simian t-cell leukemia virus (stlv) infection in wild ......baboons, african green monkeys, guenons,...

JOURNAL OF VIROLOGY, May 2004, p. 4700–4709 Vol. 78, No. 90022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.9.4700–4709.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Simian T-Cell Leukemia Virus (STLV) Infection in Wild PrimatePopulations 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,1Bernadette Abela,1,3 Severin Loul,3 Eitel Mpoudi-Ngole,3

Annemieke Vandamme,2 Eric Delaporte,1and Martine Peeters1*

UR36, IRD, and University of Montpellier I, Montpellier, France1; Department of Clinical and EpidemiologicalVirology, 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 calledprimate T-cell leukemia viruses [PTLVs]) have been characterized, with evidence for zoonotic origin fromprimates for HTLV type 1 (HTLV-1) and HTLV-2 in Africa. To assess human exposure to STLVs in westernCentral Africa, we screened for STLV infection in primates hunted in the rain forests of Cameroon. Blood wasobtained from 524 animals representing 18 different species. All the animals were wild caught between 1999and 2002; 328 animals were sampled as bush meat and 196 were pets. Overall, 59 (11.2%) of the primates hadantibodies cross-reacting with HTLV-1 and/or HTLV-2 antigens; HTLV-1 infection was confirmed in 37animals, HTLV-2 infection was confirmed in 9, dual HTLV-1 and HTLV-2 infection was confirmed in 10, andresults for 3 animals were indeterminate. Prevalences of infection were significantly lower in pets than in bushmeat, 1.5 versus 17.0%, respectively. Discriminatory PCRs identified STLV-1, STLV-3, and STLV-1 andSTLV-3 in HTLV-1-, HTLV-2-, and HTLV-1- and HTLV-2-cross-reactive samples, respectively. We identifiedfor the first time STLV-1 sequences in mustached monkeys (Cercopithecus cephus), talapoins (Miopithecusogouensis), 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) andenv sequences revealed that the strains belonged to different PTLV-1 subtypes. A high prevalence of PTLVinfection 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 agilemangabeys. Phylogenetic analyses of partial LTR sequences indicated that the agile mangabey STLV-3 strainswere 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 ababoon in Senegal. Our study documents for the first time that (i) a substantial proportion of wild-livingmonkeys 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 differentSTLV-1 and STLV-3 subtypes through handling primates as bush meat.

Simian T-cell leukemia viruses (STLVs) are the simiancounterparts of human T-cell leukemia viruses (HTLV), andthese viruses are collectively called primate T-cell leukemiaviruses (PTLVs). HTLVs are separated into two serologicallyand genetically distinct types, HTLV type 1 (HTLV-1) andHTLV-2, and both types have simian counterparts, STLV-1and STLV-2 (8, 36, 37). A third type, STLV-3, was isolatedfrom several African nonhuman primates such as hamadryasbaboons (Papio hamadryas) from east and west Africa andred-capped mangabeys (Cercocebus torquatus) and greaterspot-nosed monkeys (Cercopithecus nictitans) from Cameroon(22, 23, 35, 39). STLV-1 has been isolated from a wide varietyof Old World monkeys in Asia and Africa, including macaques,

baboons, African green monkeys, guenons, mangabeys, oran-gutans, and chimpanzees, whereas STLV-2 has been identifiedonly in captive bonobos (Pan paniscus) from the DemocraticRepublic of Congo (12, 13, 15, 24, 29, 40). The close relation-ship between HTLV-1 and STLV-1 suggests a simian origin forHTLV-1. Moreover, phylogenetic analyses of African HTLV-1and STLV-1 strains revealed that some HTLV-1 strains aremore closely related to STLV-1, suggesting the occurrence ofmultiple cross-species transmissions between primates and hu-mans and also between different primate species (18).

