and 1.00 for STLV-1/SFV, respectively).
Phylogenetic analyses. To infer the phylogenetic relation-
ships of the newly described SIV, STLV-1, and SFV strains
with previously characterized strains, we used ML and Bayes-
ian methods. The main pattern of SIV host specificity was
retrieved by these analyses (Fig. 1). SIVwrc sequences from P.
b. badius were found to form a clade with SIVwrc strains
previously identified from P. b. badius from the same area and
Piliocolobus badius temminckii from Gambia, the branch de-
fining the bipartition receiving reasonable statistical support
(bootstrap value [Bp], 57; pp, 0.99) (Fig. 1). The sister taxon to
this group appeared to be the strain identified from Kibale P.
r. tephrosceles, which also grouped with other SIVwrc with
reasonable statistical support (Bp of 66; pp of 0.97) (Fig. 1).
SIVolc, a strain identified from an olive colobus monkey [Pro-
colobus (Procolobus) verus] appeared as completing a big colo-
bine clade in the ML analysis (Bp of 58) but was found to
VOL. 84, 2010 RETROVIRUSES IN WESTERN RED COLOBUS MONKEYS7429
at ROBERT KOCH-INSTITUT July 8, 2010
group with the clade comprised of SIV in L’Hoest’s monkey
(Cercopithecus lhoesti) and the sun-tailed guenon (Cercopithe-
cus solatus) (SIVlho/sun) in Bayesian analyses, though with
very low branch support (pp of 0.57).
The general design of PTLV-1 strains clustering into geo-
graphical subtypes was retrieved by the analyses whose results
are summarized in Fig. 2. Into that scheme, our STLV-1wrc
sequences from P. b. badius did not form one monophyletic
group. Sequences were distributed in three distinct clades,
corresponding to subtype I or nested into subtype J (Fig. 2), as
defined by Junglen et al. (21). In all cases, these clades were
well supported and comprised chimpanzee strains (Bp of 98 to
99; pp of 1) (Fig. 2). The P. r. tephrosceles strain did not exhibit
a close relationship to any of the STLV-1wrc strains found in
the Taï forest, nor did it show particular affinity to any of the
previously described PTLV-1 subtypes (Fig. 2).
SFV phylogeny exhibited the expected pattern of marked
host specificity together with plausible long-term cospeciation
(Fig. 3). SFVwrc from P. b. badius respected that rule, forming
a monophyletic group supported by reasonable Bp and pp
values (Bp of 54; pp of 1) (Fig. 3). P. r. tephrosceles strains
clustered together with high support (Bp of 79; pp of 1) (Fig.
3). The existence of a colobine clade was also reasonably sup-
ported (Bp of 54: pp of 1) though its inner branching order
could not be determined (Fig. 3).
Prevalence of SIV, STLV-1, and SFV. Most data on retrovi-
ruses in wild primates are derived from studies based on bush-
meat samples or confiscated and captive populations and might
therefore not be representative for the situation in the wild.
Therefore, the main data we use here for comparison are
based on a study by Goldberg et al. (15) using samples ob-
tained from wild primates under anesthesia.
We estimate that the prevalence of SIVwrc in P. b. badius in
TABLE 1. Overview of red colobus PCR results for SIV, STLV-1, and SFVa
Remaining 19 animals
Spleen, lymph node
Spleen, lymph node
Spleen, lung, muscle
Lung, unidentified tissue
Spleen, kidney, muscle
Lymph node, intestine
Muscle, bone marrow
Blood in RNA later
Trachea, lymph node
Muscle, bone marrow
Muscle, bone marrow
Muscle (7), blood (7), other tissue (7)d
aFor SIV and SFV two different primer sets were used; where results agree only one result appears in the table.
bCMR, chimpanzee meal remain.
cSamples were tested with specific primers for red colobus virus (wrc) and with generic primers for virus.
dNumbers in parentheses are numbers of samples tested.
ePartial result; no material was available to test four individuals with generic primers for SIV.
fPartial result; no material was available to test five individuals with generic primers for SFV.
