Centers for Disease Control and Prevention, Mail Stop G18, 1600 Clifton Road, Atlanta, GA 30333, USA1
Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan2
Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait3
Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan4
Hospital Italiano, Buenos Aires, Argentina5
Author for correspondence: Philip E. Pellett. Fax +1 404 639 0049. e-mail ppellett{at}cdc.gov
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Abstract |
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Introduction |
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Methods |
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PCR amplification and DNA sequencing.
An 886 bp segment representing the nearly complete ORF K1 coding region (nt 76961 of the strain BC-1 genomic sequence; Russo et al., 1996 ) was amplified directly from DNA samples by nested PCR as described previously (Meng et al., 1999
). For some DNA specimens from which the longer product could not be amplified, such as from formalin-fixed, paraffin-embedded tissue, a shorter ORF K1 segment (nt 183545 of the BC-1 sequence) was amplified by nested PCR with primer sets as follows: forward outer primer, 5' GACCTTGTTGGACATCCTGTA 3'; reverse outer primer, 5' GAGTTTCTGGAGTTATATTG 3'; forward inner primer, 5' TTGTGCCCTGGAGTGATT 3'; and reverse inner primer 5' CA(G/T)CGTAAAATTATAGTA 3' (the G/T degeneracy at position 3 was used because of known variation at this position). The annealing temperatures for the outer and inner primer sets were 53 and 48 °C, respectively. Molecular subtype characterization of the right hand side (RHS) of the HHV-8 genome was done as described previously (Poole et al., 1999
). Two different PCR strategies were used for this: a single PCR with a pair of primers unique to the M subtype of ORF K15 and a triple primer PCR set from ORF K14.1 covering the divergent junction of the two subtypes of ORF K15 genes.
Methods for PCR contamination control and direct sequencing of uncloned PCR amplicons were described previously (Meng et al., 1999 ). Both strands were sequenced in their entirety by using a variety of primer-directed strategies.
Alignments and phylogenetic analysis.
DNA sequences of amplified products, excluding the primer regions, were assembled with GelAssemble (GCG, Madison, WI, USA). Alignments were performed with both Pileup and Clustal W (Thompson et al., 1997 ). Aligned sequences resulting from both methods were used for phylogenetic analysis. Analyses were done that included and excluded gapped regions internal to the alignments. To evaluate the suitability of the data for phylogenetic reconstruction, likelihood-mapping analysis (Strimmer & von Haeseler, 1997
) was performed with PUZZLE 4.0. Maximum-likelihood tree construction for phylogenetic relationships was done by quartet puzzling (Strimmer et al., 1997
; Strimmer & von Haeseler, 1996
) with PUZZLE 4.0 and distance methods (GCG).
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Results |
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In previous work (Meng et al., 1999 ; and unpublished data), we found that concatenation of two segments of nucleotide sequence totalling 246 bp, which span the two most variable regions of the K1 gene, VR1 and VR2, allowed derivation of phylogenies that were essentially equivalent to those obtained with nearly complete K1 sequences. We attempted to develop a typing system based on amplification and sequencing of these restricted regions, but were unsuccessful, because the combination of G+C content and predicted hairpin structures in the DNA sequence, plus the highly variable sequence, precluded the design of useful primers targetting the VR2 region. As an alternative, we designed primer sets that spanned the region encoding the amino-terminal half of the extracellular domain of the K1 gene, including VR1. We were able to amplify this 363 bp segment from some clinical specimens that were either unsuitable for amplification of longer segments, such as formalin-fixed, paraffin-embedded tissue, or from which we were otherwise unable to obtain longer K1 PCR products. Phylogenetic inferences derived from the nucleotide and amino acid sequences of the 363 bp segment had topologies similar to trees derived from nearly complete K1 sequences (not shown). In the one exception, three sequences that were identical across the 363 bp segment differed elsewhere in their K1 genes (J14 compared to Au8 and BCBL-B); this can lead to loss of resolution at the lower levels of phylogenetic trees, but does not affect higher level classification (Fig. 1A
).
