Recombination in human herpesvirus-8 strains from Uganda and evolution of the K15 gene

Dorothy N. Kakoola1, Julie Sheldonb,2, Naomi Byabazairec,3, Rory J. Bowden4, Edward Katongole-Mbidde3, Thomas F. Schulzb,2 and Andrew J. Davison1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human herpesvirus-8 (HHV-8) is believed to be the aetiological agent of Kaposi’s sarcoma (KS). KS accounts for half the reported cancer cases in Uganda, and occurs in endemic and epidemic [human immunodeficiency virus (HIV)-associated] forms. We confirmed a high prevalence (74%) of HHV-8 antibodies in 114 HIV-negative Ugandan blood donors, and characterized the genomes of HHV-8 strains present in 30 adult Ugandan KS patients. Phylogenetic analysis of the uniquely variable K1 gene indicated that the majority of KS patients were infected by the B subtype of HHV-8, several by the A5 subtype, and one by a variant of the C subtype. Sequence analysis of nine strains at several other genome loci spaced out across the genome indicated that five are recombinants between subtypes when considered independently of previously published definitions of parental (unrecombined) genotypes. When previously published parental genotypes were taken into account, seven of the nine strains appeared to be recombinants. Analysis of the K15 gene, which exists in HHV-8 in two highly diverged alleles, indicated that the P allele predominates, with only a single strain bearing the M allele. Divergence between the M allele in the latter strain and that in the previously sequenced BC1 strain is at least as great as that between representatives of the P allele. This indicates that introduction of the M allele into extant HHV-8 subtypes did not occur by a single, relatively recent recombination event as was concluded from a previous study in which very limited variation in the M allele was reported.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human herpesvirus-8 (HHV-8), also called Kaposi’s sarcoma-associated herpesvirus (KSHV), is associated with all forms of Kaposi’s sarcoma (KS), and is considered to be the infectious cause of this disease. Classical KS, which affects mainly elderly men in the Mediterranean region, usually presents as benign paranodular skin lesions, but endemic KS of human immunodeficiency virus (HIV)-negative individuals in equatorial (East and Central) Africa and KS associated with HIV infection or post-transplantation immunosuppression can be clinically aggressive, affecting internal organs. KS accounts for approximately 50% of reported cancer cases in Uganda (Wabinga et al., 1993 ). Epidemic (HIV-associated) and endemic KS occur in Uganda, each afflicting both adults and children.

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 (2–28%) and then Northern Europe, Southeast Asia and the Caribbean countries (2–4%) (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 Epstein–Barr 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 20–30% 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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Blood donor samples.
Blood samples were collected in EDTA tubes from 116 donors (98 male and 18 female) visiting Nakasero blood bank (NBB) in Kampala, the capital city of Uganda, during April–August 1998. NBB is the only blood bank facility serving the central Uganda region. The majority of donors were residents of Kampala with an age range of 18–54 years (median age 27 years). As part of routine procedures, samples were tested for HIV using two commercially available ELISA kits (Burroughs Wellcome and Welcozyme). Two HIV-1-positive samples were excluded from further analysis. Plasma was harvested from blood samples and stored at -70 °C at the Makerere University–Johns Hopkins University (MU–JHU) collaborative laboratory at Mulago Hospital, Kampala, until shipped to the UK.

{blacksquare} 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.

{blacksquare} 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 April–July 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 16–70 years (median age 38 years). HIV testing was performed at the MU–JHU 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 MU–JHU 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 {beta}-globin gene was also amplified.

{blacksquare} 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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Table 1. Locations of the genes, PCR products and sequences analysed in this and previous studies

 
PCR was performed using Taq polymerase (Boehringer Mannheim or Sigma) in a total volume of 50 µl containing 20–200 ng of template DNA. The conditions used to amplify K1 (primary amplification) and K15 products were 94 °C for 45 s, 47·5 °C for 30 s (to maximize annealing to partially mismatched sequences) and 72 °C for 1 min over 30 cycles. These conditions were also employed for all other products and for nested PCR of K1, except that the annealing temperature was increased to 60 °C and 57 °C, respectively. Stringent measures were undertaken to prevent PCR contamination: all original DNA samples were extracted in a laboratory in Uganda; separate rooms were used for pre-PCR, PCR and post-PCR steps; and the premix was UV-irradiated. Each experiment involved a sizeable batch of PCR reactions, and incorporated appropriate negative (water and uninfected cellular DNA) and positive controls.