7430 LEENDERTZ ET AL.J. VIROL.
at ROBERT KOCH-INSTITUT July 8, 2010
Taï National Park in Cote d’Ivoire is 82%. This is somewhat
higher than previously estimated for this population (26%
based on noninvasive samples from 53 individuals and 50%
based on 10 blood samples), which could be due to differences
in test material and sample sizes (7, 35). However, our results
confirm that this population has one of the highest prevalences
of this virus found in wild nonhuman primates to date. Further,
the prevalence in P. r. badius is more than three times higher
than that in the closely related species of red colobus monkey,
P. r. tephrosceles, living in Kibale, Uganda. The fact that there
is no overlap in the 95% CI of the estimated SIV prevalence in
the Kibale study and that in the present study (8 to 37% and 66
to 98% for Kibale and Taï, respectively) shows that there is a
significant difference between these two populations/species
(15). Previous studies have shown that SIV prevalence varies
greatly between species and that some populations, such as
mandrills in Cameroon and sooty mangabeys in Co ˆte d’Ivoire,
have a high frequency of SIV infection (estimated prevalence
of 79% [95% CI, 54 to 99%] and 59% [95% CI, 35 to 88],
respectively) (46, 51, 56). The effect that the high rate of SIV
occurrence might have on the P. b. badius population is un-
known. In general, natural SIV infections have been consid-
FIG. 1. Maximum-likelihood tree based on the analysis of SIV partial pol sequences (152 bp). The topologies of Bayesian maximum clade
credibility trees obtained under two different tree priors were similar when shallow evolutionary depths were considered (deep branching patterns
were not similar). Branches leading to strains isolated from red colobus monkeys in Co ˆte d’Ivoire are blue, those leading to strains isolated from
Ugandan red colobus monkeys are red, and the one branch leading to a Gambian strain is green. Major SIV groups are represented graphically
to improve readability: in every case the number of strains represented is indicated in parentheses after group names. Numbers above branches
represent bootstrap values (Bp); italicized numbers below branches represent posterior probability values (pp) obtained using the birth-death
model. Bp and pp are indicated only where Bp is ?50 and pp is ?0.95. Asterisks indicate strains identified in the present study. Note that this tree
is mid-point rooted due to the lack of information regarding the position of the root in SIV phylogeny. Tal, talapoin monkey (Miopithecus talapoin);
den, Dent’s monkey (Cercopithecus denti); deb, De Brazza’s guenon (Cercopithcus neglectus); syk, Sykes’monkey (Cercopithecus mitis); mon, mona
monkey (Cercopithecus mona); gsn, greater spot-nosed guenon (Cercopithecus nictitans); mus, mustached guenon (Cercopithecus cephus); asc,
red-tailed guenon (Cercopithecus ascanius); col, mantled guereza (Colobus guereza); agm, African green monkey (Chlorocebus aethiops); cpz,
chimpanzee (Pan troglodytes); rcm, red-capped mangabey (Cercocebus torquatus); smm, sooty mangabey monkey (Cercocebus atys); lho, L’Hoest’s
monkey (Cercopithecus lhoesti); sun, sun-tailed guenon (Cercopithecus solatus); mnd, mandrill (Mandrillus sphinx); drl, drill (Mandrillus leucopha-
eus); olc, olive colobus monkey (Procolobus verus); wrc, Western red colobus (P. b. badius); krc, Eastern red colobus (P. r. tephrosceles); wrcPBT,
Western red colobus (P. b. temminckii).
VOL. 84, 2010 RETROVIRUSES IN WESTERN RED COLOBUS MONKEYS7431
at ROBERT KOCH-INSTITUT July 8, 2010
ered nonpathogenic and even asymptomatic throughout the
course of infection (49). However, it has recently been discov-
ered that SIV in wild chimpanzees (SIVcpz) has a negative effect
on health and fertility and causes AIDS-like immunopathology in
possible that a similar effect will be found in other primate species
when additional prospective, long-term follow-up studies of SIV-
infected wild primates become available. It should be underlined
that this type of study requires continuous investigation, and the
discovery of SIV pathogenicity in the Gombe chimpanzees was
only possible because good demographic data had been obtained
from a long-term field study.
Also for STLV-1 we found that the prevalence is much
higher in P. b. badius than in P. r. tephrosceles monkeys, with no
overlap found in the 95% CI: 50% in P. b. badius (95% CI, 29
to 71%) and 6.4% in P. r. tephrosceles (95% CI, 0 to 15%) (15).
It appears that there is a great deal of variation in the esti-
mated prevalence of STLV-1 in wild primates, ranging from 0
to 89%, depending on species and region (9, 25, 37, 50). In-
terestingly, with the comparisons possible from our study, it is
striking to observe such extreme differences also between
closely related primate species. This probably means that both
genetic diversity and retroviral prevalence are determined by
geographical location for STLV-1s. However, further studies
on additional populations are needed to adequately assess if
the prevalence in P. b. badius is unusually high or if that of P.
r. tephrosceles is extraordinarily low.
In contrast to SIV and STLV-1, there was not much differ-
ence in the prevalences of SFV in our study (86%; 95% CI, 72
to 100%) and in P. r. tephrosceles (97%; 95% CI, 90 to 100%)
FIG. 2. Maximum-likelihood tree based on the analysis of PTLV-1 partial LTR sequences (422 bp). The topologies of Bayesian maximum clade
credibility trees obtained under two different tree priors were similar when shallow evolutionary depths were considered (deep branching patterns
were not similar). Branches leading to strains isolated from red colobus monkeys in Co ˆte d’Ivoire are blue, and those leading to strains isolated
from Ugandan red colobus monkeys are red. Major PTLV-1 groups are represented graphically to improve readability: in every case the number
of strains represented is indicated in parentheses after group names. Numbers above branches represent Bp values, italicized numbers below
branches represent pp values obtained using the birth-death model. Bp and pp are indicated only where Bp is ?50 and pp is ?0.95. Asterisks
indicate strains identified in the present study. Three of these strains were actually identified in more than one individual: wrc15 (published as
STLVwrc ) is identical to wrc129* and wrc212* (plus ptr-Dorry); wrc66* is identical to wrc72*; and wrc126* is identical to wrc211*. Sm, sooty
mangabey monkey (Cercocebus atys); ptr, chimpanzee (Pan troglodytes); mnd, mandrill (Mandrillus sphinx); cae, African green monkey (Chloro-
cebus aethiops); msy, Barbary macaque (Macaca sylvanus); wrc, Western red colobus (P. b. badius); krc, Eastern red colobus P. r. tephrosceles).
