Novel human herpesvirus 8 subtype D strains in Vanuatu, Melanesia.

We show human herpesvirus 8 with diverse molecular subtype D variants to be highly endemic among the NiVanuatu 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.

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Human herpesvirus 8 (HHV-8), or Kaposi Sarcoma Associated Herpesvirus (KSHV), is the etiologic agent of Kaposi sarcoma (KS). HHV-8 is not a widespread ubiquitous virus; its presence is mainly restricted to areas where classic or endemic KS is highly prevalent, i.e., estimates of HHV-8 seroprevalence in the general adult population range from 5% to >50% (1).

Exploiting the highly genetic variability of the HHV-8 K1 gene, molecular epidemiology led to the identification of 5 major K1 subtypes (A-E), some of which appear to be strongly linked to the geographic origin of the samples. Thus, the few known subtype D strains have been reported only in inhabitants from the Western Pacific region (2).

For people of Oceanian ancestry (including Melanesian, Polynesian, and Micronesian), very little data are available on the clinical and molecular epidemiology of HHV-8 and its associated diseases (3-9). Thus, we studied HHV-8 in the Vanuatu, an archipelago in the Southwest Pacific region, formerly named New Hebrides, which contains >80 islands (6 provinces) (Figure 1). Indigenous Melanesians, also called Ni-Vanuatu, constitute 98% of the current population of [approximately equal to] 210,000. A recent study suggested that HHV-8 was rare in the Ni-Vanuatu population (10).

[FIGURE 1 OMITTED]

Our goal for this cross-sectional study was to evaluate the prevalence of HHV-8 in the Vanuatu archipelago by using stringent serologic criteria and to characterize its genetic diversity.

The Study

Our work was performed on a large collection of [approximately equal to] 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 65 96 years; 182 men and 194 women) originating from the 6 provinces of the archipelago (online Appendix Figure, available from www.cdc.gov/EID/ content/13/11/1745-appG.htm). 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% (65-69 years) to 57.1% (>80 years) (Figure 1, panel A) (p = 0.0005 trend [X.sup.2] test). This high level of HHV-8 seroprevalence was present in all 6 provinces (online Appendix Figure).

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 1-9 years of age to 28.2% in adults 50 years of age, followed by a new increase in persons [greater than or equal to] 51 years of age (55.9%) (p<[10.sup.-4] trend [chi square] test). These results demonstrate that HHV-8 infection is endemic, widespread, and circulates in the Ni-Vanuatu population.

We then characterized these HHV-8 strains molecularly. All DNA samples (1 [micro]g), extracted from the PBBC, were first amplified by PCR for human 13-globin sequences to control amplifiability. 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). All PCR products were purified from gel, cloned, and sequenced. Sequences were verified on both DNA strands. ORFK1 amplification was obtained from 32 (21.6%) of the 148 HHV-8-seropositive samples tested but in none of the 26 HHV-8 seronegative samples. Sequences were obtained for only 30 of the 32 ORFKl-positive PCRs (Table).

Comparative sequence analysis indicates that the 30 new sequences differed from each other. Furthermore, among them, 3 groups can be clearly identified. 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. These sequences include 3 strains from Japan (J24, J25, and J26), 1 from Australia (3Aul), 1 from Yaiwan (TKS10), 1 from New Zealand (ZKS3), and 1 from Wallis (WalKS 1) (2,4, 6-8). 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 TKS 10 (8). The last 3 sequences from Motalava were nearly identical to the only strain from Australia (6).

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).

Conclusions

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 Taiwaneselike 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).

Acknowledgments

We thank the Vanuatu Family Health Association, a local nongovemmental association, for its continuous support and interest in this work and Helene Walter and Woreka Mera for their remarkable field work.

This study was financially supported by the Institut Pasteur, the Institut Pasteur de Nouvelle-Caledonie, and by the Centre National de la Recherche Scientifique. Renan Duprez was supported by a fellowship from the Caisse Nationale d'Assurance Maladie et Matemite des Travailleurs Non Salaries des Professions Non Agricoles.