Similar to HTLV, other simian retroviruses such as humanimmunodeficiency virus type 1 (HIV-1) and HIV-2 are of zoo-notic origin, with their closest simian relatives in the commonchimpanzee (Pan troglodytes) and the sooty mangabey (Cerco-cebus atys), respectively (6, 11). First recognized in the early1980s, HIV-1 has spread to most parts of the world, and todayit is estimated that more than 40 million individuals live withHIV infection or AIDS (34). HTLV is less pathogenic than

* Corresponding author. Mailing address: Laboratoire Retrovirus,UR036, IRD, 911 Avenue Agropolis, BP 64 501, 34394 MontpellierCdx 1, France. Phone: 33-4 67 41 62 97. Fax: 33-4 67 41 61 46. E-mail:[email protected].

4700

HIV, but HTLV-1 is known to be associated with lymphoma,leukemia (adult T-cell leukemia), and some neurological dis-orders such as tropical spastic paraparesis (9, 17, 33).

Given that humans come in frequent contact with primatesin many parts of sub-Saharan Africa, the possibility of addi-tional zoonotic transfers of retroviruses from primates has tobe considered. Prevalences of HTLV infection are high inAfrica, with the highest values in equatorial Africa and moreprecisely in the tropical forest region (5). In central Africa,prevalences of HTLV-1 infection increase with age, and inrural areas women and pygmies are more frequently infected(4, 21). All these epidemiological observations, together withthe phylogenetic relationships between HTLV and STLV, arein favor of zoonotic transmissions. The risk for acquiring suchinfections is expected to be the highest in individuals who huntprimates and who prepare their meat for consumption, as wellas in people who keep primates as pet animals. Therefore, it isimportant to study the prevalence, diversity, and geographicdistribution of these infections in wild primate populations. Ina similar way, it was previously shown that humans are exposedto a plethora of simian immunodeficiency viruses in Cameroon(26).

In the present study, we tested wild-caught primates fromCameroon for STLV infection. Cameroon is known to harbora diverse set of primate species which are extensively huntedfor food and trade at various levels (3). Our study indicatesthat in addition to frequent contamination with simian immu-nodeficiency virus, a considerable proportion of primate meatsold for consumption is contaminated with STLV-1 andSTLV-3. These data also provide an approximation of themagnitude of exposure and the variety of STLVs to whichhumans are exposed and permit estimation of the prevalencesof STLV infection in wild primate populations in Cameroon.In addition, our study further documents coinfection withSTLV-1 and STLV-3 in wild primate populations.

MATERIALS AND METHODS

Collection of primate tissue and blood samples. Blood was obtained from 524monkeys all wild caught in Cameroon between January 1999 and July 2002.Species were determined by visual inspection according to The Kingdon FieldGuide to African Mammals (14) and by use of the taxonomy described by ColinGroves (10). Three hundred twenty-eight animals were sampled as bush meatupon arrival at markets in Yaounde, the capital city, in surrounding villages, orat logging concessions in southeastern Cameroon; the other 196 animals sampledwere pets from these same areas. Table 1 summarizes the numbers of eachprimate species collected. All primate samples were obtained with governmentapproval from the Cameroonian Ministry of Environment and Forestry. Thebush meat samples were obtained by employing a strategy specifically designednot to increase the demand for bush meat, i.e., women preparing and preservingthe meat for subsequent sale and hunters already involved in the trade wereasked for permission to sample blood and tissues from carcasses which were thenreturned to their owners; animals and bush meat confiscated by the nationalprogram against poaching were also sampled. For the bush meat animals, bloodwas collected by cardiac puncture. Information provided by the owners indicatedthat most of the animals had died 12 to 72 h prior to sampling. For the petmonkeys, blood was drawn by peripheral venipuncture after the animals weretranquilized with ketamine (10 mg/kg). Plasma and cells were separated on siteby Ficoll gradient centrifugation. All samples, including peripheral blood mono-nuclear cells, plasma, whole blood, and other tissues, were stored at �20°C.

