Molecular and Phylogenetic Analyses of 16 Novel Simian T Cell Leukemia

Virology 287, 275–285 (2001) doi:10.1006/viro.2001.1018, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Molecular and Phylogenetic Analyses of 16 Novel Simian T Cell Leukemia Virus Type 1 from Africa: Close Relationship of STLV-1 from Allenopithecus nigroviridis to HTLV-1 Subtype B Strains

Laurent Meertens,* Jacques Rigoulet,† Philippe Maucle`re,*,‡ Monique Van Beveren,* Guo Min Chen,* Ousmane Diop,§ Guy Dubreuil,¶ Marie-Claude Georges-Goubot,¶ Jean-Luc Berthier,† John Lewis,࿣ and Antoine Gessain*,1

*Unite´d’Epide´miologie et Physiopathologie des Virus Oncoge`nes, De´partement du SIDA et des Re´trovirus, Institut Pasteur, 25–28 rue du Dr. Roux, 75724 Paris Cedex 15, France; †Muse´um National d’Histoire Naturelle, Paris, France; ‡Centre Pasteur du Cameroon, B.P. 1274, Yaounde´, Cameroon; §Institut Pasteur de Dakar, Dakar, Se´ne´gal; ¶Centre International de Recherches Me´dicales, Franceville, Gabon; ࿣International Zoo Veterinary Group, Keigthley, West Yorkshire BD21 1AG, United Kingdom Received February 8, 2001; returned to author for revision April 6, 2001; accepted May 21, 2001; published online August 6, 2001

A serological survey searching for antibodies reacting with human T-cell leukemia virus type 1 (HTLV-1) antigens was performed on a series of 263 sera/plasma obtained from 34 monkey species or subspecies, originating from different parts of Africa. Among them, 34 samples exhibited a typical HTLV-1 Western blot pattern. Polymerase chain reaction was performed with three primer sets specific either to HTLV-1/STLV-1 or HTLV-2 and encompassing gag, pol, and tax sequences, on genomic DNA from peripheral blood mononuclear cells of 31 animals. The presence of HTLV-1/simian T-cell leukemia virus type 1 (STLV-1) related viruses was determined in the 21 HTLV-1 seropositive animals tested but not in the 10 HTLV-1 seronegative individuals. Proviral DNA sequences from the complete LTR (750 bp) and a portion of the env gene (522 bp) were determined for 16 new STLV-1 strains; some of them originating from species for which no STLV-1 molecular data were available as Allenopithecus nigroviridis and Cercopithecus nictitans. Comparative and phylogenetic analyses revealed that these 16 new sequences belong to five different molecular groups. The A. nigroviridis STLV-1 strains exhibited a very strong nucleotide similarity with HTLV-1 of the subtype B. Furthermore, four novel STLV-1, found in Cercocebus torquatus, C. m. mona, C. nictitans, and Chlorocebus aethipos, were identical to each other and to a previously described Papio anubis STLV-1 strain (PAN 503) originating from the same primate center in Cameroon. Our data extend the range of the African primates who could be permissive and/or harbor naturally STLV-1 and provide new evidences of cross-transmission of African STLV-1 between different monkey species living in the same environment and also of STLV-1 transmissions from some monkeys to humans in Central Africa. © 2001 Academic Press Key Words: Africa; monkeys; primates; retrovirus; STLV-1; HTLV-1; molecular epidemiology; phylogeny.

INTRODUCTION logic studies in monkeys indicate that many Old World species such as macaques, chimpanzees, African green The primate T lymphotropic viruses type 1 (PTLV-1) monkeys, and baboons are STLV-1 carriers (Ibrahim et (Guo et al., 1984; Watanabe et al., 1985, 1986) include al., 1995; Ishikawa et al., 1987; Mahieux et al., 1998b). human T-cell leukemia virus type 1 (HTLV-1) (Poiesz et Molecular epidemiologic studies have revealed the al., 1980; Yoshida et al., 1982) and the simian T-cell remarkable genetic stability of HTLV-1 proviruses, ex- leukemia virus type 1 (STLV-1) (Miyoshi et al., 1982). plained by the viral amplification via clonal expansion of Human T-cell leukemia virus type 1 (HTLV-1) is the etio- the infected cells rather than by reverse transcription logic agent of a malignant CD4 lymphoproliferation (adult (Wattel et al., 1995). Furthermore, such studies have also T-cell leukemia, ATL) (Yoshida et al., 1982) and of a demonstrated that the few nucleotide substitutions ob- chronic progressive neuromyelopathy (tropical spastic served among strains are specific to the geographical paraparesis/HTLV-1 associated myelopathy, TSP/HAM) origin of the patient and are unrelated to the viral pathol- (Gessain et al., 1985). STLV-1 can cause an ATL-like pathology in infected monkeys. Several seroepidemio- ogy (Daenke et al., 1990; Gessain et al., 1992; Komurian et al., 1991; Komurian-Pradel et al., 1992; Mahieux et al., 1995, 1997). A consensus of phylogenetic sequence analyses and/or restriction fragment length polymor- 1 To whom correspondence and reprint requests should be ad- phism (RFLP) data from pol and env genes as well as dressed at Unite´d’Epide´miologie et Physiopathologie des Virus Onco- ge`nes, De´partement du SIDA et des Re´trovirus, Institut Pasteur, 25–28 from the long terminal repeat (LTR) of more than 300 rue du Dr. Roux, 75724 Paris Cedex 15, France. Fax: ϩ 33 (0)1 40 61 34 different HTLV-1 strains has established four major geo- 65. E-mail: [email protected]. graphical subtypes (genotypes): Cosmopolitan, HTLV-1

