AIDS Virus Research Unit, National Institute for Virology and Department of Virology, University of the Witwatersrand, Private Bag X4, Sandringham 2131, Johannesburg, South Africa1
Cancer Registry, South African Institute for Medical Research, Johannesburg, South Africa2
Author for correspondence: Lynn Morris. Fax +27 11 321 4234. e-mail lynnm{at}niv.ac.za
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Abstract |
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Introduction |
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KS is classified into four forms; classic (Mediterranean), endemic (African), iatrogenic (immunosuppressed) and AIDS-associated (epidemic) (Fife & Bower, 1996 ; Martin et al., 1993
; Oettle, 1962
; Wahman et al., 1991
). All of these forms have been shown to be infectious (Wahman et al., 1991
). Classic KS is a sporadic tumour found in elderly Mediterranean or Eastern European men and presents as paranodular skin lesions. Endemic KS is common in equatorial Africa, predominantly in young males and prepubescent children, and is often associated with rapid disease progression. Iatrogenic KS occurs in solid-organ transplant recipients under immunosuppressive therapy. AIDS-associated KS occurs in HIV-positive individuals and is the most fulminant form of the disease, possibly as a result of co-infection with HIV (Wahman et al., 1991
). These different forms of KS are not histologically distinguishable, although genetic studies of HHV-8 sequences have revealed that there are nucleic acid differences associated with the different forms (Boralevi et al., 1998
). However, other studies have not confirmed this (Fouchard et al., 2000
).
The HHV-8 genome contains approximately 170 kb and shows little overall nucleotide variation (Decker et al., 1996 ; Moore et al., 1996a
; Russo et al., 1996
). Recent studies have identified ORF75 as useful in delineating different strains, as this region shows 1·51·9% variation (Zong et al., 1997
). Analysis of this region has shown that subgroup A predominates in classic KS, subgroups B and C are found in Africa and all subgroups have been found in AIDS-related KS in the USA (Zong et al., 1997
). Whether these differences in the HHV-8 genome underpin differences in clinical presentation remains to be determined. Previous studies have shown the presence of HHV-8 in South Africa (Bourboulia et al., 1998
; Engelbrecht et al., 1997
; Sitas et al., 1997
, 1999
; Wilkinson et al., 1999
). We therefore undertook an analysis of the phylogenetic relationships of HHV-8 in South Africa and found that, in addition to the previously identified A, B and C subgroups, a novel subgroup is circulating in this region.
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Methods |
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Phylogenetic analysis.
The ORF75 PCR products were cloned into a TA vector (pMOSBlue T vector, Amersham, or a pGEM-T vector, Promega) and both the forward and reverse strands from one clone from each patient were sequenced. Sequencing was performed by the dideoxy chain-termination method using the Cy5 AutoRead sequencing kit (Pharmacia Biotech), the Thermo Sequenase fluorescent-labelled primer cycle sequencing kit (Amersham) or the dRhodamine cycle sequencing ready reaction kit (PE Applied Biosystems).
Sequences from the 41 samples were compared with 14 distinct A, B and C reference strains (Zong et al., 1997 ). Alignment was performed by using CLUSTAL W (Thompson et al., 1994
). A matrix of genetic distances between subgroups was determined by performing pairwise comparisons between all the sequences using the DNADIST program from the PHYLIP phylogenetic inference package (Felsenstein, 1993
). Genetic relationships between HHV-8 strains were determined by constructing phylogenetic trees based on the neighbour-joining method of Saitou & Nei (1987)
from CLUSTAL W. An unrooted tree was constructed in order to represent graphically the amount of genetic variability between the strains and subgroups. Kimuras two-parameter model was used to generate pairwise distance matrices (Kimura, 1980
) and the CONSENSE program from PHYLIP was used to determine bootstrap values for each node. Analyses were performed on 100 consecutive replicates to calculate the probability that a group of strains would cluster together. Bootstrap values of
70% correspond to a probability of
95% and were considered significant (Hillis & Bull, 1993
). The 41 sequences have been deposited in GenBank (accession numbers AF243797AF243837).
Single-strand conformational polymorphism (SSCP) analysis.
SSCP analysis was performed by using the 804 bp fragment amplified from the ORF75 region. From the PCR product, 5 µl was added to 45 µl formamide. The DNA was denatured by heating at 96 °C for 2 min. The entire reaction volume (50 µl) was loaded onto a 5% polyacrylamide gel and run at 200 V for 6 h in 1x TBE buffer. The specimens were run with samples of known subgroups, as determined by sequencing, for comparison.