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.

{blacksquare} 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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Fig. 1. HHV-8 K1 phylogenetic tree incorporating 52 almost complete K1 DNA sequences (840 bp, codons 6–285), 17 from this study (highlighted by black dots) and 35 from previous studies (Cook et al., 1999 ; Zong et al., 1999 ; Lacoste et al., 2000a ). The tree was rooted at the midpoint between the sum of the longest branches (Ugd2 and ZKS3). Four subtypes (A–D) and the A5 variants are indicated. Selected bootstrap values are included.

 

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Table 2. Published sequences used in this study

 

   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
HHV-8 seroprevalence in blood donors
Overall HHV-8 antibody prevalence was 74% (84/114 HIV-negative blood donors). Fifty-one samples (45%) reacted in the LANA/ORF73 IF test, and 76 (67%) and 61 (54%) had antibodies in the ELISA (confirmed by Western blot analysis) to the ORF65 and ORF73 recombinant proteins, respectively. Ninety-seven samples (85%) reacted in at least one of the assays, and 54 (47%) in only one assay (46 in the ELISA and eight in the IF test). Of the 46 samples that reacted only in the ELISA, 33 were confirmed by Western blot analysis, ten were not confirmed and three were indeterminant. There was little difference between prevalence in males (70/96; 73%) and females (14/18; 77%). Also, prevalence was similar in all age groups: 18–23 years (18/26; 69%), 24–29 years (34/46; 74%), 30–35 years (20/26; 77%), 36–41 years (9/12; 75%) and 42 years and above (3/4; 75%). These results indicate that HHV-8 antibodies are highly prevalent in male and female HIV-1-negative blood donors in Kampala.

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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Fig. 2. Nucleotide changes within the (A) HHV-8 K15 M and (B) K15 P alleles. Sequences of Ugandan strains (bold) were analysed together with those of BC1, BCBL-R and GK18 (Table 2). K1 subtypes are indicated. The results are presented in the context of variation from the BC1 (A) or BCBL-R (B) sequences, hyphens denoting identity. The alignments in each case consist of the entire K15 gene oriented from left to right (opposite to the genomic orientation; nucleotides 1 and 2102 in K15 M are equivalent to 136772 and 134672, respectively, in the BC1 genome). The BC1 sequence was modified before alignment by replacing the missing T residue at 136610 (see text). Nucleotides 1 and 2086 in K15 P are equivalent to 3652 and 1578, respectively, in the BCBL-R sequence and to 21490 and 19415, respectively, in the GK18 sequence. The position in the final alignment is indicated in the ‘nucleotide’ row. The ‘exon/intron’ row shows the number of the exon (E1–8) or intron (I1–7) in which each substitution or deletion is located. Amino acid differences are depicted in the ‘residue’ row, asterisks and exclamation marks indicating synonymous substitutions in exons and non-coding substitutions in introns, respectively. Deletions (Del) are represented by dots.

 
The entire K15 P gene (up to 2086 bp), with flanking sequences (about 200 bp on each side, with the downstream region extending to the beginning of ORF75), was sequenced from eight samples (Ugd2, Ugd4, Ugd12, Ugd15, Ugd16, Ugd19, Ugd23 and Ugd29) selected as representatives of the K1 subtypes. The sequences were analysed together with the corresponding regions of GK18 and BCBL-R (Table 2). A total of 34 substitutions (as well as a 10 bp insertion in the first intron of Ugd2, Ugd12 and Ugd19) was identified in the gene collection (Fig. 2B). In coding regions, nine substitutions are synonymous and 16 are non-synonymous; nine substitutions are within introns. Two substitutions are located in the downstream flanking region and none in the upstream region. Overall, 13 substitutions and a 10 bp insertion divided the P strains into two distinct groups: Ugd2, Ugd12 and Ugd19; and Ugd4, Ugd15, Ugd16, Ugd23, Ugd29, BCBL-R and GK18. Pairwise sequence comparisons revealed a maximum divergence of 0·9% (e.g. Ugd2 and Ugd16).