0042-6822/01 $35.00 275 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 276 MEERTENS ET AL. subtype A (Gessain et al., 1992; Miura et al., 1994); The goals of this study were therefore to gain new Central African, HTLV-1 subtype B (Fukasawa et al., 1987; insights into the natural distribution of the different mo- Gessain et al., 1992; Liu et al., 1994; Paine et al., 1991; lecular subtypes of STLV-1 in Africa and the possible Ratner et al., 1985); Melanesian, HTLV-1 subtype C (Ges- interspecies transmissions of STLV-1 not only between sain et al., 1991, 1993; Saksena et al., 1992; Song et al., monkeys but also from monkeys to humans in this con- 1994; Yanagihara, 1994); and the more recently discov- tinent. ered subtype D present in Central Africa mainly in Pyg- The studied series comprise 263 sera/plasma from 34 mies (Chen et al., 1995; Mahieux et al., 1997; Mboudjeka monkey species or subspecies originating from different et al., 1997). The origin of HTLV-1 geographic subtypes areas of west, central, east, and south Africa with several appears to be linked with episodes of interspecies trans- samples being obtained from wild-caught monkeys. Fur- mission between STLV-1-infected monkeys and humans, thermore, some of the novel characterized STLV-1 strains followed by variable periods of evolution in the human (both in the LTR and in the env gene) originated from host (Franchini, 1995; Giri et al., 1997; Ibrahim et al., 1995; species for which no STLV-1 molecular data were yet Kelsey et al., 1999; Koralnik et al., 1994; Liu et al., 1996; available as Allenopithecus nigroviridis and Cercopithe- Mahieux et al., 1997; Saksena et al., 1994; Slattery et al., cus nictitans. 1999; Song et al., 1994; Vandamme et al., 1994, 1998; Watanabe et al., 1985, 1986). However, clear evidence of RESULTS interspecies transmission consists only of a described affiliation between HTLV-1 subtype B and subtype D from HTLV/STLV serological results Central African inhabitants and few STLV-1 from chim- Among the 263 sera/plasma of African monkeys, indi- panzees and mandrills, respectively (Koralnik et al., 1994; rect immunofluorescence assay (IFA) screening revealed Mahieux et al., 1997, 1998a; Voevodin et al., 1997b). Re- 39 clearly positive samples and 11 uncertain, when using cently, two other African HTLV-1 variants from Zaire the MT2 HTLV-1 producing cells, 33 positives, and 5 (Efe1) and Gabon (Lib2) were discovered and may be uncertain, when using the C19 HTLV-2 producing cells. considered as the sole human prototypes of new sub- Furthermore, by the particle agglutination test, 42 of the types (E and F) (Salemi et al., 1998). Furthermore, one 263 samples scored positive and 6 scored uncertain. All STLV-1 strain from a mandrill from Gabon (MSP-Mnd9) the positive or uncertain samples, detected by one or was recently found to be related to the Lib2 strain sup- both of these screening tests, were further tested by a porting the notion of interspecies transmission (Mahieux confirmatory Western blot assay. Among them, 34 sam- et al., 2000; Slattery et al., 1999; Vandamme et al., 1998). ples exhibited a classical HTLV-1 pattern with antibody Sub-Saharan Africa is considered the largest endemic reactivities against both the gag p19 and the p24 anti- area for HTLV-1 and nearly 200 strains of African HTLV-1, gens, the recombinant protein GD21 and the HTLV-1- mainly from the A and B molecular subtypes, have been specific gp46 peptide MTA-1. It is worthwhile to note that partially characterized (Mahieux et al., 1997). Regarding in the majority of the STLV-1-positive samples, the p24 STLV-1, serological studies have demonstrated that sev- reactivity is often stronger than that found in HTLV-1 eral African monkey (sub)species are infected by STLV-1 infected samples (Fig. 1). The 34 HTLV-1/STLV-1 sero- (Englebrecht et al., 1996; Mahieux et al., 1998a,b; Mone positive animals (Table 1) included four A. nigroviridis, et al., 1992; Nerrienet et al., 1998; Saksena et al., 1993, two Cercocebus torquatus, seven Chlorocebus aethiops, 1994; van Rensburg et al., 1999; Voevodin et al., 1996a, two Cercopithecus m. mona, one C. nictitans, one Cer- 1997a,b), including different subspecies of baboons (Pa- coptithecus preussi,15Papio anubis, and two Papio pio anubis, P. hamadryas, P. cynocephalus, P. ursinus, P. cynocephalus. papio), African green monkeys (Chlorocebus aethiops pygerythus, Chlorocebus a. sabaeus, Chlorocebus a. tan- HTLV/STLV molecular screening talus, Chlorocebus a. vervet, Chlorocebus a. givet), Cer- copithecus (C. patas, C. mitis, C. ascartus, C. alboguro- Peripheral blood mononuclear cells or buffy coats lis), and Erythrocebus patas, Miopithecus talapoin, com- were available for 21 of the 34 HTLV/STLV-1 seropositive mon chimpanzees (Pan troglodytes), mandrills animals. High molecular weight DNA was extracted from (Mandrillus sphynx), drills (Mandrillus leucocephaeus), these 21 samples as well as from 10 HTLV/STLV sero- and Cercocebus atys. However, only around 50 African negative monkey samples (as controls for the polymer- STLV-1 strains have been molecularly and partially char- ase chain reaction (PCR)). DNA samples were then sub- acterized (Englebrecht et al., 1996; Koralnik et al., 1994; jected to PCR using the gag, pol, and tax primer pairs. Mahieux et al., 1998a,b, 2000; Saksena et al., 1993, 1994; The amplified products were analyzed by Southern blot van Rensburg et al., 1999; Voevodin et al., 1996a, 1997a,b; with 32P-radiolabeled probes, either specific for HTLV-1 Watanabe et al., 1986) either in a small portion (120 bp) of (gag, pol, and tax)orHTLV-2(pol) or both (tax) (Table 2). the pol gene or in fragments (300–600 bp) of the LTR All the 21 samples from HTLV-1/STLV-1 seropositive an- and/or of the env gene (300–522 bp). imals scored positive with the HTLV-1 probes but not MOLECULAR EPIDEMIOLOGY OF STLV-1 IN AFRICA 277