7432LEENDERTZ ET AL.J. VIROL.
at ROBERT KOCH-INSTITUT July 8, 2010
(15). High SFV prevalence has also been found in other wild
primate populations, where infections can reach 100% (3, 33).
The fact that this generally high prevalence is unique to SFV
(compared to SIV and STLV-1) might be explained by a lesser
sensitivity to the behavioral differences that exist between spe-
cies and even populations.
The results from our study, as well as those from the red
colobus study in Uganda, are based on relatively small sample
sizes (15). However, samples from wild red colobus monkeys
and primates in general are difficult to obtain, and one should
not refrain from discussing possible reasons for differences in
viral prevalence between populations, based on the data we
have to date. Virus biology can be one possible reason for the
observed difference in SIV and STLV-1 prevalences since
these viruses in the Taï and Kibale populations are all distinct.
Behavioral differences should also be considered although no
further demographic data are available for our study animals,
and the routes of retroviral transmission in wild primates are
not fully understood (20, 38). In general, the intense social
behavior of red colobus monkeys could give the opportunity
for frequent retrovirus transmission. The monkeys live in pro-
miscuous multimale groups of about 50 individuals, the males
fight each other to mate receptive females throughout their
adult lives, and frequent aggressive harassment of mating cou-
ples occurs (36, 52). There are, however, behavioral differences
between the red colobus monkey populations/species found in
Taï and Kibale that might explain, at least partly, the difference
in the prevalences of SIV and STLV-1 at these sites. First, the
Taï P. b. badius population has a defined breeding season of 5
to 6 months every year, whereas the Kibale P. r. tephrosceles
population breeds all year round (A. Korstjens, personal com-
munication). During the intense breeding season in Taï, the
receptive females mate with virtually all males available. In
Kibale, there are receptive females available all year round,
and the average number of males in the group is lower than in
Taï (3.5 versus 10), which makes monopoly by dominating
males easier (36, 40, 52). This means that the P. b. badius
females in Taï overall mate with a larger number of partners,
which is a risk factor in the spread of sexually transmitted
diseases (41). Second, although a precise comparison is diffi-
FIG. 3. Maximum-likelihood tree based on the analysis of SFV partial pol sequences (252 bp). The topologies of Bayesian maximum clade
credibility trees obtained under two different tree priors were similar. Branches leading to strains isolated from red colobus monkeys in Co ˆte
d’Ivoire are blue, those leading to strains isolated from Ugandan red colobus monkeys are red. Noncolobine tips are represented graphically to
improve readability: in every case the number of strains represented is indicated between parentheses after family, subfamily or genus names.
Numbers above branches represent bootstrap values (Bp), italicized numbers below branches represent posterior probability values (pp) obtained
using the birth-death model. Bp and pp are indicated only where Bp is ?50 and pp is ?0.95. Asterisks indicate strains identified in the present
study. Two of these strains were actually identified in more than one individual: wrc125 is identical to wrc3*, wrc12, wrc45*, wrc68*, wrc71*, wrc126,
wrc127, wrc128, wrc129, wrc130, wrc131, wrc213*, wrc236*, and wrc276* (15 wrc sequences plus ptr-Leo); wrc133 is identical to wrc132*. Ptr,
chimpanzee (P. troglodytes); krc, Eastern red colobus (P. r. tephrosceles); wrc, Western red colobus (P. b. badius).
VOL. 84, 2010 RETROVIRUSES IN WESTERN RED COLOBUS MONKEYS 7433
at ROBERT KOCH-INSTITUT July 8, 2010
cult, it appears that there is more aggression associated with
breeding in Taï than in Kibale because of the intense male
competition over access to females during a restricted breeding
period (A. Korstjens, personal communication). Frequent
fighting facilitates close contact between individuals and hence
represents a risk of viral transmission. Finally, the P. b. badius
males in Taï were also more frequently seen with red, ulcer-
ated penises than the males in Kibale (A. Korstjens, personal
communication). This could be a sign of other sexually trans-
mitted diseases which could ultimately make retroviral trans-
mission easier. Further studies are required to diagnose and
determine the extent of sexually transmitted diseases in this
red colobus population as well as to investigate their possible
effect on retrovirus transmission.
Coinfection and correlation of viruses. There was a high
degree of coinfection of the retroviruses in the P. b. badius
monkeys, as nearly half of the individuals were triple infected
and nearly one-third were dually infected with SIV and one of
the other retroviruses. This is not surprising considering the
relatively high prevalences of all the individual viruses. In com-
parison, the level of coinfections in the P. r. tephrosceles pop-
ulation was substantially lower (3% of the monkeys had triple
infections, and 23% had dual infections, all of which included
SFV), as would be expected with the relatively lower preva-
lences of both SIV and STLV-1 (15). Interestingly, in the Taï
P. b. badius population, no individual included in the coinfec-
tion analysis was positive for STLV-1 alone or in combination
with SFV; all the STLV-1-positive individuals were at the same
time infected with SIV. It is possible that STLV-1 is frequently
transmitted together with SIV; however, there was no signifi-
cant correlation among any of the viruses in the Taï P. b. badius
population. Also in the Kibale P. r. tephrosceles population, no
correlation was found among these viruses (15).
