MRC Virology Unit, Church Street, Glasgow G11 5JR, UK1
Molecular Virology Group, Department of Medical Microbiology and Genitourinary Medicine, The University of Liverpool, Liverpool L69 3GA, UK2
Uganda Cancer Institute, Old Mulago Hospital, PO Box 3935, Kampala, Uganda3
Department of Statistics, University of Oxford, 1 South Parks Road, Oxford OX1 3TG, UK4
Author for correspondence: Andrew Davison. Fax +44 141 337 2236. e-mail a.davison{at}vir.gla.ac.uk
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Abstract |
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Introduction |
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The highest seroprevalence of HHV-8, with estimates ranging from 11 to 77%, has been reported for sub-Saharan Africa, followed by the Mediterranean countries (228%) and then Northern Europe, Southeast Asia and the Caribbean countries (24%) (reviewed by Schulz, 1998 ; Chatlynne & Ablashi, 1999
). Estimates of seroprevalence in the USA range from 0 to 20% (Chatlynne & Ablashi, 1999
). In spite of high HHV-8 seroprevalence in most of sub-Saharan Africa, endemic KS is confined to East and Central Africa (Beral, 1991
), suggesting the presence of pathogenic variants or the involvement of co-factors. The mode by which HHV-8 is transmitted is not known, but, in countries where KS is non-endemic, HIV-1-infected homosexual men are mainly affected, indicating that transmission most probably occurs via sexual means (Simpson et al., 1996
; Schulz, 1998
). In contrast, in countries where KS is endemic, such as Uganda and Zambia, transmission frequently occurs in early childhood, suggesting a non-sexual route (Kasolo et al., 1998
; Mayama et al., 1998
).
The HHV-8 genome consists of a unique region of approximately 140·5 kbp flanked by multiple 801 bp terminal repeats. Two almost complete genome sequences are available, one from an AIDS primary effusion lymphoma cell line, BC1 (Russo et al., 1996 ), and one from an AIDS KS lesion (Neipel et al., 1997
), as well as sequences of specific regions for other strains. The great majority of the genome is highly conserved, but genes at both ends of the unique region exhibit striking variation.
The K1 gene, at the left end of the genome, encodes a type 1 membrane protein (Kasolo et al., 1998 ; Cook et al., 1999
; Meng et al., 1999
; Zong et al., 1999
). The amino acid sequence shows up to 44% divergence, with variability concentrated in two extracellular domains, VR1 and VR2 (Cook et al., 1999
; Meng et al., 1999
; Zong et al., 1999
; Lacoste et al., 2000
a). Variability is driven by positive selection (Cook et al., 1999
), an evolutionary phenomenon in which non-synonymous nucleotide changes out-number synonymous changes, with striking alterations in amino acid sequence. The K1 protein has cell-transforming potential (Lee et al., 1998
) and is a constitutive signal transducer (Lagunoff et al., 1999
). Analyses of K1 gene variation have defined five HHV-8 subtypes, generally called A, B, C, D and E (Cook et al., 1999
; Meng et al., 1999
; Zong et al., 1999
; Biggar et al., 2000
). Subtype B appears to predominate in Africa, together with a variant (A5) of the A subtype so far seen only in African samples from Zambia (Kasolo et al., 1998
), Uganda and Tanzania (Cook et al., 1999
; Meng et al., 1999
; Zong et al., 1999
; Lacoste et al., 2000
a). Subtypes A and C occur in Europe and the USA, subtype C in Northern Asia, subtype D in Southern Asia, Australia and New Zealand, and subtype E in Brazilian Amerindians. Hayward (1999)
proposed that HHV-8 branched into its various K1 subtypes over the last 105 years, correlating with the migratory patterns of human populations out of Africa.