Sequences analyses of the fragment of the gp21 env A comparison of the aligned 522-bp fragments indicate no deletions nor insertions as compared to the ATK reference strain. Genetic comparison of the 16 new STLV-1 strains between themselves indicate that they could be grouped in five molecular groups. The three A. nigroviridis sequences were nearly identical to each other (99.8 to 100% of nucleotide similarity) as were the three sequences from P. anubis that exhibited 99.6 to 100% of similarity between themselves. Furthermore, the four sequences from C. torquatus, Cercopithecus m. mona, C. nictitans, and C. aethiops, all from the same area in Cameroon, were identical. Last, the three Senegalese Chlorocebus aethiops sabaeus sequences were also identical to each other as the three from Kenyan Chlorocebus aethiops pygerythrus that exhibited 99. 4 to 100% of similarity between themselves.

TABLE 1 African Monkey Species Serologically Tested for HTLV-1/STLV-1 Antibodies

Species HTLV-1ϩ/testeda

Pan troglodytes 0/29 Gorilla gorilla 0/13 Allenopithecus nigroviridis 4/11 Cercocebus agilis 0/2 Cercocebus (a.) chrysogaster 0/6 Cercocebus torquatus 2/3 Cercocebus (t.) atys 0/1 Cercocebus galeritus 0/1 Cercocebus lunulatus 0/11 FIG. 1. Western blot (WB) analysis using WB from Diagnostic Bio- Chlorocebus aethiops 7/9 technology (HTLV blot 2.4 version). Lane 1 indicates HTLV-1-positive Cercopithecus (c.) cephus 0/2 control; lane 2, HTLV-2-positive control; lane 3, HTLV-negative control; Cercopithecus (m.) campbelli 0/2 lane 4, plasma from a STLV-1-seropositive Cercopithecus nictitans Cercopithecus (m.) lowei 0/1 (CNI-1001); lane 5, plasma from a STLV-1-seropositive Chlorocebus Cercopithecus (d.) roloway 0/4 aethiops aethiops (CAE-AGM03); lane 6, plasma from a STLV-1-sero- Cercopithecus (c.) erythrogaster 0/1 positive Chlorocebus aethiops sabeus (CAE-89032); lane 7, plasma Cercopithecus hamlyni 0/5 from a STLV-1-seropositive Allenopithecus nigroviridis (ANG–VEN). Cercopithecus (lh’) l’hoesti 0/3 Cercopithecus (m.) mitis 0/1 with the HTLV-2 probe (Table 2). Furthermore, all the Cercopithecus (m.) mona 2/3 Cercopithecus neglectus 0/7 control samples (HTLV-1, HTLV-2, and HTLV negative) Cercopithecus (m.) nictitans 1/9 reacted according to their viral genetic specificities. Cercopithecus (lh’) preussi 1/2 Such data strongly suggest that the 21 seropositive Af- Hylobates (n.) concolor 0/1 rican monkeys were infected by STLV-1 strains related to Hylobates (h.) lar 0/1 the HTLV-1 prototype strains. Hylobates (h.) moloch 0/1 Lophocebus albigena 0/5 Lophocebus aterrimus 0/6 DNA sequences analyses from STLV-1 infected Papio hamadryas 0/7 monkeys Papio (h.) anubis 15/25 Papio (h.) cynocephalus 2/8 Two genomic fragments, the complete LTR (755 bp) Papio (h.) papio 0/60 and a 522-bp fragment of the env gene, were sequenced Mandrillus leucophaeus 0/9 for 16 different STLV-1 infected animals (Table 2). The Mandrillus leucophaeus/Sphinx 0/2 complete LTR was also obtained from a DNA sample Mandrillus Sphinx 0/12 from a C. mitis (originating from Zaire) for which the Total 34/263 divergent gp21 was already available (Koralnik et al., a Number of animals with HTLV-1 positive Western profile for each 1994). species. 278 MEERTENS ET AL.

