Pygmy living in a remote area of Cameroon: An ancient origin for HTLV-II in Africa

We report characterization of a human T-cell lymphotropic virus type II (HTLV-II) isolated from an interleukin 2-dependent CD8 T-cell line derived from peripheral blood mononuclear cells of a healthy, HTLV-1I-seropositive female Bakola Pygmy, aged 59, living in a remote equatorial forest area in south Cameroon. This HTLV-II isolate, designated PYGCAM-1, reacted in an indirect immunofluorescence assay with HTLV-I1 and HTLV-I polyclonal antibodies and with an HTLV-I/II gp46 monoclonal antibody but not with HTLV-1 gag p19 or p24 monoclonal antibodies. The cell line produced HTLV-I/II p24 core antigen and retroviral particles. The entire env gene (1462 bp) and most of the long terminal repeat (715 bp) of the PYGCAM-1 provirus were amplified by the polymerase chain reaction using HTLV-IIspecific primers. Comparison with the long terminal repeat and envelope sequences of prototype HTLV-II strains indicated that PYGCAM-1 belongs to the subtype B group, as it has only 0.5-2% nucleotide divergence from HTLV-II B strains. The finding of antibodies to HTLV-II in sera taken from the father of the woman in 1984 and from three unrelated members of the same population strongly suggests that PYGCAM-1 is a genuine HTLV-II that has been present in this isolated population for a long time. The low genetic divergence of this African isolate from American isolates raises questions about the genetic variability over time and the origin and dissemination of HTLV-II, hitherto considered to be predominantly a New World virus. The origin and the routes of the worldwide dissemination of primate T-cell lymphotropic viruses (PTLVs), which include human and simian T retroviruses [HTLV-I (1), HTLV-II (2), STLV-I (3), and STLV-II (4)] and the recently isolated PTLV-L (5), PTLV-PP1664 (6), and STLVpp (7), are under active investigation. Epidemiological and phylogenetic analyses indicate that HTLV-I and STLV-I have been present in the Old World (Africa and Asia) for several millenia (8-11), whereas HTLV-II, which is highly endemic in certain native Amerindian tribes (12-17), has been considered to be a New World virus, brought from Asia into the Americas some 10,000-40,000 years ago during the migration of HTLV-IIinfected populations that occurred over the Bering land bridge (12-17). Recent serological and molecular evidence of sporadic cases of HTLV-II infection in western (18, 19) and central Africa (20-26), however, raises the possibility that HTLV-II or a related retrovirus has also been present in Africa for a long time. This hypothesis was first suggested after the detection of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. HTLV-II-like serological markers (not verified by sequencing) in Pygmies in remote areas of Zaire (22, 24) and Cameroon (25), who are considered to belong to the oldest ethnic groups in central Africa, having lived in those areas for at least 20,000 years (27, 28). We report here the isolation, molecular characterization, and phylogenetic analysis of an HTLV-II from a healthy, 59-year-old Pygmy woman of the Bakola population, an ethnic group living in a remote area of the equatorial forest of south Cameroon in which HTLV-II-like serological markers were found 10 years ago in four individuals (25).** MATERIALS AND METHODS Population Studied. The Bakola Pygmies are an ethnic group of about 400 individuals, living in the area of Akok (Campo district) (2030 N, 100 E), near Kribi in the Ocean Department of southern Cameroon. This group, traditionally hunter-gatherers, is scattered throughout the forest; they now practice rudimentary agriculture but also exchange game for staple foods and manufactured goods with neighboring farmers (27, 28). In February 1994, 41 Bakola Pygmies (19 men and 22 women; mean age, 39 years) were visited, given a medical examination, and then bled (5 ml) after informed consent had been obtained. They were either relatives of the four individuals found to have HTLV-II-like serological markers in 1984, three of whom are now dead and the fourth untraceable, or people living in the same area. Serological Tests. The sera were screened for HTLV-I/Il by an enzyme-linked immunosorbent assay (ELISA; Platelia HTLV-I new; Sanofi, Paris) and an immunofluorescence assay involving HTLV-I (MT2) or HTLV-II (C19) producing cell lines. All positive or borderline samples were tested in a Western blot assay (HTLV 2.3 Diagnostic Biotechnology, Singapore) (29). Cell Culture and Virus Isolation. In March 1994, heparinized blood specimens were collected from the individuals identified as HTLV-I/II-seropositive in the pilot survey and rushed to the Centre Pasteur in Yaounde, where peripheral blood mononuclear cells (PBMCs) were separated and stored in liquid nitrogen. The frozen cells were transported 2 weeks later to Paris and thawed, and 10 million cells were placed in a 50-ml culture flask containing 5 ml of RPMI 1640 medium Abbreviations: PTLV, primate T-cell lymphotropic virus; HTLV, human T-cell lymphotropic virus; STLV, simian T-cell lymphotropic virus; PBMC, peripheral blood mononuclear cell; LTR, long terminal repeat. 'To whom reprint requests should be addressed. **The sequences reported in this paper have been deposited in the GenBank data base [accession nos. Z46888 (LTR) and Z46889 (env gene)].