The K15 gene, at the right end of the genome, occurs as two highly diverged alleles known as P (predominant or prototype) and M (minor) that show only 30% amino acid sequence identity (Glenn et al., 1999 ; Poole et al., 1999
; Choi et al., 2000
). Each allele comprises eight exons specifying a protein with 12 membrane-spanning domains and a C-terminal cytoplasmic domain. This structure resembles that of EpsteinBarr virus LMP2, except that LMP2 possesses an N-terminal, rather than a C-terminal, cytoplasmic domain (Sample et al., 1989
). The K15 proteins possibly play a role in signal transduction (Hayward, 1999
). The P allele is the more frequent among HHV-8 genomes characterized, and has been found in association with all five K1 subtypes (Poole et al., 1999
). The rarer M allele has thus far been found in association with the A, B and C subtypes. It has been reported in various parts of the world, including some parts of Africa, but not East Africa (Poole et al., 1999
; Lacoste et al., 2000
a, b
; Meng et al., 2001
).
Poole et al. (1999) showed that patterns of divergence at various loci are generally correlated with the K1 subtype. However, the type of K15 allele appeared to be essentially unrelated to the K1 subtype. Furthermore, evidence for recombination was observed in 2030% of strains examined. Almost half of the African strains analysed in this study displayed mosaic genomes that probably reflect a history of recombination within and among subtypes.
In this paper, we describe the first analysis focused on HHV-8 strains in Uganda, a country in which KS is a prominent disease. We investigated the prevalence of HHV-8 antibodies in Ugandan blood donors and characterized the genomes of HHV-8 strains present in Ugandan KS patients.
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Methods |
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Serological tests.
Serological tests were performed in Liverpool, UK. Plasma samples (diluted 1:100) were screened for antibodies to HHV-8 ORF65 and ORF73 recombinant proteins (Simpson et al., 1996 ; Rainbow et al., 1997
) by ELISA, as described previously (Simpson et al., 1996
). An immunofluorescence (IF) test for the latency-associated nuclear antigen (LANA/ORF73) (Rainbow et al., 1997
) was then performed on paraformaldehyde-fixed BCP-1 cells (Boshoff et al., 1998
) at a plasma dilution of 1:50. Samples that reacted only in the ELISA (one or both antigens) were subjected to confirmation by Western blot analysis against recombinant ORF65 protein. Overall, samples were interpreted as being positive if they were positive in both the IF test and the ELISA (one or both antigens), or in the IF test alone or in the ELISA alone followed by a positive Western blot result.
KS DNA samples.
HHV-8-positive DNA samples used for genome characterization were derived from KS skin biopsies collected from 30 (25 male, 5 female) unrelated, adult Ugandan KS patients attending the Uganda Cancer Institute (UCI) clinic at Mulago Hospital, Kampala, during AprilJuly 1998. Mulago Hospital is the major national referral hospital in Uganda, and UCI receives most KS referrals from across the country. The patients in this study came from the southern part of the country and were all diagnosed clinically and histologically, with the exception of one (Ugd30), who became unavailable after preliminary clinical diagnosis. Ethical approval for the study was obtained from the Uganda AIDS Research Committee and the Uganda National Council for Science and Technology. Informed consent was obtained from each patient before samples were taken.
A summary of the demographic and clinical presentation data is available as supplementary data in JGV Online (http://vir.sgmjournals.org). The age range of patients was 1670 years (median age 38 years). HIV testing was performed at the MUJHU laboratory using two commercial ELISA kits, and indeterminant samples were confirmed by Western blot analysis. Twenty patients were HIV-1-positive and ten were -negative. Biopsies were collected in absolute ethanol kept on liquid nitrogen to minimize DNA degradation. The ethanol was removed following overnight incubation at -80 °C, and the biopsies were stored at 4 °C.
Tumour DNA was extracted at the MUJHU laboratory as described previously for fresh frozen biopsy samples (Cook et al., 1999 ). All samples yielded a PCR product on amplification with HHV-8 ORF26-specific primers (Chang et al., 1994
; Boshoff et al., 1995
). As a control for DNA extraction, the
-globin gene was also amplified.