TABLE 2 HTLV-1/STLV-1 Antibody Titers and HTLV/STLV Molecular Screening Results from the 16 HTLV-1/STLV-1 Seropositive Animals for Which Sequences of the LTR and the env Gene Were Obtained in This Study

GAG POL TAX

HTLV-1 antibodies GAG1- SK100- KKPX1- SK43- TR101- GAG2 SK111 KKPX2 SK44 TR102 IFA titers Sampling Geographic WB Probe Probe Probe Probe Probe Probe Probe Probe Probe Names Species location origin MT-2 C19 PA titers pattern KKGAGS SK112 SK188 KKPXS SK45 KKPXS SK45 KKPXS SK45

ANG-WIS Allenopithecus MNHN, Zaire 1/1280 1/640 Ͼ1/32768 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ nigroviridis Meńageriea ANG-MER A. nigroviridis MNHN, Zaire 1/1280 1/640 Ͼ1/32768 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ Meńageriea ANG-VEN A. nigroviridis MNHN, Zaire 1/1280 1/640 Ͼ1/32768 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ Meńageriea PAN-614 Papio anubis Roussetb Kenya 1/640 1/80 1/128 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ PAN-622S P. anubis Roussetb Kenya NS 1/20 1/512 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ PAN-623S P. anubis Roussetb Kenya 1/160 1/40 1/128 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ CMO-1301 Ceocopithecus Limbe, Rescue Cameroon 1/1280 1/320 1/8192 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ mona Centerc CNI-1001 C. nictitans Limbe, Rescue Cameroon 1/640 1/320 1/16384 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ Centerc CAE-1401 Chlorocebus Limbe, Rescue Cameroon 1/2560 1/640 1/16384 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ aethiops Centerc CTO-601 Cercocebus Limbe, Rescue Cameroon 1/160 1/40 1/2048 HTLV-Iϩϩ ϩϪ ϩϩϩϩϪϪ torquatus Centerc CAE-97024 C. aethiops Institut Pasteur Senegal 1/2560 1/640 1/16384 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ Dakard CAE-97022 C. aethiops Institut Pasteur Senegal 1/1280 1/320 1/4096 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ Dakard CAE-89032 C. aethiops Institut Pasteur Senegal 1/2560 1/320 1/16384 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ Dakard CAE-535 C. aethiops CIRMFe Kenya 1/1280 1/320 1/16384 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ CAE-511 C. aethiops CIRMFe Kenya 1/2560 1/640 1/16384 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ CAE-03 C. aethiops CIRMFe Kenya 1/2560 1/640 1/32765 HTLV-Iϩϩ ϩϪ ϩϩϩϩϩϩ

Note. The gag primers (GAG1–GAG2) amplify specifically HTLV-1/STLV-1 sequences, while the pol (SK110–SK111) and tax (KKPX1–KKPX2, SK43–SK44, TR101–TR102) primers amplify both HTLV/STLV-1 and HTLV-2 sequences. The probes KKGAGS, KKPXS, and SK112 are specific for HTLV-1/STLV-1, while the probe SK188 is specific for HTLV-2. The probe SK45 detects both HTLV-1 and HTLV-2. a The three A. nigroviridis were kept at the MNHN for several years. They were the only STLV-1 seropositive monkeys in this center and were the offspring of an old A. nigroviridis female originating from Central Africa. b The three P. anubis were kept in individual cages at the CRSSA for physiological experiments. They were originally wild-caught animals from Kenya. c These four animals were wild-caught monkeys, originating most probably from Southern Cameroon, but have been, however, kept for a few months or/years in captivity, often in the same enclosure, in the Limbe rescue primate rehabilitation center of Southern Cameroon. d The three C. aethiops, kept at the Institut Pasteur of Dakar in individual cages, were originally wild-caught in Senegal. e The three C. aethiops belong to a large troop kept at the CIRMF Gabon but were originally wild-caught in Kenya.

Genetic comparison of the 16 new env 522-bp se- Comparison with all the available HTLV-1 strains indi- quences with the other 36 published STLV-1 env se- cated that most of the new STLV-1 exhibited 3 to 5% of quences indicated close similarities between some of nucleotide divergence with the different HTLV-1 strains them. The three Senegalese Chlorocebus aethiops (data not shown). However, interestingly, the three new sabaeus sequences were very similar to a group of sequences from A. nigroviridis were closely related Chlorocebus aethiops sabaeus strains also originating (more than 98% of similarity) to three HTLV-1 strains of from Senegal. The four sequences from C. torquatus, C. the Central African B subtype (pyg294, H24, and B51 mona, C. nictitans, and C. aethiops exhibited only 1.5% of strains) originating from Gabon and South Cameroon, nucleotide divergence with an STLV-1 strain obtained respectively. Furthermore, the three new P. anubis se- from a P. anubis (PAN503) originating from the same area quences exhibited only 2.5 to 2.3% of nucleotide diver- of Cameroon (Table 3) (Mahieux et al., 2000). The three gence with the lib2 strain, the prototype of HTLV-1 sub- new sequences obtained from P. anubis exhibited 99% type F (Salemi et al., 1998). similarity with the SUF1 strain obtained from a P. hamadryas. Last, the three strains from Chlorocebus aethiops Sequences analysis of the complete LTR pygerythrus originating from Kenya were related to strains from Chlorocebus aethiops aethiops and tantalus The LTR (especially the U3 and U5 regions) is a from Ethiopia and Kenya, respectively (98 and 97% sim- more variable fragment, thus more informative for ge- ilarity). netic comparisons and phylogenetic analyses. How- MOLECULAR EPIDEMIOLOGY OF STLV-1 IN AFRICA 279

