our work was performed on a large collection of 4,500 plasma and peripheral blood buffy coat ( pbbc ) samples from different islands of the archipelago , obtained in the framework of our previous studies on human t - cell lymphotropic virus ( htlv-1 ) ( 11,12 ) . the field survey , carried out from april 2003 through august 2005 , has been extensively described ( 11 ) . to detect plasma hhv-8 antibodies , an inhouse immunofluorescence assay ( ifa ) using bc-3 cells expressing only latent - associated nuclear antigens encoded by orf73 , was performed to detect plasma hhv-8 antibodies ( 13 ) . because hhv-8 seroprevalence increases with age in a virus - endemic population , we first tested a series of 376 samples , from persons > 65 years of age ( mean 72 , median 70 , range 6596 years ; 182 men and 194 women ) originating from the 6 provinces of the archipelago ( appendix figure ) . among these 376 plasma samples , 170 ( 45.2% ) were ifa positive at a 1:160 dilution , showing a clear typical nuclear spotted seroreactivity . the hhv-8 seroprevalence was similar between men ( 45.6% ) and women ( 44.8% ) . the prevalence of hhv-8 increased with age , rising from 29.6% ( 6569 years ) to 57.1% ( > 80 years ) ( figure 1 , panel a ) ( p = 0.0005 trend  test ) . this high level of hhv-8 seroprevalence was present in all 6 provinces ( appendix figure ) . a ) age - dependent herpesvirus 8 ( hhv-8 ) seroprevalence rates in 376 ni - vanuatu persons > 65 years and living in 18 islands representative of the 6 provinces of the vanuatu archipelago . seropositivity was based on strict criteria , and only samples clearly reactive at a dilution > 1:160 were considered hhv-8 positive . b ) age - dependent hhv-8 seroprevalence rate in 283 ni - vanuatu persons from 13 families originating from 4 islands ( 3 from loh , 2 from tanna , 4 from ambae , and 4 from esperitu santo ) of the vanuatu archipelago . c ) pedigrees of 2 families from loh island in which the presence of hhv-8 was examined in members of 3 generations . numbers within circles and squares indicate ages of the patients ; nt , not tested . a second serologic survey that used 237 plasma samples taken from 13 families with genealogic trees was performed ( figure 1 , panels b and c ) . among these 237 samples , 12 originated from elderly persons included in the 376 samples tested before . the hhv-8 seroprevalence was clearly age - dependent , rising from 6.9% among children 19 years of age to 28.2% in adults 50 years of age , followed by a new increase in persons > 51 years of age ( 55.9% ) ( p<10 trend  test ) . these results demonstrate for the first time , to our knowledge , that hhv-8 infection is endemic and circulates in the ni - vanuatu population . we then characterized these hhv-8 strains molecularly . all dna samples ( 1 g ) , extracted from the pbbc , hhv-8 infection was determined by a nested pcr to obtain a 737-bp fragment of the open reading frame of the k1 gene ( orfk1 ) . the first pcr was performed with the primer set k1ag75s / k1ag1200as ( 14 ) and followed by a nested pcr with a second set of primers vr1s / vr2as1 ( 15 ) . orfk1 amplification was obtained from 32 ( 21.6% ) of the 148 hhv-8seropositive samples tested but in none of the 26 hhv-8seronegative samples . * ifa , immunofluorescence assay ; lana , hhv-8specific antibody directed against latent nuclear antigen ; pcr k1 , pcr for amplification of a 737-bp fragment of the orfk1 genomic region of hhv-8 ; na , not available . the first group comprises most strains ( 23/30 ) and corresponds to the sequences found in persons from the south central islands of the archipelago ( mallicolo , ambrym , epi , tongoa , emae , and tanna ) ; the second group comprises 4 sequences from persons living in the northern islands of loh ( lo13 , lo17 , lo48 ) and santo ( cesw32 ) ; and the third group involves only 3 sequences ( ml10 , ml36 , ml46 ) from persons living in the northern island of motalava . phylogenetic analyses were performed on the 30 novel sequences obtained in this study , on all subtype d available k1 sequences , and on representatives of the different hhv-8 subtypes / subgroups , as described ( 4 ) . the phylogenetic analyses were performed with all of the sequences available to date in genbank . these sequences include 3 strains from japan ( j24 , j25 , and j26 ) , 1 from australia ( 3au1 ) , 1 from taiwan ( tks10 ) , 1 from new zealand ( zks3 ) , and 