Serology. Plasma or whole blood samples were tested for the presence ofHTLV-cross-reactive antibodies by using a commercially available enzyme-linked immunosorbent assay (ELISA), the MUREX HTLV-I�II test (AbbottLaboratories, Wiesbaden, Germany), using as antigens synthetic peptides andrecombinant proteins representing immunodominant regions of the envelope

and transmembrane regions of HTLV-1 and HTLV-2. Samples reactive in theELISA were retested with a commercially available line immunoassay, INNO-LIA HTLV I/II (Innogenetics, Ghent, Belgium), which discriminates betweenHTLV-1- and HTLV-2-cross-reactive antibodies. This test configuration includesHTLV-1 and HTLV-2 recombinant proteins and synthetic peptides that arecoated as discrete lines on a nylon strip. The antigenicity exhibited by theseproteins and peptides is either common to HTLV-1 and HTLV-2 or specific toone 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-IIand gp21-I or gp21-II) bands are applied as non-type-specific antigens, which areused to confirm the presence of antibodies against HTLV-1 and HTLV-2. Thetype-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 infec-tions. In addition to these HTLV antigens, control lines are present on each strip:one sample addition line (3�) containing anti-human immunoglobulin (Ig) andtwo test performance lines (1� and �/�) containing human IgG. Values rep-resent reaction intensity. All assays were performed and interpreted according tothe manufacturer’s instructions.

PCR. DNA was isolated from whole blood or peripheral blood mononuclearcells using Qiagen DNA extraction kits (Qiagen, Courtaboeuf, France). Toconfirm the presence of PTLVs in samples with HTLV-cross-reactive antibodies,a previously described diagnostic tax-rex PCR allowing generic as well as type-specific detection of PTLVs was done (38). The generic PCR proved to be highlysensitive in detecting PTLV strains, and the discriminatory PCRs had highsensitivities and specificities.

For a subset of STLV-1- and STLV-3-positive samples, we also sequenced partof 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 thecarboxyl-terminal region of gp46, was amplified and sequenced with previouslydescribed primers (20). For STLV-3, a 540-bp fragment in the LTR region wasamplified with a combination of previously described and newly designed prim-ers: AV51 (38) and pX-LTRAS (5�-TTTATAGGACCCAGGGTTCTT-3� [po-sitions 8450 to 8470 in PH969]) for the first round and pX-LTRS (5�-CRGGCACACRGGYCTACTCCC-3� [positions 7932 to 7952 in PH969]) and pX-LTRAS for the second round. R represents A or G; Y represents C or T. PCRsfor both rounds were performed using the Expand High Fidelity PCR kit (RocheMolecular Biochemicals, Mannheim, Germany) and included a hot start (94°Cfor 2 min) with the following cycle conditions: 10 cycles of denaturation at 94°Cfor 15 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min followed by25 cycles with extension at 72°C for 1 min in the first cycle and for timesincreasing by an increment of 5 s per cycle thereafter. Amplification was com-pleted by a final extension at 72°C for 7 min. PCR products were sequenced usingcycle sequencing and dye terminator methodologies (ABI PRISM Big Dye Ter-minator Cycle Sequencing Ready Reaction kit with AmpliTaq FS DNA poly-merase [PE Biosystems, Warrington, England]) on an automated sequencer(ABI 373, stretch model; Applied Biosystems) either directly or following cloninginto the pGEM-T vector (Promega, Lyon, France).