TABLE 3 Sequence of Oligonucleotide Primers Used for the Nested-Polymerase Chain Reaction to Ampilfy the Complete LTR and the 522-bp Fragment of the env Gene

Sense Antisense PCR Region Primer product Annealing amplified set Name Primer sequence 5Ј 3 3Ј Name Primer sequence 5Ј33Ј size temperature

5ЈLTR outer 8255Not 8224-TTGAAGAATACACCAACATCCCC-8246 LTRU5E 8737-CGCAGTTCAGGAGGCACCAC-8718 514 bp 55.5°C 5ЈLTR inner 8255Not 8224-TTGAAGAATACACCAACATCCCC-8246 420PLTR 8702-GAACGCGACTCAACCGGCGTGGAT-8679 479 bp 55.5°C 5ЈLTR outer 8200LA 8174-CTCACACGGCCTCATACAGTACTC-8197 LTRU5E 8737-CGCAGTTCAGGAGGCACCAC-8718 564 bp 55.5°C 5ЈLTR inner 8200LA 8174-CTCACACGGCCTCATACAGTACTC-8197 420PLTR 8702-GAACGCGACTCAACCGGCGTGGAT-8679 529 bp 55.5°C 5ЈLTR outer 8200LA 8174-CTCACACGGCCTCATACAGTACTC-8197 3VLTRext 8737-CGCAGTTCAGGAGGCACCRM-8718 564 bp 54°C 5ЈLTR inner 8200LA 8174-CTCACACGGCCTCATACAGTACTC-8197 3VTLRint 8702-GAACGCRACTCAACCGGCRYGGATGG-8677 529 bp 54°C 3ЈLTR outer Enh280 257-TGACGACAACCCCTCACCTCAA-278 5PLTR 774-TCCCGGACGAGCCCCCAA-757 518 bp 55.5°C 3ЈLTR outer 5VLTRext 212-AACCACCCATTTCCTCCCCATG-233 5PLTR 774-TCCCGGACGAGCCCCCAA-757 563 bp 54.5°C 3ЈLTR inner TATAbox 317-CAGGAGTCTATAAAAGCGTGG-337 5PLTR 774-TCCCGGACGAGCCCCCAA-757 458 bp 54.5°C ENV gp21 outer ENV1 6023-TCAAGCTATAGTCTCCTCCCCCTG-6046 ENV22 6774-GGCGAGGTGGAGTCCTTGGAGGC-6752 752 bp 55.5°C ENV gp21 inner ENV1 6023-TCAAGCTATAGTCTCCTCCCCCTG-6046 ENV2 6592-GGGAGGTGTCGTAGCTGCAGGAGG-6569 570 bp 55.5°C

Note. The position of the primers is given according to the HTLV-1 ATK subtype A prototype sequence. The corresponding codes for the degenerated nucleotides are the following: R ϭ A and G; Y ϭ C and T; M ϭ A and C. ever, it is important to note that, in contrast to the strains were examined with two different sequence seg- nearly 200 HTLV-1/STLV-1 sequences available in the ments: the complete LTR and the 522-bp fragment of the gp21 env gene fragment, there are only around 50 env gene. The resultant alignments of 69 complete LTR complete and 90 partial available HTLV-1/STLV-1 LTR (750–755 bp) sequences and 99 sequences of the gp21 sequences. env gene (522 bp) were analyzed by neighbor joining Comparison of the 16 new STLV-1 sequences with the (NJ), maximum parsimony (MP) phylogenetic methods. In HTLV-1 prototypes indicated that the important regula- both analyses, Mel 5 strain, the prototype of the Melane- tory elements of the LTR such as the three tax respon- sian HTLV-1 subtype C, was used as out-group to root sive elements (TRE), the c-ets responsive element, the the tree. rex responsive element, and the rex binding region, were Phylogenetic reconstruction with the set of the com- highly conserved, suggesting that their functions were plete LTR sequences supported eight well-defined maintained. evolutionary clusters (or clades) (Fig. 2) highly sup- The complete LTR analyses showed a distribution of ported with strong bootstrap values in both NJ and MP the 16 new STLV-1 in five molecular groups, similar to (values ranging from 78 to 97%). They consisted of those found for the env gene, with a very low level of three major and five minor clades. The first one con- nucleotide divergence among strains belonging to the sists of the cosmopolitan subtype A clade and com- same group (less than 1%). By contrast there were 3 to prises only HTLV-1 strains of several disparate geo- 6% divergence among strains of different groups. Ge- graphical origins. The second clade (Central African netic comparison indicates close similarities between subtype B) comprises several HTLV-1 strains from some of the already published STLV-1 strains and some Central Africa but also one STLV-1 strain from P. trog- of the new ones obtained in this study, especially the four lodytes (PTR-CH) and our three novel sequences from new strains originating from Cameroon and the PAN 503 Allen’s swamp monkeys (ANG). The third one consists strain from the same geographical area. of HTLV-1/STLV-1 subtype D from Central Africa. The Regarding the comparison with the available HTLV-1 five other clades could be generally separated accord- LTR sequences (data not shown), the only close similar- ing to the geographical origin of the viral strains. They ities concern the three new STLV-1 from A. nigroviridis include, respectively, the HTLV/STLV subtype F and and some HTLV-1 from subtype B (96 to 97% nucleotide four other clades comprising only STLV-1 strains. One, similarity) as well as the three African green monkeys the south African STLV-1 subtype, joins four STLV-1 STLV-1 strains from Kenya (CAE-AGM03, CAE-AGM511, CAE-AGM535) with the Efe-1 strain (97% similarity) orig- from baboons and C. aethiops. This clade is flanked by inating from a pygmy from eastern Zaire and considered the east African STLV-1 subtype (joining four STLV-1 as the subtype E prototype strain (Salemi et al., 1998). from C. aethiops). Another clade includes a group of four baboon strains originating from east Africa. The last one corresponds to the central and west African Phylogenetic analyses STLV-1 subtype, a larger group of eight sequences The evolutionary relationships of the 16 new STLV-1 joining the three new Senegalese strains (C. aethiops sequences relative to previously described genetic sabaeus) and the five STLV-1 from Cameroon. 280 MEERTENS ET AL.