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 K3–ORF26, K9–T0.7/K12, ORF75 and K15. This suggests recombination between K1 and K3. Secondly, Ugd10, which does not cluster with BC1 in K1 and K3–ORF26, appears to be linked to this strain in K9–T0.7/K12, ORF75 and K15, suggesting recombination between ORF26 and K9-T0.7/K12. Thirdly, the K1 C variant (Ugd23) has a distinct sequence at K3–ORF26, 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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Fig. 3. Networks displaying relationships among DNA sequences at the (A) K3–ORF26, (B) K9–T0.7/K12, (C) ORF75 and (D) K15 P loci (Table 1). Alignments were made consisting of sequences of the Ugandan strains and those of BC1, BCBL-R and GK18 (Table 2). The length of the alignments and the number of substitution sites used to construct the networks are indicated. All substitution sites were used with the exception of one independently mutated site in T0.7/K12. Samples are represented by circles. Each subdivision represents a single substitution site; the asterisk indicates the single site in K9. Deletions are not represented.

 
Comparisons with published data
The ORF26, T0.7/K12 and ORF75 sequences of the nine Ugandan strains were also compared with sequences obtained from GenBank or published literature (Table 2; Zong et al., 1997 ; Poole et al., 1999 ; Alagiozoglou et al., 2000 ). Where possible, all the groups previously identified at each locus were represented. This analysis enabled the Ugandan sequences to be categorized according to the genotypes established by Poole et al. (1999) .

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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Table 3. Subtype designations at each locus and overall genotypes of Ugandan strains

 


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Fig. 4. Nucleotide changes within the (A) T0.7/K12 and (B) ORF75/UPS75' loci. Sequences of Ugandan strains (bold) were compared with selected published sequences (Table 2). K1 subtypes are indicated. The results are presented in the context of variation from BCBL-R sequences; hyphens and dots denote identical and deleted residues, respectively. The T0.7/K12 segment is in the orientation opposite to that of the genomic sequence, and the ORF75/UPS75' segment is in the genomic orientation. The number at the top of each site indicates the position in the alignment. Subtype designations are those defined by Poole et al. (1999) . A single synonymous site (Fig. 4A, position 90) was most likely a result of repeat mutations and was excluded from the K9–T0.7/K12 network shown in Fig. 3(B). PS, Subtypes published previously; DS, subtypes determined in this study.

 
Divergence values within and between the eight P and two M strains for T0.7/K12, ORF75, UPS75' and K15 are shown in Table 4. The sequences of the M allele in Udg10 and BC1 differ by 1·2%, a value similar to that (0·9%) for the two most widely diverged representatives of the P allele for which sequence data are available. As is also evident in the network (Fig. 3C), Ugd10 and BC1 show greater divergence (almost fourfold) within ORF75 compared to strains that possess the P allele. Divergence in ORF75 between Ugd10 and the K15 P strains is less than that between Ugd10 and BC1, indicating that Ugd10 is more closely related at this locus to the K15 P strains, in particular the B subtype (Ugd2/12/19), than to BC1. K15 P strains show minimal variation in UPS75', but diverge by up to 15% from Ugd10 and BC1.


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Table 4. Pairwise divergence values within and between K15 P and M strains

 
Analysis of the patterns identified in ORF26, T0.7/K12 and ORF75 together with those of K1 and K15 resulted in the overall genotypes summarized in Table 3. Only two Ugandan strains (Ugd2 and Ugd19) appear to have subtype B genomes throughout. The rest appear to be recombinants between subtypes, but nonetheless show features of subtype B in at least one locus. With the exception of the A5 variant of the K1 gene, which is particularly pertinent to this study, Table 3 is limited to subtypes. Further subdivisions have been described in previous studies, but the resulting classifications are not always phylogenetically justified and have not been included. It is probable that some genomes have resulted from recombination between members of the same subtype, but evidence for this is speculative.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Previous studies reported a prevalence of 11–77% for HHV-8 antibodies in Ugandan blood donors or in the general population (reviewed by Chatlynne & Ablashi, 1999 ). We tested blood donors residing in Kampala for HHV-8 antibodies by ELISA against lytic (ORF65) and latent (ORF73/LANA) antigens and by LANA IF test. The ORF65-encoded capsid protein reacts with 80–85% of KS sera and approximately 3–5% of UK blood donors, and LANA IF detects antibodies in 80–90% of KS patients and 0–3% of UK or US blood donors (Mayama et al., 1998 ).