Mr Cassar is a PhD candidate with primary research interest in the clinical and molecular epidemiology and physiopathology of dengue viruses. He is working on the epidemiology of HTLV-1 and HHV-8 in Melanesian populations.

References

(1.) Dukers NH, Rezza G. Human herpesvirus 8 epidemiology: what we do and do not know. AIDS. 2003;17:1717-30.

(2.) Hayward GS, Zong JC. modern evolutionary history of the human KSHV genome. Curr Top Microbiol Immunol. 2007;312:1-42.

(3.) Sengupta SK, Edwards K, Blair A, Hamilton DR, Niblet JS. Childhood Kaposi's sarcoma in Papua New Guinea. Aust Paediatr 3. 1986;22:301-4.

(4.) Duprez R, Cassar O, Hbid O, Rougier Y, Morisse L, Bassot S, et al. Cutaneous disseminated endemic Kaposi's sarcoma in a Polynesian man infected with a new divergent human herpesvirus 8 subtype D. J Clin Virol. 2006;37:222-6.

(5.) Rezza G, Danaya RT, Wagner TM, Sarmati L, Owen IL, Monini P, et al. Human herpesvirus-8 and other viral infections, Papua New Guinea. Emerg Infect Dis. 2001 ;7:893-5.

(6.) Meng YX, Spira TJ, Bhat GJ, Birch CJ, Druce JD, Edlin BR, et al. Individuals from North America, Australasia, and Africa are infected with four different genotypes of human herpesvirus 8. Virology. 1999;261:106-19.

(7.) Meng YX, Sata T, Stamey FR, Voevodin A, Katano H, Koizumi H, et al. Molecular characterization of strains of human herpesvirus 8 from Japan, Argentina and Kuwait. J Gen Virol. 2001 ;82:499-506.

(8.) Zong JC, Ciufo DM, Alcendor D J, Wan X, Nicholas J, Browning PJ, et al. High-level variability in the ORF-KI membrane protein gene at the left end of the Kaposi's sarcoma-associated herpesvirus genome defines four major vires subtypes and multiple variants or clades in different human populations. J Virol. 1999;73:4156-70.

(9.) Suligoi B, Danaya RT, Sarmati L, Owen IL, Boros S, Pozio E, et al. Infection with human immunodeficiency virus, herpes simplex vires type 2, and human herpesvirus 8 in remote villages of southwestern Papua New Guinea. Am J Trop Med Hyg. 2005;72:33-6.

(10.) Satoh M, Kaneko A, Kokaze A, Katano H, Sata T. Seroprevalence of human herpesvirus 8 in the Vanuatu islands in eastern Melanesia. Jpn J Infect Dis. 2006;59:63-5.

(11.) Cassar O, Capuano C, Bassot S, Charavay F, Duprez R, Afonso PV, et al. HTLV-1 Subtype C Melanesian genetic variants of Vanuatu Archipelago and Solomon Islands share a common ancestor. J Infect Dis. 2007:196:510-21.

(12.) Cassar O, Capuano C, Meertens L, Chungue E, Gessain A. Human T-cell leukemia virus type 1 molecular variants, Vanuatu, Melanesia. Emerg Infect Dis. 2005;11:706-10.

(13.) Plancoulaine S, Abel L, van Beveren M, Gessain A. High titers of anti-human herpesvirus 8 antibodies in elderly males in an endemic population. J Natl Cancer Inst. 2002;94:1333-5.

(14.) Lacoste V, Judde JG, Briere J, Tulliez M, Garin B, Kassa-Kelembho E, et al. Molecular epidemiology of human herpesvirus 8 in Africa: both B and A5 K1 genotypes, as well as the M and P genotypes of K14.1/K15 loci, are frequent and widespread. Virology. 2000;278:60-74.

(15.) Kadyrova E, Lacoste V, Duprez R, Pozharissky K, Molochkov V, Huerre M, et al. Molecular epidemiology of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 strains from Russian patients with classic, posttransplant, and AIDS-associated Kaposi's sarcoma. J Med Virol. 2003;71:548-56.