PCR products.
HHV-8 genomes were characterized in Glasgow, UK. PCR products (sizes in parentheses) were generated from seven loci: K1 (1277 bp), K3 (635 bp), ORF26 (571 bp), K9 (594 bp), T0.7/K12 (648 bp), ORF75 (749 bp for genomes with the K15 P allele and 1487 bp for the genome with the K15 M allele), K15 P (285 bp to identify the allele and 2494 bp to sequence it completely) and K15 M (298 bp to identify the allele and two overlapping regions of 1374 and 1483 bp to sequence it completely) (Table 1). The sizes of PCR products and their corresponding primer pairs are listed in JGV Online (http://vir.sgmjournals.org). Primary PCR products (i.e. not nested) were used, except for the K1 locus in three samples (Ugd4, Ugd12 and Ugd24), which was obtained as a 874 bp product by nested PCR.
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PCR products were purified directly from solution or from agarose gels using a commercial kit (Hybaid). K1, K15 and certain ORF75 products were cloned into pGEM-T (Promega) and sequenced using universal and gene-specific primers. Three independent clones were sequenced for each product to exclude PCR-induced errors. All other products were sequenced directly using the appropriate PCR primers. Samples were sequenced using an ABI PRISM 377 DNA sequencer. Data were derived from both DNA strands for all producs.
Sequence analysis.
DNA sequences were assembled and edited using SAP or Pregap4/Gap4 (Staden, 1987 ; Staden et al., 1998
) and aligned using Pileup and Pretty (GCG, Madison, WI, USA). K1 sequences were analysed phylogenetically together with published sequences (Cook et al., 1999
; Zong et al., 1999
; Lacoste et al., 2000
a) by the neighbour joining method, using Seqboot, Dnadist, Neighbor and Consense from the PHYLIP package version 3.572 (University of Washington, Seattle, USA). Network analysis (Bandelt et al., 1995
) was used to summarize relationships between the sequences at all other loci. A list of published sequences used in this study (apart from most of those in Fig. 1
) is given in Table 2
.
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Results |
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K1 subtypes in KS patients
PCR products and K1 sequences were obtained for 17 samples, including two (Ugd1 and Ugd2) described previously (Cook et al., 1999 ). A phylogenetic analysis of these strains and 35 published K1 sequences (Cook et al., 1999
; Zong et al., 1999
; Lacoste et al., 2000
a) is presented in Fig. 1
. The majority (11) of the Ugandan samples cluster with the B subtype, five (Ugd4, Ugd12, Ugd16, Ugd18 and Ugd24) with the A subtype and only one (Ugd23) with the C subtype. Within the B subtype, Ugandan strains fall into three clusters, and Gambian samples into two others. DNA sequence divergence within and between the B variants was up to 2·6% and 5%, respectively. All the Ugandan A strains cluster closely with the previously identified A5 variant, Ug374 (Cook et al., 1999
), and differ from other A strains by up to 5%. Ugd23 and its close relative K1-8/Dem, which originated from the Central African Republic (Lacoste et al., 2000
a), branch early in the C lineage. The relatively low bootstrap value (76) for this node implies that inclusion of these strains in the C subtype is tentative. Indeed, in addition to unique differences, characteristics of the A and C subtypes are present throughout the Ugd23 sequence, indicating that the K1 gene in this strain is not a recombinant between the A and C subtypes.
In summary, analysis of the K1 gene identified three HHV-8 subtypes (A5, B and an unusual C variant) in Ugandan KS patients, subtype B being predominant.
K15 genotypes in KS patients
To identify the K15 alleles present in 30 KS patients, tumour DNA samples were amplified using K15 M- and P-specific primers. Details of the samples analysed and primers used are available as supplementary data on JGV Online (http://vir.sgmjournals.org). Twenty-six samples gave products with P- but not M-specific primers, and one (Ugd10) gave a product with M- but not P-specific primers. Three samples gave no product with either primer pair, probably because of low total DNA concentration or low viral DNA copy number.
