A novel, divergent simian T-cell lymphotropic virus type 3 in a wild-caught red-capped mangabey (Cercocebus torquatus torquatus) from Nigeria

L. Meertens1, V. Shanmugam2, A. Gessain1, B. E. Beer3, Z. Tooze4, W. Heneine2 and W. M. Switzer2

1 Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, Département d'Ecosystème et Epidémiologie des Maladies Infectieuses, Batiment Lwoff, Institut Pasteur, 25–28 rue du Dr Roux, 75724 Paris Cedex 15, France
2 HIV and Retrovirology Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd, MS G-19, Atlanta, GA 30333, USA
3 Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD 20852, USA
4 Cercopan, Calabar, CRS, Nigeria

Correspondence
William M. Switzer
bis3{at}cdc.gov.


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We present here a novel, distinct simian T-cell lymphotropic virus (STLV) found in a red-capped mangabey (Cercocebus torquatus) (CTO-NG409), wild-caught in Nigeria, that showed an HTLV-2-like Western blot (WB) seroreactivity. The complete genome (8920 bp) of CTO-NG409 STLV was related to but different from STLV-3/PHA-PH969 (13·5 %) and STLV-3/PPA-F3 (7·6 %), and STLV-3/CTO604 (11·3 %), found in Eritrean and Senegalese baboons, and red-capped mangabeys from Cameroon, respectively. Phylogenetic analysis of a conserved tax (180 bp) sequence and the env gene (1482 bp) confirmed the relatedness of STLV-3/CTO-NG409 to the STLV-3 subgroup. Molecular clock analysis of env estimated that STLV-3/CTO-NG409 diverged from East and West/Central African STLV-3s about 140 900±12 400 years ago, suggesting an ancient African origin of STLV-3. Since phylogenetic evidence suggests multiple interspecies transmissions of STLV-1 to humans, and given the antiquity and wide distribution of STLV-3 in Africa, a search for STLV-3 in human African populations with HTLV-2-like WB patterns is warranted.

The genome sequence of STLV-3/CTO-NG409 has been deposited in GenBank, accession no. AY222339.


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Three types of primate T-cell lymphotropic viruses (PTLVs) have been described (Gessain et al., 2002; Slattery et al., 1999; Van Brussel et al., 1999). While two of them, PTLV-1 and PTLV-2, include both human T-cell lymphotropic viruses (HTLV-1 and HTLV-2) and their simian T-cell lymphotropic virus counterparts (STLV-1 and STLV-2, respectively), the third type (STLV-3) comprises only simian viruses (Giri et al., 1994; Goubau et al., 1994; Kalyanaraman et al., 1982; Miyoshi et al., 1982; Poiesz et al., 1980; Vandamme et al., 1996). In contrast to STLV-1, which has been found in more than 20 Old World monkey species (Meertens et al., 2001), STLV-2 has been characterized in only two captive troops of bonobos (Pan paniscus) (Giri et al., 1994; Vandamme et al., 1996). Until the end of 2001, the only known STLV-3 isolate (STLV-3/PH969; previously called STLV-L or PH969) had been obtained from a captive Eritrean sacred baboon (Papio hamadryas) (Goubau et al., 1994). In 2002, several studies reported serological and molecular detection and characterization of novel STLV-3s in monkeys originating from different ecosystems of the African continent. These monkeys included Cameroonian red-capped mangabeys (Cercocebus torquatus torquatus; strains CTO-604 and CTO-602) (Meertens et al., 2002) and spot-nosed guenons (Cercopithecus nictitans; strains CNI-217 and CNI-227) (Van Dooren et al., 2001), several Ethiopian sacred and hybrid baboons (P. hamadryasxP. anubis) (Takemura et al., 2002), and, more recently, Senegalese olive baboons (P. papio; strains PPA-F3/F4/F5/F9) (Meertens & Gessain, 2003).

Recently, 13 red-capped mangabeys (rcm) from a monkey sanctuary in Nigeria were tested for retroviral infections (Beer et al., 2001). Twelve of these animals were captured in the wild and came to the sanctuary as orphans. While in captivity, all red-capped mangabeys were housed together in enclosures separated from other monkeys. Blood specimens were obtained on an opportunistic basis as part of the monkeys initial or annual physical examinations in accordance with the animal use and care committee at this institution. Two orphaned monkeys were found to be infected with SIVrcm, and one was also seropositive for STLV antibodies. We present here the characterization of the STLV found in this animal, which appears to be a novel and unique STLV-3.

Serologically, the plasma of this animal reacted in an immunofluorescence assay in an equivalent manner to the HTLV-1- and HTLV-2-producing cell lines, MT2 and C19, respectively, with antibody titres of 1/320. However, by Western blot (WB) analysis, this plasma specimen exhibited an ‘HTLV-2 like’ pattern showing strong reactivities to GD21, p24 and K55 by using the HTLV blot version 2.4 (Genelabs Diagnostics, Singapore) (Fig. 1). This WB profile is very similar to the findings present in most other known STLV-3s. Total DNA was extracted from uncultured peripheral blood lymphocytes of this animal, and an STLV-3-like infection was confirmed by using a PCR assay that differentiates the PTLVs by using generic tax gene primers and type-specific probes as described previously (data not shown) (Busch et al., 2000). The complete proviral sequence of this STLV was then obtained by PCR amplification and sequence analysis of overlapping genomic regions as previously described (Meertens et al., 2002). The entire genome of this novel STLV-3 (called STLV-3/CTO-NG409) was 8920 bp (GenBank accession no. AY222339) in length. STLV-3/CTO-NG409 had a genomic organization very similar to that of other PTLVs and included the structural, enzymatic and regulatory proteins. Interestingly, as with other PTLV-3s, the LTR (698 bp) was smaller than in PTLV-1 (756 bp) and PTLV-2 (764 bp) by having two and not three of the 21 bp repeats (Meertens et al., 2002). Comparison of the complete genome of STLV-3/CTO-NG409 showed a nucleotide sequence similarity of only 61 % to both HTLV-1/ATK and HTLV-2/Mo. In contrast, this new STLV-3 was closer genetically to other STLV-3s but still highly divergent with nucleotide identities of only 88·7 % with STLV-3/CTO-604, 86·5 % with STLV-3/PHA-PH969 and 92·4 % with STLV-3/PPA-F3. Nonetheless, the LTR of STLV-3/CTO-NG409 was 11–14 % divergent from the three other STLV-3s, further demonstrating the uniqueness of this new virus.



