Molecular epidemiology of simian T-lymphotropic virus (STLV) in wild-caught monkeys and apes from Cameroon: a new STLV-1, related to human T-lymphotropic virus subtype F, in a Cercocebus agilis

Eric Nerrienet1, Laurent Meertens2, Anfumbom Kfutwah1, Yacouba Foupouapouognigni1 and Antoine Gessain2

Centre Pasteur du Cameroun, BP 1274, Yaoundé, Cameroon1
Unité d’Epidémiologie et Physiopathologie des Virus Oncogènes, Département du SIDA et des Rétrovirus, Institut Pasteur, 25–28 rue du Dr Roux, 75724 Paris Cedex 15, France2

Author for correspondence: Antoine Gessain. Fax +33 1 40 61 34 65, e-mail agessain{at}pasteur.fr


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A serological survey for human T-lymphotropic virus (HTLV)/simian T-lymphotropic virus (STLV) antibodies was performed in 102 wild-caught monkeys and apes from 15 (sub)species originating from Cameroon. Two animals (a Mandrillus sphinx and a Cercocebus agilis) exhibited a complete HTLV-1 seroreactivity pattern while two others lacked either the p24 (a Mandrillus sphinx) or the MTA-1/gp46 bands (a Pan troglodytes). Sequence comparison and phylogenetic analyses, using a 522 bp env gene fragment and the complete LTR, indicated that the two mandrill STLV strains belonged to the HTLV/STLV subtype D clade while the chimpanzee strain clustered in the HTLV/STLV subtype B clade. The Cercocebus agilis STLV strain, the first one found in this species, was closely related to the two HTLV/STLV subtype F strains. Such data indicate that the African biodiversity of STLV-1 in the wild is far from being known and reinforces the hypothesis of interspecies transmission of STLV-1 from monkeys and apes to humans leading to the present day distribution of HTLV-1 in African inhabitants.


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The primate T-lymphotropic viruses type 1 (PTLV-1) (Guo et al., 1984 ; Watanabe et al., 1985 , 1986 ) include human T-lymphotropic virus type 1 (HTLV-1) and simian T-lymphotropic virus type 1 (STLV-1) (Miyoshi et al., 1982 ). HTLV-1 is the aetiologic agent of a malignant CD4 lymphoproliferation [adult T cell leukaemia (ATL)] and of a chronic progressive neuromyelopathy [tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)]. STLV-1, which is endemic in many Old World monkey species (Ibrahim et al., 1995 ; Ishikawa et al., 1987 ), can cause an ATL-like pathology in some infected monkeys. HTLV-1 possesses a remarkable genetic stability and the few nucleotide substitutions observed among viral strains are specific to the patient’s geographical origin rather than to the pathology (Gessain et al., 1992 ; Mahieux et al., 1997 ). Indeed, four major geographical subtypes (genotypes) have been reported: cosmopolitan, HTLV-1 subtype A; Central African, HTLV-1 subtype B; Melanesian, HTLV-1 subtype C; and the more recently discovered subtype D present in Central Africa, mainly in Pygmies. Recently, two other African HTLV-1 variants from Zaire (Efe1) and Gabon (Lib2) were discovered and may be yet considered as the sole human prototypes of new subtypes (E and F) (Mahieux et al., 1997 ; Van Dooren et al., 2001 ).

The origin of most HTLV-1 geographical subtypes appears to be linked with multiple episodes of interspecies transmission between STLV-1-infected monkeys and humans, followed by variable periods of evolution in the human host (Salemi et al., 2000 ; Slattery et al., 1999 ; Van Dooren et al., 2001 ; Watanabe et al., 1985 ). However, clear evidence of interspecies transmission consists only of a described affiliation between HTLV-1 subtype B and subtype D from Central African inhabitants and few STLV-1 isolates from chimpanzees and mandrills respectively (Koralnik et al., 1994 ; Mahieux et al., 1998a ; Voevodin et al., 1997b ). Furthermore, one STLV-1 strain, from a mandrill from Gabon (MSP-Mnd9), was recently found to be closely related to the Lib2 strain supporting the notion of interspecies transmission (Mahieux et al., 1998a ).

Sub-Saharan Africa is considered the largest HTLV-1 endemic area. Regarding STLV-1, serological studies have demonstrated that several African monkey and ape (sub)species are infected by STLV-1, including different subspecies of Papio, Chlorocebus aethiops, Cercopithecus, Erythrocebus patas, Miopithecus talapoin, Pan troglodytes, Mandrillus sphinx, Mandrillus leucophaeus and Cercocebus atys. However, only about 50 African STLV-1 strains have been molecularly partially characterized (Englebrecht et al., 1996 ; Koralnik et al., 1994 ; Mahieux et al., 1998a , b , 2000 ; Nerrienet et al., 1998 ; Saksena et al., 1993 , 1994 ; van Rensburg et al., 1999 ; Voevodin et al., 1996a , 1997a , b ; Watanabe et al., 1986 ), either in a small portion (120 bp) of the pol gene and/or in fragments (300 to 600 bp) of the LTR and/or of the env gene (300 to 522 bp). Some of these STLV-1 strains originate from monkeys or apes kept in captivity, a situation which is known to be associated with some nosocomial interspecies transmissions (Voevodin et al., 1996b ). Furthermore, several of the other strains originate from monkeys living in South or East Africa. Thus, the data regarding African wild-caught monkeys and Apes remain rare, especially from the Western part of Central Africa (Cameroon, Congo, Gabon) where the still-present deep rain forest represents a very important focus and sanctuary for wildlife biodiversity. Thus, our goal was to look for STLV-1 in a wild-caught population of several species/subspecies of monkeys and apes from Cameroon in order to gain new insights into the natural distribution and frequency of the STLV-1 molecular subtypes in such an ecosystem and their relationships with the HTLV-1 present in the same area.