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Strains in subtype I/A were divided further into two major clades (enlarged in Fig. 1B), in which minor clades previously designated as I-A and I-B, subgroups A1, A2 and A4 and large subgroup A' fell into one clade and subtypes I-C, I-D, I-E and I-F and subgroup A3 fell into the other. Similarly, strains in subtype II/C segregated into two major clades (Fig. 1B
), with subgroups C1 and C in one clade and subgroups C3 and C' in the other.
Nearly all of the newly identified strains were different from each other. J1, J9 and J16, as well as J19, J20 and J21, were identical to each other (J19 and J20 were obtained from the same patient). The Kuwaiti and Argentine strains fell into subtypes I/A and II/C. The two subtype I/A Argentine strains were more divergent within the subtype, with one falling into each of the two main subtype I/A clades. The three subtype II/C Argentine strains clustered relatively closely in one of the two main II/C clades.
The Japanese strains were more diverse, with 12 and 18% maximum pairwise distances at the nucleotide and amino acid levels, respectively (Table 2). Five and eight Japanese strains fell into genotypes I/A and II/C, respectively (Fig. 1
). However, within these genotypes, the Japanese sequences tended to cluster. Strains J1, J8, J9, J14 and J16 clustered within subtype I/A among strains previously designated as I-A or A' (3% maximum pairwise nucleotide distance). Strains J2J5, J7, J17 and J19J21 clustered into the major subtype II/C clade previously designated as C3 or C' (4% maximum pairwise nucleotide distance). Most interestingly, three Japanese strains, all from elderly classic KS patients originally from Hokkaido, were relatively closely related to subtypes D1, D2, III/D3 and E (Table 3
). Furthermore, the pairwise distances between Hokkaido strains J24/J26 and J25 were 6 and 7% at the nucleotide and amino acid levels, respectively; this is approximately equivalent to the distance between subtypes I/A and II/C (Tables 2
and 3
). To acknowledge the distinctions among the Hokkaido strains and to avoid creating more nomenclatural confusion in the interim before a robust nomenclature is established, strains J24 and J26 are provisionally designated subtype Hok1 and strain J25 is designated Hok2.
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PCR-based studies of the K14.1/K15 genomic region at the RHS of the HHV-8 genome revealed that, among 16 Japanese strains, four had the M allele and 12 had the P allele (Table 1). Of the four with the M allele, one had a subtype I/A K1 sequence while three had subtype II/C sequences. Of the 12 strains with the P allele, four were of K1 subtype I/A and five were genotype II/C. All three Hokkaido strains had the P allele.
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Discussion |
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About 5000 Japanese people are infected with HIV-1, one third of whom have haemophilia (National AIDS Surveillance Committee, 2000 ; Kondo et al., 2000
). According to a recent survey, the seroprevalence of HHV-8 in Japan is 1·4% (Katano et al., 2000a
). KS was identified in only 5% of AIDS autopsy cases (National AIDS Surveillance Committee, 2000
; Kondo et al., 2000
) and classic KS cases are very rare, with only 39 certified classic KS cases reported as of 2000 (T. Sata and others, unpublished). Thus, the collection of specimens included in this study represents a large proportion of the known Japanese KS cases spanning many years. To our knowledge, this is the first report of Japanese K1 sequences and genotyping. The routes of HHV-8 transmission in Japan are unknown; we found no relationship between the HIV-1 and HHV-8 genotypes in individuals (Table 1
), which runs counter to co-transmission theories for HIV-1 and HHV-8.