Phylogeny. With data accumulating, the mechanisms of ret-
rovirus evolution have taken more precise and distinctive
shapes. SFV and SIV mostly show species-specific distribution,
either as a result of host-parasite cospeciation or preferential
host switching (5, 53, 61). In contrast to SFV and SIV, the
distribution of the genetic diversity of STLV-1 seems to be
more complex, which most likely reflects more frequent cross-
species transmission of this virus between different primate
species than for lenti- and spumaretroviruses (21).
Also in our study, SIV and SFV phylogenies both exhibited
the pattern of host-specific association of these retroviruses.
This was especially true for SIVwrc and SFVwrc sequences
determined from P. b. badius. In the SIV tree, SIVwrc se-
quences from P. b. badius are clustered together with those
determined from habituated P. b. badius in the same area and
in P. b. temminckii in Gambia (34, 35). Of note, these red
colobus populations are both called Western red colobus but
are believed to belong to different subspecies (54). In the
phylogeny of mitochondrial DNA, P. b. temminckii appears as
being nested into the genetic diversity of P. b. badius. SIVwrc
therefore conforms to the same pattern. In the SFV tree,
SFVwrc strains cluster into one clade but are at this time
associated with one strain previously identified as coming from
a chimpanzee, most probably as the result of a cross-species
transmission event linked to chimpanzee hunting behavior
(28). For both SIV and SFV, strains identified from P. r. te-
phrosceles in Kibale grouped with strains of P. b. badius in Taï
but were never interspersed with them, suggesting reciprocal
monophyly. As this conforms to the host phylogeny (54), this
might be explained by host-parasite cospeciation for SFVwrc, a
common process for retroviruses of this genus (53). For
SIVwrc, preferential host switching (5) would seem a much
more likely explanation given the presumed overall short time
scale of primate lentivirus evolution (62).
In contrast to SIV and SFV, the phylogeny of STLV-1 is
thought to be linked to geography rather than to host species
(57). Given this geographical component and the slow evolu-
tionary rate of STLV-1 (29), one could expect relatively low
genetic variation on small geographical scales. However, we
found that the novel strains of STLV-1wrc in P. b. badius living
in a relatively small area of the Taï rainforest showed high
genetic variability, being distributed into three distinct lineages
(of which one shows some affinity to STLV-1sm described from
sooty mangabeys from Sierra Leone, whereas STLV-1krc from
Kibale P. r. tephrosceles show no affinity to any of these lin-
eages). Thus, Taï strains were found in the two main lineages
constituting the recently described subtype J as well as in the
group formed by subtype I sequences (21). This is a consider-
able extension of their known genetic diversity as only two Taï
STLV-1wrc strains have been described so far (27). Impor-
tantly, all new STLV-1wrc strains were interspersed with those
found from P. t. verus living in the area. This confirms previous
findings of cross-species transmission of STLV-1 from the red
colobus monkeys to the chimpanzees and underlines that the
major part of the diversity of STLV-1 strains found in those
chimpanzees possibly stems from hunting and eating P. b.
badius (27). To the authors’ knowledge a comparable pattern
of genetic diversity has so far been found only in two other
herbivore primate species (C. nictitans and Cercopithecus
cephus) (31). Under the hypothesis that geographical proximity
rather than host specificity is the main determinant of the
presence of a given strain in a given species, it is tempting to
make the assumption that Taï P. b. badius monkeys are in-
fected with a wide variety of STLV-1 strains because the Taï
rainforest is a place of high endemicity for STLV-1. This might
in turn indicate that STLV-1 has been circulating in that forest
zone for a longer period than in other regions or that it has not
been submitted to similar degrees of a bottleneck effect. It
would therefore be interesting to investigate in more detail the
overall STLV-1 diversity in Taï and also other areas of tropical
Africa since, at the moment, only a two-point comparison is
possible. Increasing the sampling of P. b. badius as well as
getting samples from other Taï primate species, some of which
live in close contact with red colobus monkeys (36, 39), could
be a first step into the process.
Conclusion. This study shows that retroviral infections with
SIV, STLV-1, and SFV are common in red colobus monkeys
(P. b. badius) in the Taï National Park, Co ˆte d’Ivoire. Com-
paring our results with those obtained from a study of a sister
species (P. r. tephrosceles) in Uganda shows that the prevalence
of these retroviruses in wild primates can vary dramatically,
even between closely related species. We further demonstrate
a high genetic variability of STLV-1 in this herbivore monkey
species, which might be taken as an indication that Taï is a hot
spot of diversity for this retrovirus.