To test for DNA degradation, a 1,151-bp region of the glucose-6-phosphatedehydrogenase gene was amplified using the primers GPD-F1 (5�-CATTACCAGCTCCATGACCAGGAC-3�) and GPD-R1 (5�-GTGTTCCCAGGTGACCCTCTGGC-3�) in a single-round PCR with the following conditions: 94°C for 2 minand 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 werealigned with reference sequences from GenBank using CLUSTAL W (32) withminor manual adjustments. Gaps in the alignment were omitted from furtheranalyses. The STLV-1 LTR and env phylogenetic trees were constructed usingthe neighbor-joining (NJ) method and/or the maximum likelihood (ML) methodwith the Tamura Nei substitution model using PAUP*4.0b10 software (30). Thereliability of branching orders was tested using the bootstrap approach (1,000replicates) for the NJ tree, whereas P values were obtained for the ML tree withthe zero branch length test. The STLV-3 LTR (463 nucleotides) and PTLV tax(180 nucleotides) phylogenies were investigated with the software packagePAUP* version 4.0b10 (30). NJ and ML trees were constructed under the mostappropriate evolutionary model tested with MODELTEST 3-06 (27). For theLTR and tax sequences, the transitional model and transversional model, re-spectively, each allowing five different substitution rate categories includinggamma distribution rate heterogeneity, provided the best fit to the data. The NJtrees were constructed by iterative optimization of the model parameters, fol-lowed by a bootstrap analysis of 1,000 replicates. The ML trees were constructedby starting from the NJ tree with optimized parameters by using a heuristicsearch with the nearest-neighbor interchange and the subtree-pruning-regrafting

VOL. 78, 2004 STLV INFECTION IN WILD PRIMATES 4701

branch-swapping algorithm (28). Additionally, the P values were estimated forthe branches of the ML trees with the branch length confidence test.

Nucleotide sequence accession numbers. The new sequences have been de-posited in GenBank under the following accession numbers: AY496626 toAY496638 (LTR from STLV-1), AY496596 to AY496606 (env from STLV-1),AY496607 to AY496618 (tax from STLV-1), AY496588 to AY496595 (tax fromSTLV-3), and AY496619 to AY496625(LTR from STLV-3).

RESULTS

Estimates of prevalence of STLV infection in bush meat andpet monkey samples. Blood specimens were obtained from atotal of 524 nonhuman primates representing 18 different spe-cies. All the animals were wild caught in Cameroon. Wholeblood was collected from 328 animals that were sold as bushmeat at markets in the capital city of Yaounde, in nearbyvillages, and at logging concessions in southeastern Cameroon.The great majority (86.4%) of these animals were adults. Wealso 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 coun-try.

In order to detect PTLV infection in nonhuman primates,we used commercially available HTLV-1–HTLV-2 assays sinceall previously reported STLV infections were also identifiedthrough cross-reactivity with HTLV antigens. A total of 59(11.2%) of 524 samples tested reacted strongly in the HTLV-1-HTLV-2 ELISA (Table 1). All ELISA reactive samples wereretested with the INNO-LIA HTLV I/II confirmatory assay,and the results are summarized in Table 1. Among the 59samples, 37 were confirmed as HTLV-1 positive, 9 were con-firmed as HTLV-2 positive, 10 were confirmed as HTLV-1 andHTLV-2 positive, and results for 3 were indeterminate. Figure1 illustrates the kind of INNO-LIA reactivities that were typ-ically observed. Overall, HTLV-cross-reactive antibodies weredetected in 9 of the 18 primate species tested, and the preva-lences of seroreactivity (positive or indeterminate results) inthe different species ranged from 0.8 to 66%. Moreover, weidentified for the first time STLV sequences in talapoins, mus-tached monkeys, and gorillas. As expected, prevalences weresignificantly lower in pet animals, which were mainly infants orjuveniles, than in bush meat primates, which were predomi-nantly adult animals (1.5 versus 16.9%, respectively; data notshown). Surprisingly, corresponding to differences in species,extreme differences in prevalences of HTLV-cross-reactive an-tibodies were observed: up to 89% of agile mangabey bushmeat was infected with STLV, whereas only 0.96% of mus-tached monkeys were infected. For the majority of the 9 out of18 primate species without HTLV antibodies, the numbers ofadult animals tested were low, which may explain the lack ofreactivity. For example, we did not observe a positive reactionin samples from red-capped mangabeys (Cercocebus torquatus)and mona monkeys (Cercopithecus mona), although STLV-3and STLV-1 infections, respectively, were previously docu-mented in these species (23, 24). Interestingly, we observed 10agile mangabeys with antibodies that cross-reacted withHTLV-1- and HTLV-2-specific antigens.