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 20–30% 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.


   Acknowledgments
 
We are indebted to Duncan McGeoch for extensive discussion, for help with phylogenetic analysis and for critical reading of the manuscript. We also thank Charles Cunningham and Kathleen Wright for assistance with DNA sequencing, the MU–JHU laboratory for technical support and storage of samples, and the NBB staff and director (Peter Kataaha) for access to blood donors.

D.N.K. was supported by the Commonwealth Scholarship Commission.


   Footnotes
 
The GenBank accession numbers of sequences reported in this study are AF130292, AF130293, AY042940AY042942 and AY042944AY043009.

b Present address: Department of Virology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany.

c Deceased.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Alagiozoglou, L., Sitas, F. & Morris, L. (2000). Phylogenetic analysis of human herpesvirus-8 in South Africa and identification of a novel subgroup. Journal of General Virology 81, 2029-2038.[Abstract/Free Full Text]

Bandelt, H. J., Forster, P., Sykes, B. C. & Richards, M. B. (1995). Mitochondrial portraits of human populations using median networks. Genetics 141, 743-753.[Abstract/Free Full Text]

Beral, V. (1991). Epidemiology of Kaposi’s sarcoma. In Cancer, HIV and AIDS (Cancer Surveys, vol. 10) , pp. 5-22. Edited by V. Beral, H. W. Jaffe & R. A. Weiss. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory.

Biggar, R. J., Whitby, D., Marshall, V., Linhares, A. C. & Black, F. (2000). Human herpesvirus 8 in Brazilian Amerindians: a hyperendemic population with a new subtype. Journal of Infectious Diseases 181, 1562-1568.[Medline]

Boshoff, C., Whitby, D., Hatziioannou, T., Fisher, C., van der Walt, J., Hatzakis, A., Weiss, R. & Schulz, T. (1995). Kaposi’s sarcoma-associated herpesvirus in HIV-negative Kaposi’s sarcoma. Lancet 345, 1043-1044.[Medline]

Boshoff, C., Gao, S. J., Healy, L. E., Matthews, S., Thomas, A. J., Coignet, L., Warnke, R. A., Strauchen, J. A., Matutes, E., Kamel, O. W., Moore, P. S., Weiss, R. A. & Chang, Y. (1998). Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice. Blood 91, 1671-1679.[Abstract/Free Full Text]

Chang, Y., Cesarman, E., Pessin, M. S., Lee, F., Culpepper, J., Knowles, D. M. & Moore, P. S. (1994). Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266, 1865-1869.[Medline]

Chatlynne, L. G. & Ablashi, D. V. (1999). Seroepidemiology of Kaposi’s sarcoma-associated herpesvirus (KSHV). Seminars in Cancer Biology 9, 175-185.[Medline]

Choi, J.-K., Lee, B.-S., Shim, S.-N., Li, M. & Jung, J. U. (2000). Identification of the novel K15 gene at the rightmost end of the Kaposi’s sarcoma-associated herpesvirus genome. Journal of Virology 74, 436-446.[Abstract/Free Full Text]

Cook, P. M., Whitby, D., Calabro, M.-L., Luppi, M., Kakoola, D. N., Hjalgrim, H., Ariyoshi, K., Ensoli, B., Davison, A. J., Schulz, T. F. & the International Collaborative Group (1999). Variability and evolution of Kaposi’s sarcoma-associated herpesvirus in Europe and Africa. AIDS 13, 1165–1176.[Medline]

Glenn, M., Rainbow, L., Auradé, F., Davison, A. & Schulz, T. F. (1999). Identification of a spliced gene from Kaposi’s sarcoma-associated herpesvirus encoding a protein with similarities to latent membrane proteins 1 and 2A of Epstein–Barr virus. Journal of Virology 73, 6953-6963.[Abstract/Free Full Text]