7434 LEENDERTZ ET AL.J. VIROL.
at ROBERT KOCH-INSTITUT July 8, 2010
We thank the Ivorian authorities for long-term support, especially
the Ministry of Environment and Forests as well as the Ministry of
Research, the directorship of the Taï National Park, the Office Ivoirien
des Parcs et Re ´serves, and the Swiss Research Centre in Abidjan. We
also thank S. Metzger, W. Rietschel, and field assistants and students
of the Taï Chimpanzee Project for assistance in sample collection and
U. Thiesen, A. Blasse, A. Kopp, S. Handrick, J. Hinzmann, and A.
Hu ¨bner for assistance in the laboratory. For sequencing we thank J.
Tesch. We thank A. Korstjens, K. Zuberbu ¨hler, and P. Beziers for
useful discussions on red colobus monkey behavior.
The study was funded by the Max Planck Institute for Evolutionary
Anthropology, Leipzig, Germany, and the Robert Koch Institute, Ber-
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
2. Boesch, C., and H. Boesch-Achermann. 2000. The chimpanzees of the Taï
forest: behavioural ecology and evolution. Oxford University Press, Oxford,
3. Calattini, S., E. Nerrienet, P. Mauclere, M. C. Georges-Courbot, A. Saib,
and A. Gessain. 2004. Natural simian foamy virus infection in wild-caught
gorillas, mandrills and drills from Cameroon and Gabon. J. Gen. Virol.
4. Cardini, A., and S. Elton. 2009. The radiation of red colobus monkeys
(Primates, Colobinae): morphological evolution in a clade of endangered
African primates. Zool. J. Linn. Soc. 157:197–224.
5. Charleston, M. A., and D. L. Robertson. 2002. Preferential host switching by
primate lentiviruses can account for phylogenetic similarity with the primate
phylogeny. Syst. Biol. 51:528–535.
6. Clewley, J. P., J. C. Lewis, D. W. Brown, and E. L. Gadsby. 1998. A novel
simian immunodeficiency virus (SIVdrl) pol sequence from the drill monkey,
Mandrillus leucophaeus. J. Virol. 72:10305–10309.
7. Courgnaud, V., P. Formenty, C. Akoua-Koffi, R. Noe, C. Boesch, E. Dela-
porte, and M. Peeters. 2003. Partial molecular characterization of two simian
immunodeficiency viruses (SIV) from African colobids: SIVwrc from West-
ern red colobus (Piliocolobus badius) and SIVolc from olive colobus (Pro-
colobus verus). J. Virol. 77:744–748.
8. Courgnaud, V., X. Pourrut, F. Bibollet-Ruche, E. Mpoudi-Ngole, A. Bour-
geois, E. Delaporte, and M. Peeters. 2001. Characterization of a novel simian
immunodeficiency virus from guereza colobus monkeys (Colobus guereza) in
Cameroon: a new lineage in the nonhuman primate lentivirus family. J. Vi-
9. Courgnaud, V., S. Van Dooren, F. Liegeois, X. Pourrut, B. Abela, S. Loul, E.
Mpoudi-Ngole, A. Vandamme, E. Delaporte, and M. Peeters. 2004. Simian
T-cell leukemia virus (STLV) infection in wild primate populations in Cam-
eroon: evidence for dual STLV type 1 and type 3 infection in agile manga-
beys (Cercocebus agilis). J. Virol. 78:4700–4709.
10. Douady, C. J., F. Delsuc, Y. Boucher, W. F. Doolittle, and E. J. Douzery.
2003. Comparison of Bayesian and maximum likelihood bootstrap measures
of phylogenetic reliability. Mol. Biol. Evol. 20:248–254.
11. Drummond, A. J., and A. Rambaut. 2007. BEAST: Bayesian evolutionary
analysis by sampling trees. BMC Evol. Biol. 7:214.
12. Fedigan, L. M. 8 March 2010, posting date. Ethical issues faced by field
primatologists: asking the relevant questions. Am. J. Primatol. doi:10.1002/
13. Gao, F., E. Bailes, D. L. Robertson, Y. Chen, C. M. Rodenburg, S. F.
Michael, L. B. Cummins, L. O. Arthur, M. Peeters, G. M. Shaw, P. M. Sharp,
and B. H. Hahn. 1999. Origin of HIV-1 in the chimpanzee Pan troglodytes
troglodytes. Nature 397:436–441.
14. Gillespie, T. R., C. L. Nunn, and F. H. Leendertz. 2008. Integrative ap-
proaches to the study of primate infectious disease: implications for biodi-
versity conservation and global health. Am. J. Phys. Anthropol. Suppl. 47:
15. Goldberg, T. L., D. M. Sintasath, C. A. Chapman, K. M. Cameron, W. B.
Karesh, S. Tang, N. D. Wolfe, I. B. Rwego, N. Ting, and W. M. Switzer. 2009.
Coinfection of Ugandan red colobus (Procolobus [Piliocolobus] rufomitratus
tephrosceles) with novel, divergent delta-, lenti-, and spumaretroviruses.
J. Virol. 83:11318–11329.
16. Gouy, M., S. Guindon, and O. Gascuel. 2010. SeaView version 4: a multi-
platform graphical user interface for sequence alignment and phylogenetic
tree building. Mol. Biol. Evol. 27:221–224.
17. Guindon, S., and O. Gascuel. 2003. A simple, fast, and accurate algorithm to
estimate large phylogenies by maximum likelihood. Syst. Biol. 52:696–704.