Confirmation of STLV infection by confirmatory and dis-criminatory PCR analysis of the tax gene. In order to confirmwhether animals with HTLV-cross-reactive antibodies wereinfected with a PTLV and to determine with which type of

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4702 COURGNAUD ET AL. J. VIROL.

PTLV, we performed PCR using highly cross-reactive taxprimer pairs previously shown to amplify sequences from avariety of divergent HTLV and STLV strains and known tohave a high specificity in characterizing the PTLV type. Amongthe 59 samples with a positive or indeterminate serology, 41samples for which sufficient additional material was availablewere tested by generic PCR followed by type-specific PCR todiscriminate between STLV-1, STLV-2, and STLV-3. Amongthe 41 samples, 24 were positive for HTLV-1, 9 were positivefor HTLV-2, 5 were positive for HTLV-1 and HTLV-2, and 3had an indeterminate serology in the INNO-LIA HTLV I/IIassay. The two HTLV-2-positive samples from greater spot-nosed monkeys have been previously described and were iden-tified as being infected with a new SLTV-3 variant (39). The taxPCR results are summarized in Table 2. Among the 24 HTLV-1-positive samples, 5 were negative by PCR, 18 were positivefor STLV-1, and in 1 sample (from animal 01CM-1135)STLV-1 and STLV-3 were detected. This dual infection wasconfirmed by sequence analysis of the tax fragments. All sevenHTLV-2-seropositive samples were reactive with the STLV-3-specific tax primers only, which was confirmed by sequenceanalysis of four samples (Fig. 2). Among the five samplesreactive with HTLV-1- and HTLV-2-specific antigens in theline immunoassay, three were determined to carry bothSTLV-1 and STLV-3, and the remaining two were found tocarry only HTLV-1. In two of the three samples with indeter-minate HTLV serology, no viruses could be amplified with thegeneric primers, and in the remaining sample, STLV-3 waspresent and confirmed by sequence analysis of the tax frag-ment.

Overall, we confirmed the presence of STLVs in eight of thenine primate species in which we observed HTLV-cross-reac-tive antibodies. Only among chimpanzees, where we observedone animal with indeterminate HTLV serology, could noSTLV infection be demonstrated by PCR. All samples whichwere identified as HTLV-2 positive by serology were in factinfected with an STLV-3 strain. More interestingly, we ob-served that one primate species can be infected with two dif-ferent STLV types; more precisely, STLV-1 and STLV-3 in-fections were observed in agile mangabeys and in greater spot-nosed monkeys. In addition, we showed that the same animalcan be infected with both viruses at the same time. We iden-tified four agile mangabeys (animals 01CM-1038, 01CM-1122,01CM-1135, and 01CM-1272) that were coinfected withSTLV-1 and STLV-3. Figure 2 shows the phylogenetic treeanalysis of the tax sequences and encompasses results of thediagnostic and discriminatory tax PCRs for a subset of samples.It is clear from this figure that the previously reported taxSTLV-3 sequences from greater spot-nosed monkeys formed adistinct, well-supported (90% bootstrap support for NJ; P of�0.05 for ML) cluster within the STLV-3 group. All otherSTLV-3 strains clustered together but separately from thegreater spot-nosed monkey STLV-3 strains, with a reasonablebootstrap 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 lessaccording to geographic origins of the STLV host species. Dueto the low genetic diversity in tax and the shortness of thefragment, however, the topology among these STLV-3 strainswas not well supported. Based on this 180-bp fragment, five

FIG. 1. Detection of HTLV-1- and HTLV-2-cross-reactive antibodies in sera from agile mangabeys (Cercocebus agilis) by using a lineimmunoassay (INNO-LIA HTLV confirmation; Innogenetics). The HTLV antigens include recombinant proteins and synthetic peptides which areeither common to HTLV-1 and HTLV-2 or specific to one of the two viruses. The first three control lines contain human IgG in differentconcentrations 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-3-seropositive Cercocebus agilis. Lane 01-CM1003 represents an example of plasma with indeterminate serology.


