In February 2002, we recruited 391 women during a clinical survey for sexually transmitted diseases in various remote rural communities of western Ambae Island in the Penama Province of the Vanuatu Archipelago. Ambae Island, also known as Aoba, has a population of ≈9,500. The women participating in this survey were offered a complete clinical examination, with Papanicolaou test analysis for all women >25 years of age. For each participant, we obtained plasma and buffy coats from 5 mL of blood obtained by venipuncture. The blood samples were rapidly transferred to Institut Pasteur de Nouvelle-Calédonie, where plasma and buffy coats were isolated, frozen, and stored (at –80°C) until HTLV screening. Informed consent was obtained from each woman participating in the field survey. This study was approved by the Ministry of Health of the Republic of Vanuatu and was supported by the Vanuatu Family Health Association, a local nongovernmental organization. Samples were taken from 391 women (mean age 36 years, range 16–82 years) with the following stratification by age: 11.2% from women 15–24 years of age, 28.4% from women 25–34 years of age, 31.2% from women 35–44 years of age, 17.4% from women 45–54 years of age, and 11.8% from women ≥55 years of age.

Plasma HTLV-1 antibodies were detected by enzyme-linked immunosorbent assay (ELISA) (HTLV-I+II, Abbott-Murex, Kent, United Kingdom) with Western blot (HTLV-I/II Blot 2.4, Diagnostic Biotechnology, Singapore) used for confirmation. On Western blot, plasma samples were considered HTLV-1–positive if they reacted to the 2 Gag proteins (p19 and p24) and both env-encoded glycoproteins: the HTLV-1–specific recombinant gp46-I peptide (MTA-1) and the specific HTLV-1/HTLV-2 recombinant GD 21 protein. Plasma samples were considered negative when no band were shown and indeterminate when partially reactive (15,16).

Forty-nine of the 391 plasma samples studied tested positive or borderline by ELISA, and 4 of these samples displayed full reactivity on Western blot (Figure 1). One sample also displayed a typical HTLV gag-indeterminate profile (16), and 6 displayed weak reactivity (19 or GD 21 bands). The 4 plasma samples testing positive by Western blot had higher immunofluorescence assay titers on MT2 (HTLV-1) cells than on C19 (HTLV-2) cells and high particle agglutination titers (Table 1). We carried out a second serologic survey on 64 members of the families of the 4 women seropositive for HTLV-1. This survey identified 2 more infected women; 1 was the mother of an index patient, and the other was the sister-in-law of another index patient (Table 1). These results confirm the circulation of HTLV-1 in this population.

High molecular-weight DNA was extracted from buffy coats from the 4 HTLV-1–seropositive women, 5 HTLV-1–seronegative persons, and 6 others with indeterminate Western blot results, by using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). The 15 DNA samples studied were subjected to polymerase chain reaction with primers specific for the human β-globin gene to check that cellular DNA was amplifiable for all samples (17). We then subjected DNA samples to 2 series of polymerase chain reaction to obtain the complete long terminal repeat (LTR) (755 bp) and a 522-bp region of the env gene as previously described (18). Fragments of the appropriate size were amplified for the 4 HTLV-1–seropositive women, whereas the other 11 samples yielded negative results. The amplified products were cloned and sequenced, and phylogenetic studies were performed as previously described (18). Both the complete LTR and the 522-bp env fragment were obtained for the 4 HTLV-1–seropositive women.

The gp21 gene sequences of the 4 HTLV-1 strains involved were almost identical (99.6%–99.8 % nucleotide similarity) and were very similar to those of Melanesian strains. These strains were closely related (99.4%) to certain strains from Solomon Islanders (Mel 4, 8) but were only 97.1%–98.3% similar to strains from Papua New Guinea residents (Mel 2, 7) and from Australian aborigines (MSHR-1), respectively. Finally, the sequences of these new strains diverged from those of HTLV-1 strains from the 3 other main molecular subtypes (A, B, D) by 6% to 9%.

The 4 new HTLV-1 LTR sequences were also very closely related (98%–100% nucleotide similarity). They displayed 2% nucleotide divergence from Mel 5 (from a Solomon Islander), the only available LTR from all the HTLV-1 subtype C strains. However, they also displayed up to 11% nucleotide divergence from HTLV-1 strains from other molecular subtypes.

Phylogenetic analyses were performed on all the available env and LTR HTLV-1 sequences from Melanesia, and on several representatives of HTLV-1 and STLV-1 strains from the various subtypes/subgroups as described (18), by the neighbor-joining (NJ) method. Similar tree topologies were obtained for both genomic regions (Figure 2 and Figure A1). Analyses of these trees confirmed that the 4 novel Vanuatu HTLV-1 strains were closely related to all available HTLV-1 subtype C strains (Table 2). Indeed, in the env analysis, which included 71 HTLV-1 strains (including 12 Melanesian strains and 1 from an Australian aborigine, Table 2) and 55 STLV-1 strains, the 4 new HTLV-1 strains clustered with subtype C (Figure 2). This subtype only includes strains from Australia, Papua New Guinea, the Solomon Islands, and Vanuatu. Within this clade are at least 2 subgroups, strongly supported phylogenetically: 1 comprises the Vanuatu strains and most of the strains from the Solomon Islands (bootstrap values of 88%), and the other comprises the 3 isolates from Papua New Guinea (the Hagahai population), with a bootstrap value of 100%. Two other unique and divergent strains, the only strain available from an Australian aborigine (MSHR-1) and the other from a Solomon Islander (Mel-12), may represent prototypes of 2 other clades within the Melanesian subtype C.

In conclusion, we report, for the first time, the presence of HTLV-1 infection in a Ni-Vanuatu population living in remote villages. We also demonstrate that the viruses infecting these Ni-Vanuatu persons are novel HTLV-1 molecular variants belonging to the Melanesian divergent C subtype. This finding suggests that these viruses were introduced into Vanuatu by ancient migrations of Melanesian populations. The first people to reach Santa Cruz, Banks, Vanuatu, and the Loyalties Islands ≈3,600 years ago seem to have been Austronesian speakers (19). Epidemiologic and clinical surveys are under way in this area to determine the extent of such retroviral infection and associated neurologic and hematologic diseases. In addition, studies of viral and mitochondrial/nuclear DNA are being conducted and should provide insight into the migrations of the first settlers and the origin, evolution, and modes of dissemination of such retroviruses.