18. Guindon, S., F. Lethiec, P. Duroux, and O. Gascuel. 2005. PHYML on-
line—a web server for fast maximum likelihood-based phylogenetic infer-
ence. Nucleic Acids Res. 33:W557–W559.
19. Hahn, B. H., G. M. Shaw, K. M. De Cock, and P. M. Sharp. 2000. AIDS as
a zoonosis: scientific and public health implications. Science 287:607–614.
20. Heeney, J. L., A. G. Dalgleish, and R. A. Weiss. 2006. Origins of HIV and the
evolution of resistance to AIDS. Science 313:462–466.
21. Junglen, S., C. Hedemann, H. Ellerbrok, G. Pauli, C. Boesch, and F. H.
Leendertz. 2010. Diversity of STLV-1 strains in wild chimpanzees (Pan
troglodytes verus) from Cote d’Ivoire. Virus Res. 150:143–147.
22. Keele, B. F., J. H. Jones, K. A. Terio, J. D. Estes, R. S. Rudicell, M. L.
Wilson, Y. Li, G. H. Learn, T. M. Beasley, J. Schumacher-Stankey, E.
Wroblewski, A. Mosser, J. Raphael, S. Kamenya, E. V. Lonsdorf, D. A.
Travis, T. Mlengeya, M. J. Kinsel, J. G. Else, G. Silvestri, J. Goodall, P. M.
Sharp, G. M. Shaw, A. E. Pusey, and B. H. Hahn. 2009. Increased mortality
and AIDS-like immunopathology in wild chimpanzees infected with SIVcpz.
23. Kwok, S., G. Ehrlich, B. Poiesz, R. Kalish, and J. J. Sninsky. 1988. Enzy-
matic amplification of HTLV-I viral sequences from peripheral blood mono-
nuclear cells and infected tissues. Blood 72:1117–1123.
24. Mugisha, L., C. Ku ¨cherer, H. Ellerbrok, S. Junglen, J. Opuda-Asibo, O. O.
Joseph, G. Pauli, and F. H. Leendertz. Retroviruses in wild-born semi-
captive East African sanctuary chimpanzees (Pan troglodytes schweinfurthii)
Open Vet. Sci. J., in press.
25. Leendertz, F. H., C. Boesch, H. Ellerbrok, W. Rietschel, E. Couacy-Hymann,
and G. Pauli. 2004. Non-invasive testing reveals a high prevalence of simian
T-lymphotropic virus type 1 antibodies in wild adult chimpanzees of the Tai
National Park, Cote d’Ivoire. J. Gen. Virol. 85:3305–3312.
26. Leendertz, F. H., C. Boesch, S. Junglen, G. Pauli, and H. Ellerbrok. 2003.
Characterization of a new simian T-lymphocyte virus type 1 (STLV-1) in a
wild living chimpanzee (Pan troglodytes verus) from Ivory Coast: evidence of
a new STLV-1 group? AIDS Res. Hum. Retroviruses 19:255–258.
27. Leendertz, F. H., S. Junglen, C. Boesch, P. Formenty, E. Couacy-Hymann, V.
Courgnaud, G. Pauli, and H. Ellerbrok. 2004. High variety of different
simian T-cell leukemia virus type 1 strains in chimpanzees (Pan troglodytes
verus) of the Tai National Park, Cote d’Ivoire. J. Virol. 78:4352–4356.
28. Leendertz, F. H., F. Zirkel, E. Couacy-Hymann, H. Ellerbrok, V. A. Morozov,
G. Pauli, C. Hedemann, P. Formenty, S. A. Jensen, C. Boesch, and S.
Junglen. 2008. Interspecies transmission of simian foamy virus in a natural
predator-prey system. J. Virol. 82:7741–7744.
29. Lemey, P., O. G. Pybus, S. Van Dooren, and A. M. Vandamme. 2005. A
Bayesian statistical analysis of human T-cell lymphotropic virus evolutionary
rates. Infect. Genet. Evol. 5:291–298.
30. Lemey, P., M. Salemi, L. Bassit, and A. M. Vandamme. 2002. Phylogenetic
classification of TT virus groups based on the N22 region is unreliable. Virus
31. Liegeois, F., B. Lafay, W. M. Switzer, S. Locatelli, E. Mpoudi-Ngole, S. Loul,
W. Heneine, E. Delaporte, and M. Peeters. 2008. Identification and molec-
ular characterization of new STLV-1 and STLV-3 strains in wild-caught
nonhuman primates in Cameroon. Virology 371:405–417.
32. Ling, B., M. L. Santiago, S. Meleth, B. Gormus, H. M. McClure, C. Apetrei,
B. H. Hahn, and P. A. Marx. 2003. Noninvasive detection of new simian
immunodeficiency virus lineages in captive sooty mangabeys: ability to am-
plify virion RNA from fecal samples correlates with viral load in plasma.
J. Virol. 77:2214–2226.
33. Liu, W., M. Worobey, Y. Li, B. F. Keele, F. Bibollet-Ruche, Y. Guo, P. A.
Goepfert, M. L. Santiago, J. B. Ndjango, C. Neel, S. L. Clifford, C. Sanz, S.