sequences from agile mangabey virus strains were even iden-tical to sequences from the previously described strains fromred-capped mangabeys (CTO602 and CTO604) (23). TheSTLV-1 strains clustered with other African HTLV-1 andSTLV-1 strains, but the support here was also rather low.Moreover, we identified for the first time STLV sequences intalapoins, mustached monkeys, and gorillas. Therefore, we fur-ther investigated longer fragments from these STLV-1 andSTLV-3 strains in more divergent gene regions such as theLTR and/or env.

env and LTR sequence analysis of STLV-1 strains obtainedfrom different primate species. The complete STLV-1 LTRwas sequenced for 10 STLV-1-infected and 3 STLV-1- andSTLV-3-coinfected animals. The 10 STLV-1-infected animalswere representatives of the following primate species: agilemangabeys (four), mustached monkeys (one), crested monas(one), mandrills (two), and talapoins (two). The three coin-fected animals (01CM-1038, 01CM-1122, and 01CM-1272)were all agile mangabeys. Figure 3 shows the phylogenetic treeanalyses of the LTRs and env. Phylogenetic tree analyses of thenew sequences together with previously published STLV andHTLV sequences representing the different HTLV-1-STLV-1subtypes using both NJ and ML revealed that all sequencesfrom agile mangabeys were closely related to one another (98.9to 100% identity) and to a previously published STLV-1 se-quence obtained from an agile mangabey (25) (99.3 to 99.7%identity) captured in southeast Cameroon (NJ, bootstrap val-ues of 54 and 100%; ML, P of �0.001 and 0.008). The STLV-1LTR sequence from the only mustached monkey also clusteredwith the STLV-1 sequences from agile mangabeys (99.1 to99.3% identity) from the same area in Cameroon (25) (NJ,bootstrap value of 61%; ML, P of �0.001). The LTR se-quences from STLVs obtained from agile mangabeys and mus-tached monkeys clustered with the sequence from subtype F

identified in an individual from Gabon (96 to 96.6% identitywith the Lib2 sequence) with 86% bootstrap support for NJand a P value of �0.001 for ML. The STLV-1 sequences fromthe mandrills from our study and from the crested mona clus-tered with high support values with sequences from the centralAfrican subtype D in both trees (96% identity between se-quences from strains 1228 and H23 and 99.5% identity be-tween sequences from strains ML4 and H23). The sequencesobtained from talapoins clustered with those of STLVs fromwestern Africa and western central Africa (NJ, bootstrap valueof 60%; ML, P of �0.001). The STLV-1 strain obtained froma gorilla clustered with HTLV-1 subtype B (98.2% identitywith H24; NJ, bootstrap value of 82%; ML, P of �0.001).

Partial env sequences were also obtained from the above-described samples except from that from the mustached mon-key. Phylogenetic tree analysis of the env sequences showedclustering patterns similar to those determined by analysis ofthe LTR region.

LTR sequence analysis of STLV-3 strains obtained fromSTLV-3-infected and STLV-1- and STLV-3-coinfected agilemangabeys. Fragments of 540 bp comprising the LTR regionsof STLV-3 strains were obtained from four STLV-3-infectedand three STLV-1- and STLV-3-coinfected animals. All thenew STLV-3 sequences were closely related to one another(97.8 to 100% identity) and were also most closely related to anSTLV-3 sequence (97.8 to 99.6% identity) obtained from arecently described red-capped mangabey from Cameroon (23).Even with this limited number of STLV-3 LTR sequencesavailable in the GenBank database, we observed a tendencytoward STLV-3 clustering according to the geographic originsof the viral host species. Similar to those of STLV-1, STLV-3sequences from coinfected animals did not form a separatesubcluster. For the STLV-3-infected greater spot-nosed mon-keys, for which the tax sequences were previously reported, we

TABLE 2. PTLV confirmation and discrimination by generic and type-specific tax PCR for HTLV-1 and HTLV-2 antibody cross-reactivesamples