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Fig. 1. Western blot (WB) serological pattern of a wild-caught Nigerian red-capped mangabey (CTO-NG409), obtained by using the WB kit from Genelabs Diagnostics (HTLV blot version 2.4).

 
A 180 bp tax sequence and the complete envelope (env) gene (1482 bp) of all available STLV-3 sequences were phylogenetically analysed using the PHYLIP 3.6a package and the Puzzle 4.0.2 program. Maximum-likelihood (ML) was used to estimate parameters by using the Tamura–Nei substitution model, and trees were generated by the neighbour-joining (NJ) and ML methods. In both trees, STLV-3/CTO-NG409 clustered in the PTLV-3 group and more specifically in the Central and West African STLV-3 subgroup with bootstrap values of 78·9 and 82·8 in the tax and env trees, respectively (Fig. 2a, b). Furthermore, the novel STLV-3/CTO-NG409 was clearly related to the Senegalese baboon strains, with strong bootstrap support of 97·9 (NJ) (Fig. 2b) and 99·0 (ML) in the env tree (data not shown).



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Fig. 2. Unrooted phylogenetic trees generated by neighbour-joining (NJ) analysis of the (a) tax (180 bp) and (b) env (1482 bp) sequences of STLV-3 CTO-NG409 from a red-capped mangabey with other available PTLV and HTLV sequences. 1000 bootstrap replicate values are shown on the branches of the tree, and branch lengths are proportional to the evolutionary distance (scale bar) between the taxa.

 
The third codon position (cdp) of the env polyprotein alignment and the TN93 model with discrete gamma-distributed rates among sites implemented in the Puzzle 4.0.2 program were used to test the molecular clock hypothesis and estimate the divergence dates between the STLV-3 strains, as previously described (Meertens & Gessain, 2003; Meertens et al., 2002). The TN93 model has been previously shown by the likelihood ratio test to be the best-fitting substitution model for PTLV genomes (Salemi et al., 2000). In addition, it has been shown that PTLVs evolve following a molecular clock at third cdp's when using the TN93 model (Salemi et al., 2000). The molecular clock model was found to be valid, and the evolutionary rate was estimated at around 1·68x10-6±0·19x10-6 nucleotide substitutions per site per year. Using this value and an upper limit clock calibration date of 60 000 years ago based on anthropological estimates of the migration of populations from Asia to Melanesia, we were able to determine divergence times for the PTLVs. As a starting date for the molecular clock analysis we used the separation between the HTLV-1/Mel5 strain and the African PTLV-1. Branch lengths of the ML tree and their standard errors were then used to deduce the divergence times. We estimated that the separation between the East African and the West and Central African STLV-3 subgroups occurred about 140 900±12 400 years ago. This date was anterior to the separation of the different PTLV-1 subtypes (91 000±8900 years) and the HTLV-2 subtypes (65 600±8400 years), suggesting an ancient origin for the PTLV-3. Interestingly, the novel C. torquatus STLV-3 strain from Nigeria (CTO-NG409) diverged from CTO-604, the only other C. torquatus strain from a nearby area of Cameroon, over 115 400±10 500 years ago.

This study reports the characterization of a novel STLV-3 in a C. torquatus from Nigeria, CTO-NG409 and is the second demonstration that STLV-3 can infect this species in the wild. Interestingly, these two CTO strains show 11·2 % and 13·8 % divergence over their complete genomes and LTRs, respectively. This finding agrees completely with the divergence of 8·9 % in the LTRs of the different STLV-3 strains infecting P. hamadryas strains from Ethiopia and Eritrea (Takemura et al., 2002). Nevertheless, in phylogenetic trees, these STLV-3 strains cluster together in highly supported clades corresponding to their geographical origin rather than by their host phylogeny as described for most of the PTLV-1s (Slattery et al., 1999).

Evidence for multiple interspecies transmissions among the PTLV-1s (Meertens et al., 2001; Nerrienet et al., 2001), along with the widespread distribution of STLV-3 across Africa and its probable ancient existence, raises questions about whether humans are infected with STLV-3-like viruses. The presence of such viruses in red-capped mangabeys, which are intensely hunted in both Nigeria and Cameroon, may support this possibility. So far, limited studies of 24 Africans and 325 non-Africans with HTLV seroindeterminate WB results have not detected any STLV-3-like infection in humans (Busch et al., 2000; Vandamme et al., 1997). Additional studies of larger numbers of human samples, especially from different areas of Africa, with not only indeterminate but also HTLV-2-like WB profiles, will be necessary to assess the rate of infection with STLV-3. The development and use of STLV-3-specific diagnostic reagents will facilitate screening for STLV-3-like infections in both humans and nonhuman primates.


   ACKNOWLEDGEMENTS
 
We thank Vinod Bhullar for her expert technical assistance with the Western blot analysis.

Use of trade names is for identification only and does not imply endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.


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Received 25 March 2003; accepted 3 July 2003.