Laboratoire de Virologie, CERVI, UPRES EA 2387, Hôpital Pitié-Salpétrière, 83 bld de lHôpital, 75651 Paris cedex 13, France1
Unité dEpidémiologie et de physiopathologie des virus oncogènes, Institut Pasteur, 75724 Paris cedex 15, France2
Génétique épidémiologique et structure des populations humaines, INSERM U535, 94276 Kremlin Bicêtre cedex, France3
Centre Pasteur du Cameroun, BP 1274, Yaoundé, Cameroon4
Cancer Research Center, RAMS, Kashirskaya 24, 115478, Moscow, Russia5
Department of Microbiology, Osaka University Medical School, Suita, Osaka, Japan6
Author for correspondence: Henri Agut. Fax +33 1 42 17 74 11. e-mail henri.agut{at}psl.ap-hop-paris.fr
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Abstract |
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The complete genomic characterization of two different HHV-7 strains (Megaw et al., 1998 ; Nicholas, 1996
) as well as the partial nucleotide sequencing of others (Franti et al., 1998
; Poirel et al., 1997
) indicated a very high degree of homology between these strains. Despite the overall high conservation of HHV-7 DNA, critical point changes allowed us to describe distinct alleles of the glycoprotein B (gB), phosphoprotein p100 and major capsid protein (MCP) genes. Two combinations of p100, gB and MCP alleles, designated Co1 and Co2, were tentatively interpreted as the genetic signatures of two major HHV-7 variants (Franti et al., 1998
, 1999
). Starting with preliminary data about the frequency of distinct gB alleles in African, Caribbean and European subjects (Franti et al., 1998
), the hypothesis was made that HHV-7 variants were differently distributed according to the geographical origin of the populations studied.
To explore this hypothesis, we undertook a large study including subjects from different parts of the world. Peripheral blood mononuclear cells (PBMC) were obtained from 297 different individuals, some of them having been previously studied with regard to the epidemiology of human T-cell leukaemia virus type 1 (HTLV-1) infection (Biglione et al., 1999 ; Gessain et al., 1995
; Ureta-Vidal et al., 1999
). These subjects were classified into four continental groups and nine populations subgroups according to their birthplaces. The African group (n=65) included a Bakola Pygmy population from Cameroon (n=13), and a Bantu population from Cameroon (n=44) and Zaire (n=8). The Asian group (n=100) included a South Asian population (n=13) recruited from a very large heterogeneous area including China (n=9), Vietnam (n=1), Sri Lanka (n=2) and Polynesia (n=1), a Japanese population from Osaka and Kagoshima (n=47), and a Mongol population (n=40) from Irkutsk (Siberia). Both Pygmy and Mongol subjects belonged to isolated groups thought to be aboriginal populations. The European group (n=64) consisted of Caucasian French subjects. The American group (n=68) consisted of three populations: subjects from French West Indies (n=18), Noir-marron subjects (n=40) from French Guiana and South Amerindians (n=10) from Argentina. The nested PCR amplification and characterization of HHV-7 genes from PBMC DNA were carried out as previously reported (Franti et al., 1998
, 1999
), except that, in the case of the p100 gene, the previously described p100.4 inner primer was replaced by p100.6 (5' GTAGAAGGAACGATCTTCCTT 3') located at position 1643716457 of reference strain JI DNA (Nicholas, 1996
). The risk of cross-contamination during amplification was kept under control by specific measures: strict separation of the different steps of PCR analysis, an absolute requirement for the negativity of blank reactions and negative controls, repeated testing of coded samples in independent assays.
The three DNA amplicons, related to p100, gB and MCP genes, were 463, 2491 and 351 bp long respectively. They were analysed by both endonuclease restriction and nucleotide sequencing focused on the characterization of the critical positions highlighted in Table 1. The AAT to GAT substitution at codon position 721 of the p100 gene induces an additional MboII cleavage site. Codon ATC at position 763 of the MCP gene also induces an MboII site, while codon ATA at the same position induces an SspI site. Codon ACA at position 774 of the MCP gene induces a MunI site. The GTG codon at position 119 and codon GGG at position 220 of the gB gene correspond to ApaI and BstEII sites respectively. The three other polymorphic sites at the codons 137, 185 and 406 of the gB gene were accessible to nucleotide sequencing only. Except for the AAT to GAT substitution at codon 721 of p100, which induces an aspartic acid to asparagine change, all other nucleotide modifications supporting the definition of HHV-7 alleles were silent at the protein level as previously noted (Franti et al., 1998
, 1999
).