FIG. 2. Rooted phylogenetic tree generated by the neighbor joining (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 out-group. The bootstrap analysis was applied on the NJ methods using 1000 bootstrap samples. The values on the branches indicate frequencies of occurrence for 1000 trees. The 17 new STLV-1 sequences (highlighted in bold with a star) were analyzed 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.

In the LTR analysis, the newly sequenced STLV-1 mitis, data on STLV-1 were available. This was performed both from Zaire, and the subtype E prototype Efe1 strain, from by serological and by molecular means in A. nigroviridis eastern Zaire, are not included in a specific highly sup- and C. nictitans. We thus extend the range of the African ported clade but are flanking the east African STLV-1 primates who could be permissive and/or naturally har- subtype. bor such viruses. In the env analysis, which comprises 99 sequences, Furthermore, we provide new evidence for interspe- the topology of the phylogenetic tree (Fig. 3) was roughly cies transmission of STLV-1 between primates including similar to the one obtained by the LTR analyses for most humans in Africa. The notion of interspecies transmis- of the molecular clades. However, only five of the eight sion from STLV-1 to humans leading to clades in some of LTR clades were phylogenetically supported by strong the present-day living African inhabitants is mainly bootstrap values: the HTLV-1/STLV-1 subtype D group, based on the very close nucleotide relationship between the large west and Central African STLV-1 subtype, the few STLV-1 present in chimpanzees (Voevodin et al., HTLV-1/STLV-1 subtype F, the east African STLV-1 clade, 1997b; Watanabe et al., 1986; Mahieux et al., 1997) and and the Ugandan STLV-1 clade. The 16 new strains were mandrills (Mahieux et al., 1998a) and HTLV-1 of the concordantly classified in the LTR and the env gp21 subtypes B and D, respectively. Thus, the STLV-1 derived gene. from wild-caught mandrills were shown to be genetically undistinguishable from HTLV-1 subtype D strains DISCUSSION (Mahieux et al., 1998a). Furthermore, these animals nat- In this study, we describe the presence of STLV-1 viral urally lived in the same restricted forest area of the strains in monkey species or subspecies for which no western part of Central Africa (South Cameroon, Gabon, MOLECULAR EPIDEMIOLOGY OF STLV-1 IN AFRICA 281

FIG. 3. Rooted phylogenetic tree generated by the neighbor joining method with a 522-bp fragment of the env gene encompassing most of the gp21 and the carboxyl-terminus of gp46. The HTLV-1 subtype C MEL5 prototype sequence was used as an out-group. The bootstrap analysis was applied on the NJ methods using 1000 bootstrap samples. The values on the branches indicate frequencies of occurrence for 1000 trees. The 16 new STLV-1 sequences (highlighted in bold with a star) were analyzed 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. and Central African Republic) as do the humans (mostly ical distribution of A. nigroviridis in the wild. In this pygmies) infected by the HTLV-1 subtype D isolates region, contact between humans and A. nigroviridis are (Mahieux et al., 1997). Regarding STLV-1 subtype B in- frequent, as such swamp monkeys are easily hunted by fected chimpanzees, the situation is however less clear: torchlight from boats during the night and the carcasses first the exact geographical origin and/or the subspecies are frequently sold on the steamers and the pirogues of these infected great apes are not precisely known in that ply the Zaire River (Kingdom, 1997). Our data thus several cases. Furthermore, some of them have been reexamine the hypothesis of STLV-1 transmissions from inoculated with HTLV-1 strains of unknown subtype monkeys to humans in Central Africa. (Voevodin et al., 1997b). In this study, we report the There are also several lines of evidence suggesting detection and molecular characterization of a novel the possible transmission of STLV-1 between different STLV-1 strain, present in three A. nigroviridis kept cur- monkey species: (1) macaque to baboon transmissions rently at the MNHN, Paris. These three novel STLV-1 of STLV-1 has been reported in a primate center (Voevo- strains exhibited very strong nucleotide similarity with din et al., 1996b). (2) STLV-1 clades, phylogenetically several HTLV-1 of the B subtype present in inhabitants supported by high bootstrap values, include STLV-1 from the western part of Central Africa. This virus was strains from different monkey species living in the same very probably present initially in an old wild-caught fe- geographical area. Thus, as an example, we have re- male A. nigroviridis originating from Central Africa and cently reported that STLV-1 from three wild-caught south was transmitted from such a mother to three offspring. African P. ursinus were closely affiliated with two STLV-1 Such data fit quite well with the current known geograph- from south African vervets (C. aethiops pygerythrus) 282 MEERTENS ET AL.