The entire K15 gene (2101 bp) from Ugd10, with flanking sequences (about 200 bp on each side, with the downstream region extending to the beginning of ORF75), was sequenced. The sequence is closely related to that in the BC1 genome (Fig. 2A). The two sequences are equal in length, except that BC1 lacks an A residue within exon 1 (previously identified as an error; Glenn et al., 1999
; Poole et al., 1999
) and has an additional C residue in intron 5. Both sequences exhibit major divergence from the P allele, commencing at the same position. The Ugd10 K15 gene differs from that of BC1 by 25 nucleotide substitutions, equivalent to 1·2% divergence (Fig. 2A
). In coding regions, six substitutions are synonymous and nine are non-synonymous; ten substitutions are within introns. One substitution was identified in the upstream flanking region and none in the downstream region.
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Identification of recombinants
The Ugandan samples described above were analysed using networks at five additional loci spaced out at roughly equal intervals across the genome: K3 (0·14 map units), ORF26 (0·35), K9 (0·61), T0.7/K12 (0·85) and ORF75 (0·97) (Table 1). The ORF75 locus was chosen because of its location immediately adjacent to K15. Where available, corresponding sequences from BC1, BCBL-R and GK18 were included.
Network analysis is a way of illustrating relationships between closely related sequences in a way that distinguishes single nucleotide differences and enables likely histories to be inferred by parsimonious reconstruction of ancestral states. Networks, including that for K15 P, are shown in Fig. 3. Those for K3 and ORF26 were similar, and were combined (Fig. 3A
). That for K9 contained a single variable site, and was combined with that for T0.7/K12 (Fig. 3B
). A third group for the K15 M allele (Ugd10 and BC1) is not shown because it is very distant from the P allele. Examination of the patterns, together with those identified in K1 (A BC1, Ugd4, Ugd12, Ugd16; B Ugd2, Ugd10, Ugd15, Ugd19, Ugd29; C Ugd23), showed that some strains grouped differently at different loci. Firstly, as described above, three strains (Ugd4, Ugd12, Ugd16) have A5 K1 genes and are closer to BC1 than to other Ugandan strains with B K1 genes. These, however, cluster with strains possessing B K1 genes, and not with BC1, in K3ORF26, K9T0.7/K12, ORF75 and K15. This suggests recombination between K1 and K3. Secondly, Ugd10, which does not cluster with BC1 in K1 and K3ORF26, appears to be linked to this strain in K9T0.7/K12, ORF75 and K15, suggesting recombination between ORF26 and K9-T0.7/K12. Thirdly, the K1 C variant (Ugd23) has a distinct sequence at K3ORF26, but clusters with other strains at the three remaining loci, suggesting recombination between ORF26 and K9-T0.7/K12. There is no evidence for recombination in strains Ugd2, Ugd15, Ugd19 and Ugd29, as they grouped similarly at all loci. Moreover, all strains co-segregated in K9-T0.7/K12, ORF75 and K15, thus yielding no evidence for recombination in this region. Analysis of a combined network for the ten strains comprising data for ORF26, T0.7/K12, ORF75 and K15 also revealed evidence for recombination at points between ORF26 and T0.7/K12, but not in the region encompassing T0.7/K12, ORF75 and K15 (data not shown). This network also provided no evidence for recombination within the loci investigated. The network analysis, which is independent of external categorizations of nucleotide pattern, indicated that five of the nine Ugandan strains examined (Ugd4, Ugd10, Ugd12, Ugd16 and Ugd23) are recombinants.