Species Virus identification byINNO-LIA HTLVI/II

No. ofsamplestested

No. of samples positive by tax PCR for:No. of samples negative

by tax PCRSTLV-1 STLV-3 STLV-1 andSTLV-3

Cercocebus agilis HTLV-1 14 8 0 1 5HTLV-2 7 0 7 0 0HTLV-1 and HTLV-2 5 2 0 3 0Indeterminate HTLV 2 0 1 0 1

Cercopithecus nictitans HTLV-1 2 2 0 0 0HTLV-2 2 0 2a 0 0

Cercopithecus pogonias HTLV-1 2 2 0 0 0Chlorocebus tantalus HTLV-1 1 1 0 0 0Miopithecus ogouensis HTLV-1 1 1 0 0 0Mandrillus sphinx HTLV-1 1 1 0 0 0Cercopithecus cephus HTLV-1 1 1 0 0 0Pan troglodytes Indeterminate HTLV 1 0 0 0 1Gorilla gorilla HTLV-1 2 2 0 0 0

Subtotal HTLV-1 24 16 0 1 5HTLV-2 9 0 9 0 0HTLV-1 and HTLV-2 5 2 0 3 0Indeterminate HTLV 3 0 1 0 2

Total 41 20 10 4 7

a Previously reported to be infected with STLV-3.


were not able to amplify the LTR fragment due to the de-graded nature of the DNA (39). Figure 4 shows the phyloge-netic tree analysis of the STLV-3 LTR sequences.

DISCUSSION

The majority of previous studies of STLV infection haverelied almost exclusively on surveys of captive monkeys or apesthat were either kept as pets or housed at zoos or primatecenters. The great majority of pet monkeys are acquired at avery young age, often when their parents are killed by hunters.STLV infection rates of captive monkeys may thus not accu-rately reflect STLV infection prevalence rates in the wild.

Phylogenetic tree analysis of HTLV and STLV strainsshowed that zoonotic transfers of STLV to humans have oc-curred on several occasions (37), but no study has examinedthe prevalences of STLV infection among African primatesthat are frequently hunted or kept as pets. In this paper, wecollected blood from 524 monkeys representing 18 differentspecies. All of the animals were wild caught in the rain forestsof Cameroon and sampled as either bush meat or pet animals.This approach allowed us simultaneously to identify STLVinfection prevalence rates in wild primate populations and todetermine to what extent humans are exposed to STLVs. Wedetected cross-reactive antibodies suggesting PTLV infection

in 11% of all tested animals. STLV infection was confirmed byPCR in 8 of the 18 species tested, and phylogenetic analysesrevealed the presence of STLVs clustering in different PTLVtypes. We confirmed STLV-1 infection in three species previ-ously identified as STLV carriers by serology only, namely,mustached monkeys (Cercopithecus cephus), talapoins (Mio-pithecus ogouensis), and gorillas (Gorilla gorilla). We showedalso for the first time the presence of STLV-3 infection in agilemangabeys (Cercocebus agilis), and even coinfection withSTLV-1 and STLV-3 was observed in this primate species.

Our data reveal for the first time that a considerable pro-portion of wild-living primates in Cameroon are infected withSTLV and that these primates may be a source of infection tothose who come in contact with them. Although new STLV-infected host species were identified and new STLV variantswere characterized, it is likely that our data represent onlyminimal estimates concerning STLV prevalences and STLVdiversity in Cameroon because not all native primate specieswere tested and many were undersampled because they wereeither rare or absent in the regions of Cameroon where wesampled for this study. For example, the absence of reactivesera from mona monkeys (Cercopithecus mona) and red-capped mangabeys (Cercocebus torquatus), two species knownto harbor STLV, must be due to the low numbers of bloodsamples analyzed (23, 24).

FIG. 2. PAUP* NJ tree of a 219-bp tax-rex fragment including sequences from reference strains of each PTLV type and subtype with thebootstrap values (in percentages) and P values (**, P � 0.001; *, P � 0.05) noted on the branches.