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Results |
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Subgroup N shows significant inter-subgroup variation
In order to determine the degree of difference of the N subgroup from the A, B and C reference subgroups, the inter-subgroup nucleotide distance was calculated by using the distance matrix algorithm from PHYLIP (Table 2). All eight subgroup N strains were compared with subgroup A (n=7), B (n=3) and C (n=3) strains from Zong et al. (1997)
. The N subgroup differed from the A, B and C strains by nucleic acid distances of 4·8, 4·2 and 4·5%, respectively (Table 2
), indicating that it was equally different from each of the three reference subgroups. The intra-subgroup variation for each of the South African subgroups was also determined. While the A/B strains differed from each other by approximately 0·6%, an average intra-subgroup variation of 0·2% was observed for subgroup C and 0·4% for the N subgroup (not shown). Thus, while the N subgroup showed significant variation from the established subgroups, strains within the N subgroup showed limited variation relative to each other, similar to subgroups A, B and C.
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Discussion |
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Subgroup determination based on polymorphic differences in the ORF75 and UPS75 gene regions has identified three genetic subgroups, A, B and C (Zong et al., 1997 ). According to the geographical location from which the samples were obtained, it was suggested that the A subgroup predominates in regions associated with classical KS, whereas the B and C subgroups prevail in Africa, although these associations are often tenuous (Cook et al., 1999
; Zong et al., 1997
). All subgroups have been found in the USA, and the limited variation between them has led to the suggestion that they originated from a common isolate (Zong et al., 1997
). More recent data for the highly variable ORFK1 region have indicated the complexity of HHV-8 genetic types and the presence of chimeric or recombinant viruses (Poole et al., 1999
). Isolates EKS1, C282, ST1 and AKS1/2, which were originally classified as subgroup A, were reclassified as A/C following analysis of the K1 region. Subgroup C genomes were shown to be divergent upstream of the UPS75 region in ORFK15. Two diverged alleles were identified at this site: the minor form, which includes the prototype C strains ASM72 and HBL6 (Poole et al., 1999
) shown in Fig. 1
, and the predominant form. Since this study focuses on the ORF75 region and not ORFK1 or ORFK15, we have continued to use the original subgroup designations of A, B and C (Zong et al., 1997
).
South African subgroups were determined by sequencing single clones from 41 HHV-8 DNA samples, which were analysed in the ORF75 gene region. The extent of genetic variation within a single patient or as a result of clone bias was not examined, but such variation would probably be minor and would not influence the overall groupings. Comparison of the two samples available from one patient (LN7 and PB7) supports this notion. Based on the 17 characteristic loci in the ORF75 gene, subgroups A, B and C were all found to occur in South Africa, although subgroup C was found only rarely (3/40, 8%). In addition, a fourth, newly defined, novel (N) subgroup was identified. The N subgroup was defined by 33 mutations, 32 of which were newly described. Of the original 17 loci, 15 were retained, giving a total of 47 key diagnostic polymorphisms within the 804 bp region that defined the four subgroups in South Africa.
A similar analysis carried out across the 267 predicted amino acids of the ORF75 gene product recorded 17 polymorphisms that were characteristic for the four subgroups. The majority of mutations delineated the N subgroup (n=12), with only one to three point mutations descriptive for the other three subgroups. Subgroup designation based on polymorphisms is therefore limited when using amino acids, compared with nucleic acids. In general, both methods can be used for subtyping as well as delineating strains containing mutations overlapping with A and B subgroups, as well as identifying new strains of HHV-8.
Based on the analysis of specific polymorphisms, it may be concluded that distinct A and B subgroups exist in South Africa. However, this analysis is based on only 6% (47/804 nucleotides) of the ORF75 gene. More detailed and statistically accurate analysis of this region based on phylogenetic relationships has shown that the A and B subgroups were not supported by strong bootstrap values and hence were termed A/B strains. Closer analysis of the polymorphic changes revealed that the A/B strains shared four specific loci. These loci were previously specific for subgroup C (Zong et al., 1997 ), and were subsequently lost with the addition of the N subgroup. Thus, while A and B strains show a subgroup-specific pattern, they also share common mutations that prevent their segregation as distinct subgroups on a phylogenetic tree. The A and B subgroups are also less distinct in other regions of the genome lying just outside of ORF75, where only one nucleotide difference was found between the A and B subgroups (Zong et al., 1997
). Other researchers have also experienced difficulties in identifying distinct A and B subgroups in UPS75 using the prototype A, B and C sequences (Fouchard et al., 2000
). It is interesting that these researchers were using a Zambian strain of HHV-8. Studies done with HHV-8 from Central Africa, Senegal, Cameroon and French Guyana did in fact identify subgroup A and C strains but no isolates from subgroup B. This study examined a smaller fragment (473 bp) in the ORF75 region, which may have allowed for a definitive subgroup analysis of A and C in the absence of B (Fouchard et al., 2000
).
