Laboratory of Viral Pathogenesis, Research Center for AIDS, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawahara-machi, Sakyo-Ku, Kyoto, Japan1
Department of Neuropathology, Chile University, Chile2
Hangaroa Hospital, Easter Island, Chile3
Department of Virology, Faculty of Medicine, Kagoshima University, Japan4
Division of Epidemiology, Aichi Cancer Center Research Institute, Japan5
Author for correspondence: Masahiro Yamashita.Fax +81 75 761 9335. e-mail myamashi{at}virus.kyoto-u.ac.jp
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Abstract |
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Introduction |
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The islands of the Pacific Ocean have been intensively surveyed for the presence of HTLV-I because of their geographical proximity to known endemic areas of infection. This region is not only adjacent to the two endemic foci of HTLV-I in Japan and South America, but it also includes another endemic focus, Melanesia, which is one of the three ethnogeographic regions (Melanesia, Micronesia and Polynesia) of the Pacific Ocean. In contrast, past seroepidemiological surveys have uncovered little evidence of this virus in the Micronesian and Polynesian regions (Tajima et al., 1991 ; Yanagihara, 1994
), except for the cases of HTLV-I infection among some Japanese Americans living in Hawaii (Blattner et al., 1986
; Ureta Vidal et al., 1994a
). During a recent survey of HTLV-I in Chile, we took advantage of an opportunity to conduct a seroepidemiological survey of the population of Easter Island, which is located on the eastern edge of Polynesia, thereby hoping to gain new insights into the prevalence of this virus in the Pacific region. During the survey, we identified one HTLV-I-seropositive case among the Rapa Nui, the native inhabitants of this island. A new HTLV-I isolate derived from this carrier (E-12) was phylogenetically analysed in an attempt to understand the origin and past dissemination of HTLV-I on the island.
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Methods |
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PCR.
DNA was extracted by a conventional method, using proteinase K, from peripheral blood mononuclear cells which were obtained from whole blood samples by the Ficoll gradient method. Thereafter, DNA was subjected to nested PCR to amplify a part of the long terminal repeat (LTR) region approximately 590 bp long and corresponding to positions 99 to 685 in ATK, the prototype Japanese HTLV-I strain (Seiki et al., 1983 ). The nested PCR conditions and the oligonucleotide primers for amplification were described previously (Yamashita et al., 1995
). In addition, we amplified a part of the env gene (522 bp long) which includes the carboxyl terminus of gp46 and almost the entire transmembrane protein gp21, as described previously (Mboudjeka et al., 1997
). Special care was taken in the PCR procedure to avoid contaminating the amplified products. All the genomic DNAs were manipulated in a room free from the amplified products, and a negative control was used in each PCR experiment.
Subcloning and sequencing.
The amplified LTR region fragments were subcloned into plasmid vector pUC119 using the TA cloning method. Approximately 500-bp-long sequences of the LTR region (positions 122 to 628 in ATK) were determined from the cloned PCR products. We sequenced one clone, since nucleotide sequences from different clones of a sample were virtually identical in our previous investigation. The fragment was sequenced in both directions, yielding a 507-bp-long nucleotide sequence. A fragment of the partial env gene was directly sequenced after purification of the PCR products by phenolchloroform extraction. The nucleotide sequences were determined by using an automated DNA sequencer (Applied Biosystems). The new nucleotide sequences in the present study have been deposited in GenBank (accession nos AF013221 and AF132300).
Phylogenetic analysis.
For construction of phylogenetic trees, each set of nucleotide sequences, both newly obtained and previously reported, was aligned by using the computer software CLUSTAL W (Thompson et al., 1994 ) and minor manual modifications. All phylogenetic trees in the present study were constructed by using three methods: the neighbour-joining (NJ) method (Saitou & Nei, 1987
), the maximum parsimony (MP) method and the maximum likelihood (ML) method. We used CLUSTAL W for construction of the NJ trees and PHYLIP version 3.52 (Felsenstein, 1993
), a phylogeny inference package, for construction of the MP trees. In order to ascertain the robustness of the constructed NJ trees, bootstrapping (Felsenstein, 1985
) was done to generate 100 resamplings of the original sequence alignments and pairwise genetic distances were estimated for each resampling by Kimura's two-parameter method (Kimura, 1980
). For the MP trees, 100 resamplings of the original alignment were generated using the SEQBOOT program and then the most parsimonious trees were generated from bootstrapped sequence data using the DNAPARS program. The majority-rule consensus MP tree was generated using the CONSENSE program. The ML trees were generated with the DNAML program (global rearrangements, randomized input order, and outgroup rooting options `on'). The empirical transition/transversion bias was 4·5 for the partial LTR (507 bp) analysis and 3·2 for the gp21 env (522 bp) analysis. Since the statistical evaluation of the branch length and branching nodes is a built-in feature of the DNAML program, bootstrapping was not done in the ML method. The trees were visualized with TREEVIEW (Page, 1996
). In this report, only the phylogenetic trees constructed by the NJ method are shown because the trees constructed by the MP and ML methods were virtually identical to those constructed by the NJ method.
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Results |
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Discussion |
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In contrast, the possibility for direct dissemination of HTLV-I from other Pacific islands to Easter Island must be quite low. The marked difference between the new HTLV-I from Easter Island and the HTLV-Is of the Melanesian region argues against the introduction of the new isolate from the Melanesian region. In addition, although Polynesians are considered to be the first settlers of Easter Island, a Polynesian origin of the new isolate also seems unlikely, as the two HTLV-Is of the Cosmopolitan group from Bellona Island were not closely related to the new isolate, based on the gp21 env gene sequence. Our recent study, in which HTLV-I isolate BEL1 was analysed based on the LTR region, supported the env-based phylogenetic analysis, showing that E-12 clusters with BEL1 (data not shown). Nonetheless, the present results do not completely exclude the possibility that the new isolate is a relic of a past endemic of the virus in Polynesia, since there are still few data on the prevalence of HTLV-I in Polynesia. On the other hand, sporadic infections recently identified in residents of the Marshall Islands and Nauru (Miller et al., 1998 ; Nicholson et al., 1992
), both of which are situated in the Micronesian region, leave open the possibility of some transmission of HTLV-I from Micronesia to Easter Island. In summary, our findings strongly suggest that the new isolate of HTLV-I in Easter Island originated in South America. However, it is evident that more data are needed to understand the past dissemination of this virus in the Pacific region.
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Acknowledgments |
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Footnotes |
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References |
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Received 8 April 1999;
accepted 7 May 1999.