(Mahieux et al., 1998b). In our present study, we report (GD21) protein, and an HTLV-1 specific peptide corre- that four novel STLV-1 found in C. torquatus, Cercopithe- sponding to residues 162–209 of the gp46 protein (MTA- cus m. mona, C. nictitans, and C. aethiops are identical to 1). Stringent Western blot criteria were used and a sam- a P. anubis strain (Mahieux et al., 2000). All five animals ple was considered as HTLV-1/STLV-1 positive when were wild-caught monkeys, originating most probably exhibiting antibodies against both p19 and p24 antigens from Southern Cameroon, but have been however kept as well as against both GD21 and MTA-1. for a few months or years in captivity, often in the same enclosure, in a primate rehabilitation center of Southern HTLV-1/STLV-1 molecular studies Cameroon. In this peculiar case, which provides another Genomic DNA was extracted from peripheral blood example of STLV-1 transmission between different mon- mononuclear cells or buffy coats by the QIAamp DNA key species living in the same environment, it is impos- Blood Mini Kit (Qiagen GmbH, Hilden, Germany). For all sible to exact the precise location of the interspecies polymerase chain reactions, each reaction tube, pre- transmission (in the wild and/or the rehabilitation center). pared in an isolated room, contained 1 ␮g of DNA, 0.2 In conclusion, our data provide further evidence for the mM deoxynucleoside triphosphate mix (Roche, Mann- presence of new STLV-1 in a wide diversity of African heim, Germany), 5 ␮l10ϫ reaction buffer, 2 mM MgCl , monkey (sub)species, suggesting that the biodiversity of 2 and 2.5 U of Taq gold DNA polymerase (Perkin–Elmer, such STLV-1 in the wild is far from being entirely known. Branchburg, NJ) in a total volume of 50 ␮l. For all PCR Our study also reexamines the hypotheses of cross- experiments, a total of 30 cycles (94°C for 30 s, anneal- transmission of African STLV-1 between different monkey ing temperature (ranging from 54 to 56°C according to species living in the same environment but also of the primer sets) for 30 s and 72°C for 30 s) was per- STLV-1 transmissions from some monkeys to humans in formed after a denaturation at 94°C for 9 min. An exten- Central Africa. sion of 10 min was realized after the last cycle. Further studies are ongoing to characterize new To detect the presence of STLV-1 proviruses, three STLV-1 from Central African wild-caught monkeys and primer pairs amplifying fragments of the gag, pol, and tax their possible human counterparts. Such studies per- genes, respectively, were used as previously described formed mainly on individuals with HTLV seroindetermi- (Mahieux et al., 1998b). nate reactivities on Western blot will permit us to gain For the sequences analyses, two series of PCR were new insights into the origin, evolution, and modes of performed to obtain the following. dissemination of these retroviruses, thus opening new (A) The complete LTR (755 bp). Two semi-nested PCR, avenues of research on the co-ancestry of primate ret- one for the 5ЈLTR and the second for the 3ЈLTR, were roviruses and their human hosts. performed as previously described (Mahieux et al., 1997) (Table 3). In some cases we used also new HTLV-1/ MATERIALS AND METHODS STLV-1 consensus or degenerated primers (Table 3 and Fig. 4). Animals studied (B) A 522-bp region of the env gene coding most of the Over a period of 5 years (1995–1999), we obtained 263 gp21 and part of the carboxyl-terminal region of the gp46. serum/plasma samples from wild-caught or captive mon- This region was amplified by semi-nested PCR as pre- keys living in African or French primatology centers viously described (Table 3 and Fig. 4) (Mahieux et al., (CIRMF Gabon, Limbe´ Cameroon, CRSSA, La Tronche, 1997). Rousset) and French zoos (Me´nagerie du Muse´um Na- The 33 nucleotide sequences reported here have tional d’Histoire Naturelle (MNHN), Zoo de Vincennes, been submitted to the GenBank nucleotide sequence Zoo de La Palmyre, Touroparc, Parc du Lunaret). database and have been assigned the Accession Nos. AY026826–AY026841 (env) and AY026842–AY026858 HTLV-1/STLV-1 serological tests (LTR). To detect HTLV/STLV antibodies, we first screened Phylogenetic analyses plasma or sera using two serological tests: a commercial particle agglutination (PA) test (Fujirebio, Japan) and an Multiple sequence alignment was performed with the indirect immunofluorescence assay (IFA) using MT2 ClustalW algorithm implemented in MacVector 6.5 (Ox- HTLV-1-producing cells and C19 HTLV-2-producing cells ford Molecular).The 69 LTR HTLV-1/STLV-1 strains were (Mahieux et al., 1997). IFA and PA was also used to aligned using the complete LTR sequence (ATK nt determine the titers of HTLV-1 antibodies in all the sero- 1–755). The 99 env HTLV-1/STLV-1 strains were aligned positive samples. All the positive or indeterminate sam- using a 522-bp sequence (ATK env polyprotein nt 875- ples were tested by a confirmatory Western blot assay 1398). Phylogenetic analyses were performed using the (Diagnostic Biotechnology, HTLV2.4 Singapore). This kit PHYLIP package with two different methods: neighbor contains disrupted HTLV-1 virions, a recombinant gp21 joining and maximum parsimony method. The SEQBOOT MOLECULAR EPIDEMIOLOGY OF STLV-1 IN AFRICA 283