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In the case of 177 samples among the 297 tested (60%), the alleles of the three genes p100, gB and MCP were identified concomitantly, which permitted us to determine the corresponding combinations according to their genetic definition (Table 1). Twelve different combinations, designated Co1Co12, were found. As in the previous preliminary study (Franti et al., 1999
), Co1 and Co2 were the most frequent combinations, with overall detection rates of 52% and 20% respectively (Table 2
). No other combination exceeded 10% of the samples tested. The phylogenetic relationship between the different combinations was investigated using a parsimony approach (PAUP 4.0, D. L. Swofford, Sinauer associates, Sunderland, MA, USA) and a median network analysis (Bandelt et al., 1999
) in the NETWORK software (v2.0b) (http://www.fluxus-engineering.com). Due to the low number of critical point mutations, no recombination event between the three genes could be hypothesized and further considered. In total, 102 distinct phylogenetic trees exhibiting the same parsimony rate were constructed, indicating that many different evolution profiles could explain the present distribution of HHV-7 allele combinations. However, two major groups emerged, as illustrated by network representation (Fig. 1
), and were aggregated around Co1 and Co2 respectively. The first one, surrounding Co1 and designated group 1, included Co4, Co5, Co6, Co8, Co9 and Co11. The second one, surrounding Co2 and designated group 2, included Co3, Co7, Co10 and Co12. Group 1 was predominant in Asia, Africa and American populations of African origin and was also present, albeit at a lower frequency, in European and Mongol populations. Conversely, group 2 was predominant in European and Mongol populations but almost completely absent in the populations of African origin. The differences of variant distribution between population groups were confirmed using the analysis of molecular variance model (AMOVA) (Excoffier et al., 1992
) implemented in the ARLEQUIN software (version 1.1) (http://anthropologie.unige.ch/arlequin) after withdrawal of the American group due to the presence of recently imported populations of African origin. There was a significant difference between the group consisting of European and Mongol populations on one hand, and that consisting of the other Asian and African populations on the other hand (P=0·04). The nonrandom geographical distribution of the different variants confirmed that HHV-7 may behave as a marker of human populations. Each of the two groups of HHV-7 variants was detected in aboriginal populations such as Pygmies for group 1 and Mongols for group 2, indicating the stable long-standing character of this distribution.
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The definition of HHV-7 alleles is based on a low number of crucial nucleotides, which might be considered to be a major limitation of the study. However the reproducibility of variation repertoire at these positions in the present study and previous ones (Franti et al., 1998 , 1999
) unambiguously distinguishes these nucleotide changes from strain-specific polymorphisms which probably exist concomitantly in the HHV-7 genome, as suggested by other authors (Wyatt & Frenkel, 1992
). In theory, this makes possible the use of HHV-7 alleles as genetic markers of human populations. On the other hand, the limited number of relevant positions precludes any interpretation about the mechanism of genetic HHV-7 evolution which might involve point mutations, interallelic recombination or both. This low number may also lead us to consider an unexpected genetic proximity between the HHV-7 strains of two distant populations, such as Japanese and Pygmies as well as Mongols and Europeans. It is possible that this apparently close relationship will disappear when areas of higher variability, if such regions exist in the HHV-7 genome, are investigated and reveal local patterns of genetic evolution. As an example, the original studies on human herpesvirus-8 (HHV-8) variability, focused on very conserved small fragments of two genes, led to an initial classification of HHV-8 strains into three subtypes (Zong et al., 1997
). More recent studies exploiting the significantly higher genetic variability of another gene, K1, led to a different segregation into at least four major subtypes, which was strongly supported by phylogenetic and epidemiological studies (Lacoste et al., 2000
; Poole et al., 1999
; Zong et al., 1999
). Conversely, the human retrovirus HTLV-1 exhibits a remarkably high genetic stability, explained by virus amplification via clonal expansion of infected cells rather than reverse transcription. The few nucleotide substitutions observed among different HTLV-1 strains have permitted us to understand not only virus transmission but also recent or ancient movements of human populations throughout the world (Biglione et al., 1999
; Gessain et al., 1995
; Ureta-Vidal et al., 1999
). To date, attempts to find areas of higher variability in the HHV-7 genome have been unsuccessful, at least in our hands, in particular when studying the glycoprotein L gene (Franti et al., 1999
) and the U89U90 genes (unpublished results). A notable exception is the previously known variability of genomic termini, also reported for HHV-6 (Dominguez et al., 1999
; Isegawa et al., 1999
; Wyatt & Frenkel, 1992
), which is believed to reflect mainly strain-to-strain variations rather than stable differential genetic profiles.
In that sense, it would be very interesting now to compare, within the same populations, the general distribution of HHV-7 variants to those of HHV-8, HTLV-1 and JC virus, a human papovavirus also used as a marker of human groups (Agostini et al., 1997 ). With the present state of knowledge, group 1 of the HHV-7 variants appears to be ubiquitous and more widely spread than group 2. However, the prevalence of group 2 both in Europeans and Mongols is intriguing. It would be tempting to revisit this particular distribution in the search for either local virus evolution related to population migration or specific host factors leading to the long-standing selection of pre-existing virus strains.
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References |
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Received 1 May 2001;
accepted 22 August 2001.
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