Hayward, G. S. (1999). KSHV strains: the origins and global spread of the virus. Seminars in Cancer Biology 9, 187-199.[Medline]

Kasolo, F. C., Monze, M., Obel, N., Anderson, R. A., French, C. & Gompels, U. A. (1998). Sequence analysis of human herpesvirus-8 strains from both African human immunodeficiency virus-negative and -positive childhood endemic Kaposi’s sarcoma show a close relationship with strains identified in febrile children and high variation in the K1 glycoprotein. Journal of General Virology 79, 3055-3065.[Abstract]

Lacoste, V., Judde, J.-G., Briere, J., Tulliez, M., Garin, B., Kassa-Kelembho, E., Morvan, J., Couppie, P., Clyti, E., Vila, J. F., Rio, B., Delmer, A., Mauclere, P. & Gessain, A. (2000a). 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 278, 60-74.[Medline]

Lacoste, V., Kadyrova, E., Chistiakova, I., Gurtsevitch, V., Judde, J.-G. & Gessain, A. (2000b). Molecular characterization of Kaposi’s sarcoma-associated herpesvirus/human herpesvirus-8 strains from Russia. Journal of General Virology 81, 1217-1222.[Abstract/Free Full Text]

Lagunoff, M., Majeti, R., Weiss, A. & Ganem, D. (1999). Deregulated signal transduction by the K1 gene product of Kaposi’s sarcoma-associated herpesvirus. Proceedings of the National Academy of Sciences, USA 96, 5704-5709.[Abstract/Free Full Text]

Lee, H., Veazey, R., Williams, K., Li, M., Guo, J., Neipel, F., Fleckenstein, B., Lackner, A., Desrosiers, R. C. & Jung, J. U. (1998). Deregulation of cell growth by the K1 gene of Kaposi’s sarcoma-associated herpesvirus. Nature Medicine 4, 435-440.[Medline]

Mayama, S., Cuevas, L. E., Sheldon, J., Omar, O. H., Smith, D. H., Okong, P., Silvel, B., Hart, C. A. & Schulz, T. F. (1998). Prevalence and transmission of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) in Ugandan children and adolescents. International Journal of Cancer 77, 817-820.

Meng, Y.-X., Spira, T. J., Bhat, G. J., Birch, C. J., Druce, J. D., Edlin, B. R., Edwards, R., Gunthel, C., Newton, R., Stamey, F. R., Wood, C. & Pellett, P. E. (1999). Individuals from North America, Australasia, and Africa are infected with four different genotypes of human herpesvirus 8. Virology 261, 106-119.[Medline]

Meng, Y.-X., Sata, T., Stamey, F. R., Voevodin, A., Katano, H., Koizumi, H., Deleon, M., De Cristofano, M. A., Galimberti, R. & Pellett, P. E. (2001). Molecular characterization of strains of Human herpesvirus 8 from Japan, Argentina and Kuwait. Journal of General Virology 82, 499-506.[Abstract/Free Full Text]

Neipel, F., Albrecht, J.-C. & Fleckenstein, B. (1997). Cell-homologous genes in the Kaposi’s sarcoma-associated rhadinovirus human herpesvirus 8: determinants of its pathogenicity? Journal of Virology 71, 4187-4192.[Free Full Text]

Nicholas, J., Zong, J.-C., Alcendor, D. J., Ciufo, D. M., Poole, L. J., Sarisky, R. T., Chiou, C.-J., Zhang, X., Wan, X., Guo, H.-G., Reitz, M. S. & Hayward, G. S. (1998). Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV8. Journal of the National Cancer Institute Monographs 23, 79-88.[Medline]

Poole, L. J., Zong, J.-C., Ciufo, D. M., Alcendor, D. J., Cannon, J. S., Ambinder, R., Orenstein, J. M., Reitz, M. S. & Hayward, G. S. (1999). Comparison of genetic variability at multiple loci across the genomes of the major subtypes of Kaposi’s sarcoma-associated herpesvirus reveals evidence for recombination and for two distinct types of open reading frame K15 alleles at the right-hand end. Journal of Virology 73, 6646-6660.[Abstract/Free Full Text]