FIG. 4. Positions and orientations of primers used to amplify the complete LTR and the gp21 region of the env gene. The sequences of the primers are given in Table 3. program generated 1000 data sets that are random re- virus type I strains from South Africa. JAIDS Hum. Retrovirol. 12(3), sampled versions of the aligned sequences. Distance 298–302. matrix was calculated for each data set using the DNA- Franchini, G. (1995). Molecular mechanisms of human T-cell leukemia/ lymphotropic virus type I infection. Blood 86(10), 3619–3639. DIST program with the Kimura two-parameter and em- Fukasawa, M., Tsujimoto, H., Ishikawa, K., Miura, T., Ivanoff, B., Cooper, pirical transition/transversion ratio for the LTR (4.90) and R. W., Frost, E., Delaporte, E., Mingle, J. A., Grant, F. C., et al. (1987). the env region (5.65) were used. These ratios were esti- Human T-cell leukemia virus type I isolates from Gabon and Ghana: mated from data set with the treepuzzle-50 program. The Comparative analysis of proviral genomes. Virology 161(2), 315–320. NEIGHBOR program generated a tree for each data set Gessain, A., Barin, F., Vernant, J. C., Gout, O., Maurs, L., Calender, A., and a consensus tree was constructed by using the and de The, G. (1985). Antibodies to human T-lymphotropic virus CONSENSE program with the majority rule criteria. The type-I in patients with tropical spastic paraparesis. Lancet 2(8452), 407–410. same data sets were treated with DNAPARS program, Gessain, A., Boeri, E., Yanagihara, R., Gallo, R. C., and Franchini, G. based on the maximum parsimony method, with the (1993). Complete nucleotide sequence of a highly divergent human same parameters used in NJ to test the robustness of the T-cell leukemia (lymphotropic) virus type I (HTLV-I) variant from phylogeny. melanesia: Genetic and phylogenetic relationship to HTLV-I strains from other geographical regions. J. Virol. 67(2), 1015–1023. Gessain, A., Gallo, R. C., and Franchini, G. (1992). Low degree of human ACKNOWLEDGMENTS T-cell leukemia/lymphoma virus type I genetic drift in vivo as a means of monitoring viral transmission and movement of ancient We thank the veterinarians and colleagues who provided us some of human populations. J. Virol. 66(4), 2288–2295. the monkey samples studied here. We especially thank Dr. Florence Gessain, A., Yanagihara, R., Franchini, G., Garruto, R. M., Jenkins, C. L., Oliver of Museum National d’Histoire Naturelle; Dr. Thierry Petit of the Ajdukiewicz, A. B., Gallo, R. C., and Gajdusek, D. C. (1991). Highly Zoo la Palmyre; Dr. Lacroix of the National Wild Life Research Center, Saudi Arabia; Dr. Diane Agay and Dr. Antonia Van Uye from the CRSSA; divergent molecular variants of human T-lymphotropic virus type I Dr. Yves Chaduc of Touroparc; and Dr. M. Vassart of the Parc des from isolated populations in Papua New Guinea and the Solomon Lunarets. We also acknowledge the Pandrillus (Cameroon/Nigeria) Islands. Proc. Natl. Acad. Sci. USA 88(17), 7694–7698. directors, Elizabeth Gadsby and Peter Jenkins, for great help in obtain- Giri, A., Slattery, J. P., Heneine, W., Gessain, A., Rivadeneira, E., Desro- ing some of the blood samples studied. This study was financially siers, R. C., Rosen, L., Anthony, R., Pamungkas, J., Iskandriati, D., supported by the Agence Nationale de Recherches Contre le Richards, A. L., Herve, V., McClure, H., O’Brien, S. J., and Franchini, G. SIDA. L.M. is supported by a fellowship from the Ministe`redela (1997). The tax gene sequences form two divergent monophyletic Recherche. We thank Renaud Mahieux for helpful discussions and lineages corresponding to types I and II of simian and human T-cell critical review of this manuscript. leukemia/lymphotropic viruses. Virology 231(1), 96–104. Guo, H. G., Wong-Stall, F., and Gallo, R. C. (1984). Novel viral sequences related to human T-cell leukemia virus in T cells of a seropositive REFERENCES baboon. Science 223(4641), 1195–1197. 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