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In ORF26, the Ugandan sequences fall into the B, B/C or C patterns (Table 3). The substitution sites for T0.7/K12 and ORF75 are shown in Fig. 4
. The latter locus was analysed in conjunction with the 209 bp region (UPS75') which extends upstream from ORF75 to the point where the P and M alleles become distinct, at 25 or 21 bp downstream from the K15 P or M termination codons, respectively. With the exception of Ugd10, the Ugandan strains fall into nucleotide patterns reported previously (Fig. 4
; Table 3
). In genomes with the K15 M allele, Poole et al. (1999)
noted a nucleotide pattern (designated M) that was associated with the presence of the K15 M allele and extended into ORF75 and, in some cases, T0.7/K12. Ugd10 shares characteristics with both the B and M patterns at these loci and is distinct from that of the other Ugandan strains (Fig. 4
). We have therefore denoted the Ugd10 pattern in the T0.7/K12 and ORF75 loci as M'. Ugd10 is identical to BC1 in UPS75'.
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Discussion |
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Antibodies were detected in 67% and 45% of Ugandan blood donors by ORF65 ELISA and LANA IF, respectively. The results of these two tests are not in absolute agreement, as is usually the case with African samples (Mayama et al., 1998 ). Both tests detected antibodies in all 19 Ugandan KS patients tested. Combined, the two tests detected antibodies in a high percentage of blood donors (74%), indicating that HHV-8 infection is widespread in Kampala, and probably in Uganda as a whole. Attempts to detect HHV-8 in peripheral white blood cells by PCR were largely unsuccessful, probably because HHV-8 occurs intermittently and at very low levels in healthy individuals (Whitby et al., 1995
; Simpson et al., 1996
). Consistent with previous findings (Mayama et al., 1998
), we found no significant difference in HHV-8 antibody prevalence between males and females, even though KS tends to be more common in males. This highlights the potential importance of co-factors in the development of KS.
We identified representatives of three of the five subtypes of the K1 gene in biopsy samples from 17 Ugandan KS patients: five A5 variants, 11 subtype B strains and a single subtype C variant. These results strengthen the evidence that the K1 B subtype predominates in Uganda and in Africa (Cook et al., 1999 ; Meng et al., 1999
; Zong et al., 1999
; Lacoste et al., 2000a
). A recent compilation of 75 K1 sequences originating from African patients or from individuals of African origin (but living elsewhere) indicated that A5 variants may be as prevalent and widespread in Africa as the B subtype (Lacoste et al., 2000a
). A5 variants have been detected in samples from Zambia, Tanzania, Uganda, Cameroon, the Central African Republic and in Creoles, recent African immigrants to French Guiana (Kasolo et al., 1998
; Cook et al., 1999
, Meng et al., 1999
; Zong et al., 1999
; Lacoste et al., 2000a
). Subtype A5 and B strains identified in this study were distributed throughout the region from which the KS patients came. The detection of strains in the A/C lineage belonging to neither subtype (K1-43/Ber) or only nominally to the C subtype (Ugd23 and K1-8/Dem) reduces the usefulness of the phylogenetic distinction between the A and C subtypes. This could be resolved either by abolishing the distinction, or by introducing additional subtypes to accommodate novel strains.
As in most previous studies, we noted no obvious association between K1 subtype and HIV status or clinical picture in the Ugandan KS patients. Also, no correlation was noted with ethnicity (tribe). A hint of a geographical association was noted in that all three samples from one area (Mukono; Ugd1, Ugd21 and Ugd26) were closely related B variants (Fig. 1). However, larger studies are required before definitive conclusions can be drawn.
Previous reports examining variability in the K15 gene, including an extensive study of more than 60 strains, largely used PCR assays to distinguish the P and M alleles (Poole et al., 1999 ; Lacoste et al., 2000
a, b; Meng et al., 2001
). We extended a similar PCR study to a sequence analysis of the entire K15 gene in selected strains. PCR screening of 30 Ugandan KS tumour samples confirmed that the majority contains the P allele, as predicted by Poole et al. (1999)
. The K15 M allele was identified in a single sample (Ugd10) originating from a 70-year-old male patient with endemic KS from Western Uganda, a region where endemic KS was common in older men prior to the HIV epidemic (Ziegler & Katongole-Mbidde, 1996
). To our knowledge, this is the first time the K15 M allele has been reported in East African samples. The M allele appears to be rarer in this region than in Central and West Africa (Lacoste et al., 2000a
).