The Kuwaiti and Argentine strains studied here belong to the two widely distributed and more-prevalent HHV-8 subtypes, I/A and II/C. Thus, the Argentine strains are highly diverged from the subtype E strains identified elsewhere in South America (Brazil) in an isolated population of Amerindians; the Kuwaiti strains, although from Asia, are not related to the subtypes thus far found only along the southern and western Pacific Rim. Biggar et al. (2000) proposed that adaptation of HHV-8 to the host genotype can help explain the relatively high seroprevalence of the virus in some isolated populations and suggests the possibility of barriers to transmission between populations. Consistent with this, we suggest the possibility that the subtype I/A and II/C strains have evolved to enable more efficient transmission across human genotypic boundaries. This would explain in part the worldwide distribution of these subtypes, against a background of host genotype-restricted HHV-8 subtypes.
In the current HHV-8 K1 genotyping systems, most strains collected worldwide cluster into two well-populated clades (I/A and II/C). Strains from Africa have been of subtypes IV/B or a specific subgroup of I/A. Members of all three clades can have either the M or P allele at the right-hand side (RHS) of the genome. Strains obtained predominantly from aboriginal or relatively isolated populations form clades that branch at a distance from subtypes I/A and II/C; they have been designated variously as D1, D2, III/D3 and E. The K1 sequences (subtypes Hok1 and Hok2) found in elderly KS patients from the northern Japanese island of Hokkaido are especially interesting. Hokkaido has a large population of Ainu aboriginals, who are considered to be descendants of the native Asian populations of northern Japan and outliers relative to more predominant Asian populations that are thought to have arisen in South-east Asia (Zimdahl et al., 1999 ). Although we do not know the ethnic or genetic origin of the individuals from whom the Hokkaido strains were obtained, the presence of these unusual strains in this area is further evidence that the global migration of HHV-8 paralleled that of humans, rather than it being a recent introduction (Hayward, 1999
). Molecular genetic population analysis based on HLA class II allele frequencies suggests that the Ainu are midway between other east Asian populations, including mainland Japanese, and native Americans (Bannai et al., 1996
). These observations may support the hypothesis that the Ainu are the descendants of the Upper Palaeolithic populations of north-east Asia from which Native Americans are also derived. However, the relatively close relationship between the Hokkaido HHV-8 strains and a previously studied Australian strain classified as subtype III/D3 (67% pairwise nucleotide distance) suggests that the Hokkaido strains may have originated from South-east Asia, where Austronesians originated. Further study will be required to resolve these questions.
The nucleotide distance between the Hokkaido strains and type III/D3 Australian strains (67%) is generally less than that from other D or E strains (511%) and is substantially less than that from I/A, II/C and IV/B strains (1214%) (Table 3). More importantly, the distance between the Hok1 and Hok2 strains is 6 and 8% at the nucleotide and amino acid levels, which is nearly as great as the distance between subtypes I/A and II/C (8 and 12%, respectively). Unless the Hokkaido and III/D3 strains evolved by a mechanism different from that for the I/A and II/C strains, this suggests that the Hokkaido strains may represent two distinct subtypes and that, as more strains are analysed, each of the D subgroups will emerge as independent subtypes.
The current HHV-8 genotyping designations reflect the history of the field but do not allow easy description of phylogenetic relationships that have emerged from the growing collection of strains studied (Table 4). In addition to a dilemma in defining and designating top-level clades, designations for lower levels are fuzzy and sometimes arbitrary. The differences among the current systems are due to limitations of specimen collections (numbers, ethnic background of patients and geographical origin), the analytical methods used for phylogenetic analysis, the use of amino acid versus nucleotide sequences and attempts to preserve linkages between K1 genotyping and earlier typing systems. A new and robust nomenclature and classification system is needed for HHV-8 K1 genotyping that accommodates the current dataset and allows for the incorporation of new information. We look forward to working with others toward this end.
After submission of this manuscript, Lacoste et al. (2000) published a description of K1 sequences from Russian patients with classical KS. Six were of the I/A subtype and one was II/C. These authors also found both the M and P K14.1/K15 alleles in the I/A viruses included in their study.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 August 2000;
accepted 6 November 2000.