1 from wallis ( walks1 ) ( 2,4 , 68 ) . our results demonstrate that the ni - vanuatu hhv-8 clustered in 3 different genotype d subclades , which are highly supported phylogenetically with high bootstrap values of 99% or 100% ( figure 2 ) . the first one comprising most strains corresponds to sequences closely related to each other and to the 2 polynesian strains walks1 and zks3 ( 4,8 ) . the second group ( loh / santo ) , with only 4 sequences , was closely related to the taiwanese strain tks10 ( 8) . the last 3 sequences from motalava were nearly identical to the only strain from australia ( 6 ) . unrooted phylogenetic tree generated by the neighbor joining ( nj ) method with a 624-bp fragment of the k1 gene . the phylogeny was derived by the nj method by using the gtr model in the paup program version 4.0b10 ( sinauer associates , sunderland , ma , usa ) . branch lengths are drawn to scale , with the bar indicating 0.1-nt replacement per site . numbers on each node indicate the percentage of bootstrap samples ( 1,000 ) in which the cluster is supported . only bootstrap values > furthermore , phylogenetic analysis showed a star - like tree with a long branch for the polynesian clade , which includes most ni - vanuatu strains . this finding strongly suggests a common origin or ancestor for these strains , with a possible founder effect ( figure 2 ) . based on stringent serologic and molecular analyses , our study demonstrates for the first time , to our knowledge , that hhv-8 infection is endemic in a melanesian population . our serologic findings are consistent with those found in some remote villages of papua new guinea ( 5,9 ) . on the basis of these studies , it is tempting to suggest that intrafamilial hhv-8 transmission occurs in such populations , as previously demonstrated in highly hhv-8 endemic populations of african origin ( 13 ) . from the molecular point of view , finding such a high molecular diversity of hhv-8 subtype d with some polynesian- , taiwanese- and australian - like strains was surprising . these heterogeneous findings contrast with the more homogenous situation found for htlv-1 genotypes in the same population ( 11 ) . our molecular findings suggest that hhv-8 has been introduced in the ni - vanuatu populations by different migrations of infected persons . this conclusion is strengthened by the clustering of the australian- and taiwanese - like strains in the northern islands of loh and motalava . a variety of scenarios have been proposed to explain the peopling of near and remote oceania , and our data highlight the possible multiple origins of ni - vanuatu ancestors . ongoing molecular studies on both viral and mitochondrial / nuclear dna will contribute to this debate through analyses of the variations observed . indeed , these variations are intimately linked with the dispersal of early human settlers ; analyses of the genetic variability of hhv-8 can help us reconstruct the patterns of human dispersal into oceania ( 11 ) . map of vanuatu archipelago showing the distribution of human herpesvirus 8 ( hhv-8 ) seroprevalence in persons > 65 years of age and living in different provinces . the 6 administrative divisions studied were the torba province , comprising mainly torres and banks islands ; the sanma province , comprising esperitu santo and malo islands ; the penama province , comprising pentecost , ambae , and maewo islands ; the malampa province , comprising malekula , ambrym , and paama islands ; the shefa province , comprising mainly shepherds and efate islands ; and the tafea province , comprising tanna , erromango , and aneityum islands . for each area , the number of persons tested and the number and percentage ( in parentheses ) of hhv-8-seropositive samples ( immunofluorescence assay for latent nuclear antigens ) are indicated . the mean age and the gender of the studied population are also shown . to have a good specificity , we considered as hhv-8 positive only samples that were clearly reactive at a dilution > 1:160 .
we show human herpesvirus 8 with diverse molecular subtype d variants to be highly endemic among the ni - vanuatu population . 
 most k1 genes were nearly identical to polynesian strains , although a few clustered with australian or taiwanese strains . 
 these results suggest diverse origins of the ni - vanuatu population and raise questions about the ancient human population movements in melanesia .