There is evidence for recombination in about 2030% of HHV-8 strains (including five out of 12 African strains) by the criterion of lack of co-segregation at multiple genetic loci (Poole et al., 1999 ). In agreement, our network analysis identified recombinants among the Ugandan strains. Limited to the loci examined and employing the definitions of parental genotypes established by Poole et al. (1999)
, we found that seven of nine Ugandan strains appear to be recombinants. HHV-8 recombinants are thus common in nature. However, survival of the individual products of recombination is likely to be very rare, given the relatively low number of recombination events required to explain observed genome structures (Table 3
), the lack of detectable recombination within individual loci, and the time thought to have elapsed since the subtypes diverged (of the order of 105 years; Hayward, 1999
). Our findings also support the conclusion of Poole et al. (1999)
that divergence at the right end of the genome in K15 M strains extends leftwards (at a greatly reduced degree) into ORF75 and, in some instances, into T0.7/K12.
In a model focusing on the origin of the K15 alleles, Hayward (1999) astutely proposed that an ancestor of HHV-8 diverged into two forms, one of which eventually led to extant P allele lineages and one of which obtained the M allele by recombination with a related Old World monkey, or perhaps great ape, virus. In order to accommodate the claim of Poole et al. (1999)
that the M allele in 18 strains of HHV-8 exhibited virtually no sequence variation, it was proposed that the M allele and adjacent sequences re-entered extant lineages relatively recently (approximately 35000 years ago) via a single recombination event with a K1 C subtype genome. The M allele was then passed to other subtypes, with further recombination events resulting in differences in the extent of adjacent M allele-linked sequences.
Our data indicate that the M allele and adjacent regions have been evolving in at least two forms for a period equivalent to that during which extant subtypes have diversified. In the absence of evidence for either allele undergoing unusual evolutionary processes, this implies that introduction of the M allele into modern lineages did not occur via a single, recent event. In principle, the M allele could have been introduced by a single, ancient recombination event into a lineage that gave rise to extant subtypes, or into modern lineages by two separate, possibly recent recombination events involving diverged donors of the M allele. Noting the high level of divergence in UPS75' between strains containing P and M alleles, we tend towards the former alternative and speculate as follows. Two viral lineages diverged during primate evolution (of the order of 107 years ago), continuing to evolve with their host species and eventually producing distinct viruses containing the M or P alleles. At the stage when the host was a hominid (106 years ago), the M allele was transferred to a P allele-containing genome. The M allele and adjacent UPS75' region (now diverged from its equivalent in P allele-containing genomes) were then transferred into a P allele-containing lineage in an ancestral human (105 years ago). Further divergence, recombination and extinctions as envisaged by Hayward (1999) , coupled with the vicissitudes of human survival and migration, resulted in extant HHV-8 genotypes.
The identification of diverged versions of the M allele might be taken as compromising the assumption that the P allele is indigenous to HHV-8 and that the M allele was captured. Identification of apparently non-recombinant Ugandan strains that contain the P allele (Ugd2 and Ugd19) supports the view that the P allele was indeed indigenous, but it must be recognized that this argument depends upon correct definition of parental genotypes. Further insights into the fascinating but complex aspects of HHV-8 evolution are expected to emerge from continuing examination of extant HHV-8 lineages and related primate viruses.
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Acknowledgments |
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D.N.K. was supported by the Commonwealth Scholarship Commission.
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Footnotes |
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b Present address: Department of Virology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany.
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Received 11 April 2001;
accepted 4 July 2001.