Similar to that with HIV, human infection with HTLV-1 andHTLV-2 most likely resulted from cutaneous or mucous mem-brane exposure to infected blood during the hunting andbutchering of STLV-infected primates for food or from bitesfrom STLV-infected pet animals. Although no HTLV-3 infec-tion is yet described in humans, our study shows that humans

are exposed to STLV-3-infected primate bush meat fromgreater spot-nosed monkeys and agile mangabeys and possiblyother, not-yet-identified STLV-3-harboring primate species.Samples from STLV-3-infected animals either reacted withHTLV-2 antigens in the INNO-LIA assay or had an indeter-minate HTLV serology. It will thus be important to genetically

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 relationshipswere determined using LTR (A) and env (B) sequences as described in Materials and Methods. The numbers along the branches are the bootstrapvalues (in percentages), and two asterisks indicate that the branch has a P value of �0.001 in the ML analysis. Horizontal branch lengths are drawnto scale.


characterize human samples with HTLV-2 or indeterminateHTLV serology to study whether STLV-3 cross-species trans-mission between primates and humans has already occurredand, if so, whether this infection is associated with any diseasein humans. Indeterminate HTLV Western blot patterns arefrequently observed in central Africa, and although a majorityof such patterns may be due to other environmental (viral orparetic) factors, the possibility for HTLV-3 infection has to befurther explored (19). Bush meat hunting, to provide animalproteins for the family and a source of income, has a long-standing 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 re-mote forest areas, led to human migration and the develop-ment of social and economic networks in previously inaccessi-ble forest areas. Villages around logging concessions havegrown from a few hundred to several thousand inhabitants injust a few years (2, 41). These socioeconomic changes suggestthat the magnitude of human exposure to primates infectedwith STLV has increased, as have the social and environmentalconditions that would be expected to support the emergence ofnew zoonotic infections with STLVs.

FIG. 3—Continued.


Our study shows clearly that significantly more adult mon-keys than juveniles are infected (1.5% prevalence in pets ver-sus 16.9% in bush meat samples), thus suggesting a low verticaltransmission rate and confirming that estimates of the preva-lence of STLV infection have to be made using adult animals.We also observed extreme differences in prevalence ratesamong different primate species. We tested large numbers ofgreater spot-nosed and mustached monkeys, but only a fewanimals were determined to be positive. In contrast, more than80% of adult agile mangabeys were infected with a PTLV, andeven STLV-1 and STLV-3 infections and STLV-1-STLV-3coinfections were observed among these animals in the wild.Another study among wild primate populations in Ethiopiaalso revealed discrepancies among STLV infection prevalencesamong different baboon species (31). STLV-3 and STLV-1infections were observed, and one hybrid baboon was positiveby STLV-1- and STLV-L-specific PCR, suggesting a dual in-fection (31). It is known for HTLV infection in humans thatgeographic and/or intrafamilial clusters with high prevalencesof infection exist (16). It has to be further investigated whetherthe high prevalences observed in certain monkeys are specificfor the species or whether, similar to those among humans,geographic clusters also exist among nonhuman primates.Therefore, additional prevalence studies of STLV infectionsamong wild primate populations have to be done in otherregions of Cameroon and Africa.

In conclusion, our study shows that humans are exposed toa large variety of STLV-1 and STLV-3 strains. Further studiesare needed to determine whether zoonotic transmissions ofSTLVs, especially STLV-3, from primates has occurred. In

order to understand the evolution of PTLVs, it will be impor-tant to identify and compare STLVs from primate species fromwestern, central, and eastern Africa, as well as to determineSTLV 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 humanpopulations.

ACKNOWLEDGMENTS

We thank the Cameroonian Ministries of Health and of Environ-ment and Forestry for permission to perform this study and the stafffrom project PRESICA for logistical support and assistance in thefield.

This work was supported in part by grants from the National Insti-tutes of Health (RO1 AI 50529), the Agence National de Recherchesur le SIDA (ANRS), and the Fonds voor Wetenschappelijk Onder-zoek (grant 0288.01).

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simian t-cell leukemia virus (stlv) infection in wild ......baboons, african green monkeys, guenons,...

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