Kamenya, M. L. Wilson, A. E. Pusey, N. Gross-Camp, C. Boesch, V. Smith,
K. Zamma, M. A. Huffman, J. C. Mitani, D. P. Watts, M. Peeters, G. M.
Shaw, W. M. Switzer, P. M. Sharp, and B. H. Hahn. 2008. Molecular ecology
and natural history of simian foamy virus infection in wild-living chimpan-
zees. PLoS Pathog. 4:e1000097.
34. Locatelli, S., B. Lafay, F. Liegeois, N. Ting, E. Delaporte, and M. Peeters.
2008. Full molecular characterization of a simian immunodeficiency virus,
SIVwrcpbt from Temminck’s red colobus (Piliocolobus badius temminckii)
from Abuko Nature Reserve, The Gambia. Virology 376:90–100.
35. Locatelli, S., F. Liegeois, B. Lafay, A. D. Roeder, M. W. Bruford, P. For-
menty, R. Noe, E. Delaporte, and M. Peeters. 2008. Prevalence and genetic
diversity of simian immunodeficiency virus infection in wild-living red colo-
bus monkeys (Piliocolobus badius badius) from the Tai forest, Cote d’Ivoire
SIVwrc in wild-living Western red colobus monkeys. Infect. Genet. Evol.
36. McGraw, W. S., K. Zuberbu ¨hler, and R. Noe. 2007. Monkeys of the Taï
Forest: an African primate community. Cambridge University Press, New
37. Meertens, L., J. Rigoulet, P. Mauclere, M. Van Beveren, G. M. Chen, O.
Diop, G. Dubreuil, M. C. Georges-Goubot, J. L. Berthier, J. Lewis, and A.
Gessain. 2001. Molecular and phylogenetic analyses of 16 novel simian T cell
leukemia virus type 1 from Africa: close relationship of STLV-1 from Allen-
opithecus nigroviridis to HTLV-1 subtype B strains. Virology 287:275–285.
38. Nerrienet, E., X. Amouretti, M. C. Muller-Trutwin, V. Poaty-Mavoungou, I.
Bedjebaga, H. T. Nguyen, G. Dubreuil, S. Corbet, E. J. Wickings, F. Barre-
Sinoussi, A. J. Georges, and M. C. Georges-Courbot. 1998. Phylogenetic
analysis of SIV and STLV type I in mandrills (Mandrillus sphinx): indications
VOL. 84, 2010 RETROVIRUSES IN WESTERN RED COLOBUS MONKEYS7435
at ROBERT KOCH-INSTITUT July 8, 2010
that intracolony transmissions are predominantly the result of male-to-male Download full-text
aggressive contacts. AIDS Res. Hum. Retroviruses 14:785–796.
39. Noe, R., and R. Bshary. 1997. The formation of red colobus-diana monkey
associations under predation pressure from chimpanzees. Proc. Biol. Sci.
40. Nunn, C. L. 1999. The number of males in primate social groups: a compar-
ative test of the socioecological model. Behav. Ecol. Sociobiol. 46:1–13.
41. Nunn, C. L., J. L. Gittleman, and J. Antonovics. 2000. Promiscuity and the
primate immune system. Science 290:1168–1170.
42. Peeters, M., V. Courgnaud, B. Abela, P. Auzel, X. Pourrut, F. Bibollet-Ruche,
S. Loul, F. Liegeois, C. Butel, D. Koulagna, E. Mpoudi-Ngole, G. M. Shaw,
B. H. Hahn, and E. Delaporte. 2002. Risk to human health from a plethora
of simian immunodeficiency viruses in primate bushmeat. Emerg. Infect. Dis.
43. Plantier, J. C., M. Leoz, J. E. Dickerson, F. De Oliveira, F. Cordonnier, V.
Lemee, F. Damond, D. L. Robertson, and F. Simon. 2009. A new human
immunodeficiency virus derived from gorillas. Nat. Med. 15:871–872.
44. Posada, D. 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol.
45. Refisch, J., and I. Kone. 2005. Impact of commercial hunting on monkey
populations in the Tai region, Cote d’Ivoire. Biotropica 37:136–144.
46. Santiago, M. L., F. Range, B. F. Keele, Y. Li, E. Bailes, F. Bibollet-Ruche, C.
Fruteau, R. Noe, M. Peeters, J. F. Brookfield, G. M. Shaw, P. M. Sharp, and
B. H. Hahn. 2005. Simian immunodeficiency virus infection in free-ranging
sooty mangabeys (Cercocebus atys atys) from the Tai Forest, Cote d’Ivoire:
implications for the origin of epidemic human immunodeficiency virus type
2. J. Virol. 79:12515–12527.
47. Santiago, M. L., C. M. Rodenburg, S. Kamenya, F. Bibollet-Ruche, F. Gao,
E. Bailes, S. Meleth, S. J. Soong, J. M. Kilby, Z. Moldoveanu, B. Fahey,
M. N. Muller, A. Ayouba, E. Nerrienet, H. M. McClure, J. L. Heeney, A. E.
Pusey, D. A. Collins, C. Boesch, R. W. Wrangham, J. Goodall, P. M. Sharp,
G. M. Shaw, and B. H. Hahn. 2002. SIVcpz in wild chimpanzees. Science
48. Seiki, M., S. Hattori, Y. Hirayama, and M. Yoshida. 1983. Human adult
T-cell leukemia virus: complete nucleotide sequence of the provirus genome
integrated in leukemia cell DNA. Proc. Natl. Acad. Sci. U. S. A. 80:3618–
49. Silvestri, G. 2009. Immunity in natural SIV infections. J. Intern. Med. 265:
50. Sintasath, D. M., N. D. Wolfe, M. Lebreton, H. Jia, A. D. Garcia, J. Le
Doux-Diffo, U. Tamoufe, J. K. Carr, T. M. Folks, E. Mpoudi-Ngole, D. S.