The studied series comprised 102 plasma samples from 15 monkey and apes species or subspecies (Table 1) originating from the Mvog-betsi Zoo, Yaoundé. These animals originated from different areas of Cameroon (mostly the south), where most of them were initially kept as pets for a variable period of time, after their mothers were killed by hunters. They were confiscated by the Ministry of Environment and Forestry (MINEF), and then gathered in the Zoo. For some of these (sub)species (as Cercocebus agilis, Cercopithecus neglectus, C. nictitans or C. cephus cephus) no data were available regarding the presence of STLV-1 infection.


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Table 1. Names and numbers of the different African monkey and ape (sub)species from the Yaoundé Zoo included in this study

 
Among these 102 plasma samples, the two serological screening tests [immunofluorescence (IFA) on MT2 and C19 cells (Mahieux et al., 1997 ) and ELISA (Platellia HTLV-1/2, Sanofi Diagnostic Pasteur)] revealed four positive samples and five borderline or uncertain. These nine samples were further tested by a confirmatory Western blot assay (HTLV-2-4, Diagnostic Biotechnology Singapore). Two of these samples (CAG-DJA.853 and MSP-SAN.855) exhibited a complete HTLV-1 pattern with antibody reactivities against both the Gag p19 and p24 antigens, the recombinant protein GD21 and the HTLV-1-specific gp46 peptide, MTA-1. For two other samples, the seroreactivity was nearly complete with, however, the p24 band lacking for one sample (MSP-BET.854) and the MTA-1 band for the other one (PTR-CAR.875). These four samples were clearly positive by IFA with titres on MT2 cells ranging from 1/80 to 1/640. Only a few other samples reacted, with some faint Gag reactivities (mainly p19) or GD21 alone. The four HTLV-1/STLV-1-seropositive animals (Table 1) included one Cercocebus agilis young male, one Pan troglodytes female and two Mandrillus sphinx (both young females). These four animals very probably originated from South Cameroon and were seropositive for STLV-1 since their arrival in the Zoo.

PCR was performed on high molecular mass DNA extracted from the peripheral blood mononuclear cells or buffy coats of these four animals and from several controls. Two genomic fragments were amplified and sequenced, the complete LTR (755 bp) and a 522 bp fragment of the env gene (Mahieux et al., 1997 ). The seven new LTR and env sequences determined herein were deposited in the National Center for Biotechnology Information database. The GenBank accession numbers are AF38466 to AF38472.

A comparison of the aligned 522 bp fragments of the gp21 env gene obtained for the four STLV-1-seropositive animals indicated that these sequences exhibited no deletions nor insertions as compared to the ATK HTLV-1 reference strain. Furthermore, genetic comparison of these four new sequences with the other published STLV-1 env sequences indicated close similarities between some of them. The two new sequences from Mandrillus sphinx (MSP-BET.854 and MSP-SAN.855), which exhibited only 1 bp difference, were closely related (99% similarity) to three sequences from Mandrillus sphinx from Gabon (Mnd13, 15, 18). The sequence from the Cercocebus agilis isolate (CAG-DJA.853) exhibited only 2·5% (509/522) nucleotide divergence from an STLV-1 strain obtained from another Mandrillus sphinx strain (Mnd9) also originating from Gabon. Lastly, the new sequence from Pan troglodytes (PTR-CAR.875) was closely related (97 to 98%) to three published sequences from Pan troglodytes, two of unknown geographical origin and one from Sierra Leone. Comparison with all the available HTLV-1 strains also indicated some close relationship: the two new mandrill strains were related to HTLV-1 subtype D strains, including the H2-3 strain originating from a Pygmy from South Cameroon. The CAG-DJA.853 strain exhibited only 2·7% of divergence with the Lib2 strain, the sole HTLV-1 representative of subtype F. The new chimpanzee strain (PTR-CAR.875) was also closely related (97 to 98% of similarity) to several HTLV-1 strains of subtype B (H2-4, T49, St DEN...) originating from Cameroon or Gabon respectively.