Address for correspondence: Antoine Gessain, Unit6 d'Epidemiologie et Physiopathologie des Virus Oncogenes/URA CNRS 3015, Departement de Virologie, Batiment Lwoff, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris CEDEX 15, France; email: [e-mail]

Olivier Cassar,*[dagger] Philippe V. Afonso, [dagger] Sylviane Bassot, [dagger] Sabine Plancoulaine, [dagger] [double dagger] Renan Duprez, [dagger] Corinne Capuano, [section] Myriam Abel, [paragraph] Paul M.V. Martin,* and Antoine Gessaint

*Institut Pasteur de Nouvelle-Caledonie, Noumea, France; [dagger]Institut Pasteur, Paris, France; [double dagger] Institut National de la Sante et de la Recherche Medicale USSO, Paris, France; [section] World Health Organization/Organisation Mondiale de la Sante, Port-Vila, Republic of Vanuatu; and [paragraph]Ministry of Health, Port-Vila, Republic of Vanuatu

Table. Demographic, geographic, and serologic data of HHV-8
seropositive persons from the Vanuatu Archipelago, confirmed
by molecular analysis *

Virus       Age,         Island of
strain       y     Sex     origin    Province

LO 17        70     F       Loh        TORBA
LO 13        23     F       Loh        TORBA
LO 48        64     F       Loh        TORBA
ML 10        60     F    Mota Lava     TORBA
ML 36        60     M    Mota Lava     TORBA
ML 46        61     M    Mota Lava     TORBA
ES 65        82     F      Santo       SANMA
CES W32      60     F      Santo       SANMA
MA 55        60     F      Maewo      PENAMA
MAL 4        74     M    Mallicolo    MALAMPA
MAL 24       70     M    Mallicolo    MALAMPA
RYM 31       80     F     Ambrym      MALAMPA
RYM 42       74     F     Ambrym      MALAMPA
EP 43        80     F       Epi        SHEFA
EP 58        79     F       Epi        SHEFA
EP 111       74     F       Epi        SHEFA
TON 72       75     M     Tongoa       SHEFA
CTON H54     71     M     Tongoa       SHEFA
EM 1         70     M      Emae        SHEFA
EM 4         75     F      Emae        SHEFA
CEM H2       73     M      Emae        SHEFA
EF 43        73     F      Efate       SHEFA
EF 52        74     M      Efate       SHEFA
EF 55        81     M      Efate       SHEFA
EF 56        75     F      Efate       SHEFA
EF 60        73     F      Efate       SHEFA
CEF H56      72     M      Efate       SHEFA
CEF H67      70     M      Efate       SHEFA
TA 115       77     M      Tanna       TAFEA
TA 116       70     F      Tanna       TAFEA
TA 124       70     F      Tanna       TAFEA
TA 162       70     M      Tanna       TAFEA

Virus       IFA titers                     GenBank
strain        (LANA)        PCR K1      accession no.

LO 17         10.240          +           EF589758
LO 13          160            +           EF589757
LO 48          640            +           EF589759
ML 10          640            +           EF589763
ML 36          640            +           EF589764
ML 46         1.280           +           EF589765
ES 65          160            +           EF589756
CES W32        320            +           EF589746
MA 55         2,560           +           EF589760
MAL 4          320            +           EF589761
MAL 24        1.280           +           EF589762
RYM 31          40            +           EF589766
RYM 42          80            +           EF589767
EP 43         20.480          +           EF589753
EP 58         20.480          +           EF589754
EP 111        1,280           +           EF589755
TON 72        5.120           +           EF589772
CTON H54      2.560           +           EF589747
EM 1           640       + ([dagger])        NA
EM 4          2.560           +           EF589752
CEM H2        1.280           +           EF589744
EF 43          640            +           EF589748
EF 52         1.280           +           EF589749
EF 55          640            +           EF589750
EF 56         5.120      + ([dagger])        NA
EF 60          640            +           EF589751
CEF H56         80            +           EF589742
CEF H67        640            +           EF589743
TA 115        1.280           +           EF589768
TA 116         320            +           EF589769
TA 124        5.120           +           EF589770
TA 162        5.120           +           EF589771

* IFA, immunofluorescence assay, LANA, HHV-8 specific 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.

([dagger]) Weak PCR signal.