Burke, W. Heneine, and W. M. Switzer. 2009. Simian T-lymphotropic virus
diversity among nonhuman primates, Cameroon. Emerg. Infect. Dis. 15:175–
51. Souquiere, S., F. Bibollet-Ruche, D. L. Robertson, M. Makuwa, C. Apetrei,
R. Onanga, C. Kornfeld, J. C. Plantier, F. Gao, K. Abernethy, L. J. White, W.
Karesh, P. Telfer, E. J. Wickings, P. Mauclere, P. A. Marx, F. Barre-
Sinoussi, B. H. Hahn, M. C. Muller-Trutwin, and F. Simon. 2001. Wild
Mandrillus sphinx are carriers of two types of lentivirus. J. Virol. 75:7086–
52. Struhsaker, T. T. 1975. The red colobus monkey. University of Chicago
Press, Chicago, IL.
53. Switzer, W. M., M. Salemi, V. Shanmugam, F. Gao, M. E. Cong, C. Kuiken,
V. Bhullar, B. E. Beer, D. Vallet, A. Gautier-Hion, Z. Tooze, F. Villinger,
E. C. Holmes, and W. Heneine. 2005. Ancient co-speciation of simian foamy
viruses and primates. Nature 434:376–380.
54. Ting, N. 2008. Mitochondrial relationships and divergence dates of the
African colobines: evidence of Miocene origins for the living colobus mon-
keys. J. Hum. Evol. 55:312–325.
55. Vandamme, A. M., M. Salemi, M. Van Brussel, H. F. Liu, K. Van Laethem,
M. Van Ranst, L. Michels, J. Desmyter, and P. Goubau. 1998. African origin
of human T-lymphotropic virus type 2 (HTLV-2) supported by a potential
new HTLV-2d subtype in Congolese Bambuti Efe Pygmies. J. Virol. 72:
56. VandeWoude, S., and C. Apetrei. 2006. Going wild: lessons from naturally
occurring T-lymphotropic lentiviruses. Clin. Microbiol. Rev. 19:728–762.
57. Van Dooren, S., E. J. Verschoor, Z. Fagrouch, and A. M. Vandamme. 2007.
Phylogeny of primate T lymphotropic virus type 1 (PTLV-1) including var-
ious new Asian and African non-human primate strains. Infect. Genet. Evol.
58. Van Heuverswyn, F., Y. Li, C. Neel, E. Bailes, B. F. Keele, W. Liu, S. Loul,
C. Butel, F. Liegeois, Y. Bienvenue, E. M. Ngolle, P. M. Sharp, G. M. Shaw,
E. Delaporte, B. H. Hahn, and M. Peeters. 2006. Human immunodeficiency
viruses: SIV infection in wild gorillas. Nature 444:164.
59. Verdonck, K., E. Gonzalez, S. Van Dooren, A. M. Vandamme, G. Vanham,
and E. Gotuzzo. 2007. Human T-lymphotropic virus 1: recent knowledge
about an ancient infection. Lancet Infect. Dis. 7:266–281.
60. Villesen, P. 2007. FaBox: an online toolbox for fasta sequences. Mol. Ecol.
61. Wertheim, J. O., and M. Worobey. 2007. A challenge to the ancient origin of
SIVagm based on African green monkey mitochondrial genomes. PLoS
62. Wertheim, J. O., and M. Worobey. 2009. Dating the age of the SIV lineages
that gave rise to HIV-1 and HIV-2. PLoS Comput. Biol. 5:e1000377.
63. Wolfe, N. D., P. Daszak, A. M. Kilpatrick, and D. S. Burke. 2005. Bushmeat
hunting, deforestation, and prediction of zoonoses emergence. Emerg. In-
fect. Dis. 11:1822–1827.
64. Wolfe, N. D., W. M. Switzer, J. K. Carr, V. B. Bhullar, V. Shanmugam, U.
Tamoufe, A. T. Prosser, J. N. Torimiro, A. Wright, E. Mpoudi-Ngole, F. E.
McCutchan, D. L. Birx, T. M. Folks, D. S. Burke, and W. Heneine. 2004.
Naturally acquired simian retrovirus infections in central African hunters.
65. Xia, X., and Z. Xie. 2001. DAMBE: software package for data analysis in
molecular biology and evolution. J. Hered. 92:371–373.
7436LEENDERTZ ET AL. J. VIROL.
at ROBERT KOCH-INSTITUT July 8, 2010