The LTR (especially the U3 and U5 regions) is a more variable fragment, and is thus more informative for genetic comparisons and phylogenetic analyses. The full LTR sequence was obtained from CAG-DJA.853, MSP-BET.855 and PTR-CAR.875; a partial LTR sequence was also obtained from the second mandrill strain and was nearly identical to the corresponding region in MSP-BET.855. Comparison of the three new complete LTR STLV-1 sequences with the HTLV-1 prototypes indicated that the important regulatory elements of the LTR such as the three tax-responsive elements (TRE), the c-ets-responsive element, the rex-responsive element and the rex-binding region were highly conserved, suggesting that their functions were maintained. The LTR sequence comparative analyses revealed again some close similarities between the two new mandrill strains and the HTLV-1/STLV-1 subtype D strains as well as between the GAG-DJA.853 and the HTLV1/STLV-1 subtype F strains (Mnd9 and Lib2). Lastly, comparison of the new PTR-CAR.875 strain with the few other available subtype B STLV-1 and several HTLV-1 sequences confirm its closest relationship to some human strains (98·8% with T49) rather than to the other Pan troglodytes strains (96·9 to 97·5%).

Phylogenetic analyses were performed on all the available STLV-1 sequences from Africa as well as several representatives of HTLV-1 strains of the different subtypes as previously described (Salemi et al., 1998), using both neighbour-joining (NJ) and DNA maximum parsimony (MP) methods. Similar results were obtained by both methods. Analyses of the trees (Figs 1 and 2) reinforced the close relationship of the four new STLV-1 strains with some HTLV-1 strains. Indeed, the highly supported HTLV/STLV subtype D and subtype F clades comprised the two new mandrill strains and the new Cercocebus agilis respectively. This was clear for the two genomic regions analysed (LTR and env) (Figs 1 and 2). Regarding the new chimpanzee isolate, it clustered always in the large subtype B clade, which comprise several HTLV-1 and very few STLV-1, mostly from Central Africa. However, within this clade, the new chimpanzee strain was closely affiliated (NJ and MP bootstrap=96% for LTR) to two HTLV-1 (T49, PH236) originating from South Cameroon and Gabon.



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Fig. 1. Rooted phylogenetic tree generated by the NJ method with a 522 bp fragment of the env gene encompassing most of gp21 and the carboxy-terminus of gp46. The HTLV-1 subtype C MEL5 prototype sequence was used as an outgroup. The bootstrap analysis was applied on the NJ methods with 1000 data sets. Distance matrixes were generated using the DNADIST program with the Kimura two-parameter algorithm and a transition/transversion ratio of 5·73. The values on the branches indicate frequencies of occurrence for 1000 trees. The four new STLV-1 sequences (in bold) were analysed with 83 STLV-1/HTLV-1 sequences available from the GenBank database. The branch lengths are proportional to the evolutionary distance (scale bar) between the taxa. GenBank accession numbers for the four new env sequences are AF384866 to AF84869.

 


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Fig. 2. Rooted phylogenetic tree generated by the NJ method with the complete LTR (755 bp long in the HTLV-1 strain ATK reference sequence). The HTLV-1 subtype C MEL5 prototype sequence was used as an outgroup. The bootstrap analysis was applied on the NJ methods with 1000 data sets. Distance matrixes were generated using the DNADIST program with the Kimura two-parameter algorithm and a transition/transversion ratio of 4·82. The values on the branches indicate frequencies of occurrence for 1000 trees. The three new STLV-1 sequences (in bold) were analysed with 52 STLV-1/HTLV-1 sequences available from the GenBank database. The branch lengths are proportional to the evolutionary distance (scale bar) between the taxa. GenBank accession numbers for the three new LTR complete sequences are AF384870 to AF384872.

 
In conclusion, our study demonstrates the presence of STLV-1 infection in wild-caught monkeys and apes from Cameroon, Western Central Africa. Because most of these animals were taken out from their natural ecosystem when very young, these infections most probably reflect mother-to-offspring transmission, thus suggesting that the STLV-1 infection level in a representative population of monkeys and apes in the wild might be higher. Indeed, in two large populations of wild-caught African green monkeys from Senegal, the STLV-1 seroprevalence reached 40% with a clear increase with age (Durand et al., 1995 ). Moreover, the finding of three different molecular STLV-1 subtypes in such a small monkey and ape population originating from a relatively restricted area of Africa (less than 5% of the Central African rain forest) suggests that the biodiversity of such STLV-1 infection is still far from being known. Lastly, it is interesting to note that in this monkey and ape series, originating from Southern Cameroon, the three different molecular STLV-1 subtypes found correspond to the three HTLV-1 subtypes that are endemic in the human population living in the same region. Such geographical association reinforces strongly the notion of interspecies transmission of STLV-1 from monkeys and apes to human leading to the present-day distribution of HTLV-1 in inhabitants of these areas.


   Acknowledgments
 
We greatly thank Dr Christopher Mitchell, the Ministry of Environment and Forestry (MINEF) and the Cameroon Wildlife Aid Fund (CWAF), for their support and assistance. We also thank Jermie Mfoupouendoun for his technical assistance. The study was financially supported by the Agence Nationale de Recherches contre le SIDA (ANRS) and L. Meertens is supported by a fellowship from the Ministère de la Recherche. We also thank Renaud Mahieux for helpful discussions and critical review of the manuscript.


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Received 30 May 2001; accepted 15 August 2001.