Rainbow, L., Platt, G. M., Simpson, G. R., Sarid, R., Gao, S.-J., Stoiber, H., Herrington, C. S., Moore, P. S. & Schulz, T. F. (1997). The 222- and 234-kilodalton latent nuclear protein (LNA) of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen. Journal of Virology 71, 5915-5921.[Abstract]

Russo, J. J., Bohenzky, R. A., Chien, M.-C., Chen, J., Yan, M., Maddalena, D., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y. & Moore, P. S. (1996). Nucleotide sequence of Kaposi’s sarcoma-associated herpesvirus (HHV8). Proceedings of the National Academy of Sciences, USA 93, 14862-14867.[Abstract/Free Full Text]

Sample, J., Liebowitz, D. & Kieff, E. (1989). Two related Epstein–Barr virus membrane proteins are encoded by separate genes. Journal of Virology 63, 2743-2753.

Schulz, T. F. (1998). Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8). Journal of General Virology 79, 1573-1591.[Free Full Text]

Simpson, G. R., Schulz, T. F., Whitby, D., Cook, P. M., Boshoff, C., Rainbow, L., Howard, M. R., Gao, S. J., Bohenzky, R. A., Simmonds, P., Lee, C., deRuiter, A., Hatzakism, A., Teddar, R. S., Wellar, I. V. D., Weiss, R. A. & Moore, P. S. (1996). Prevalence of Kaposi’s sarcoma-associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen. Lancet 348, 1133-1138.[Medline]

Staden, R. (1987). Computer handling of DNA sequencing projects. In Nucleic Acid and Protein Sequence Analysis: A Practical Approach , pp. 173-217. Edited by M. J. Bishop & C. J. Rawlings. Oxford:IRL Press.

Staden, R., Beal, K. F. & Bonfield, J. K. (1998). The Staden package. Computer methods in molecular biology. In Bioinformatics Methods and Protocols , pp. 115-132. Edited by S. Misener & S. A. Krawetz. Totowa, NJ:Humana Press.

Wabinga, H. R., Parkin, D. M., Wabwire-Mangen, F. & Mugerwa, J. W. (1993). Cancer in Kampala, Uganda, in 1989–91: changes in incidence in the era of AIDS. International Journal of Cancer 54, 26-36.

Whitby, D., Howard, M. R., Tenant-Flowers, M., Brink, N. S., Copas, A., Boshoff, C., Hatziouannou, T., Suggett, F. E. A., Aldam, D. M., Denton, A. S., Miller, R. F., Weller, I. V. D., Weiss, R. A., Tedder, R. S. & Schulz, T. F. (1995). Detection of Kaposi’s sarcoma-associated herpesvirus (KSHV) in peripheral blood of HIV-infected individuals predicts progression to Kaposi’s sarcoma. Lancet 346, 799-802.[Medline]

Ziegler, J. L. & Katongole-Mbidde, E. (1996). KS in childhood: an analysis of 100 cases from Uganda and relationship to HIV infection. International Journal of Cancer 65, 200-203.

Zong, J.-C., Metroka, C., Reitz, M. S., Nicholas, J. & Hayward, G. S. (1997). Strain variability among Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8): evidence that a large cohort of United States AIDS patients may have been infected by a single common strain. Journal of Virology 71, 2505-2511.[Abstract]

Zong, J.-C., Ciufo, D., Alcendor, D. J., Wan, X., Nicholas, J., Browning, P. J., Rady, P. L., Trying, S. K., Orenstein, J. M., Rabkin, C. S., Su, I.-J., Powell, K. F., Croxson, M., Foreman, K. E., Nickoloff, B. J., Alkan, S. & Hayward, G. S. (1999). High-level variability in the ORF-K1 membrane protein gene at the left end of the Kaposi’s sarcoma-associated herpesvirus genome defines four major virus subtypes and multiple variants or clades in different human populations. Journal of Virology 73, 4156-4170.[Abstract/Free Full Text]

Received 11 April 2001; accepted 4 July 2001.