Since the A and B strains are closely related, it is possible that one of these strains may represent the progenitor or that one of these subgroups may have been the original isolate.
The C and N subgroups remain distinct clusters supported by strong bootstrap values and characteristic loci. Within the N subgroup, there was limited variation between the eight sequences analysed (intra-subgroup variation). However, when compared with sequences from other subgroups (inter-subgroup variation), there was a high level of genetic diversity (4·24·8%). Previous analysis of a larger region within ORF75 (UPS75) using subgroups A, B and C showed only 1·5% variation (Zong et al., 1997 ). Thus, the addition of the N subgroup has revealed that the ORF75 gene is more divergent than was first realized. Studies in other parts of Africa on both the ORF75 and the K1 regions have not revealed the presence of the N subgroup (Fouchard et al., 2000
; Kasolo et al., 1998
). Ongoing studies in other gene regions of subgroup N, including ORF26 and T0.7/K12, have revealed that it contains sequences that do not align with previously defined subgroups (G. Hayward, personal communication), suggesting further that the N subgroup is novel. New HHV-8 subgroups are continually being identified, including subgroup D in the South Pacific (Poole et al., 1999
) and samples from Zambian children with febrile illness, which differ significantly from published subgroups (Kasolo et al., 1998
), but the relationship of these new subgroups to N is unknown. It has been hypothesized that HHV-8 is an ancient virus that branched out into its various subgroups over 100000 years ago (Hayward, 1999
). Thus, the N subgroup may represent a vestige of modern-day HHV-8 subgroups that may have been present for a long period, only coming to the fore as a result of the HIV epidemic. Alternatively, it could be a new subgroup that has evolved due to continuous reactivation of HHV-8 in AIDS-infected individuals, especially in developing countries, where HIV antiviral therapy is not used. More studies on this newly identified HHV-8 subgroup need to be done in order to determine its distribution, transmission, genetic diversity and pathogenic potential.
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Acknowledgments |
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Footnotes |
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References |
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Bourboulia, D., Whitby, D., Boshoff, C., Newton, R., Beral, V., Carrara, H., Lane, A. & Sitas, F. (1998). Serologic evidence for mother-to-child transmission of Kaposis sarcoma-associated herpesvirus infection.JAMA280, 31-32.
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 Kaposis sarcoma.Science266, 1865-1869.[Medline]
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. (1999). Variability and evolution of Kaposis sarcoma-associated herpesvirus in Europe and Africa. International Collaborative Group.AIDS13, 1165-1176.[Medline]
Corbellino, M., Poirel, L., Bestetti, G., Pizzuto, M., Aubin, J. T., Capra, M., Bifulco, C., Berti, E., Agut, H., Rizzardini, G., Galli, M. & Parravicini, C. (1996). Restricted tissue distribution of extralesional Kaposis sarcoma-associated herpesvirus-like DNA sequences in AIDS patients with Kaposis sarcoma.AIDS Research and Human Retroviruses 12, 651-657.[Medline]
Decker, L. L., Shankar, P., Khan, G., Freeman, R. B., Dezube, B. J., Lieberman, J. & Thorley-Lawson, D. A. (1996). The Kaposi sarcoma-associated herpesvirus (KSHV) is present as an intact latent genome in KS tissue but replicates in the peripheral blood mononuclear cells of KS patients.Journal of Experimental Medicine184, 283-288.[Abstract]
Engelbrecht, S., Treurnicht, F. K., Schneider, J. W., Jordaan, H. F., Steytler, J. G., Wranz, P. A. & van Rensburg, E. J. (1997). Detection of human herpes virus 8 DNA and sequence polymorphism in classical, epidemic, and iatrogenic Kaposis sarcoma in South Africa.Journal of Medical Virology52, 168-172.[Medline]
Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, WA, USA.
Fife, K. & Bower, M. (1996). Recent insights into the pathogenesis of Kaposis sarcoma.British Journal of Cancer73, 1317-1322.[Medline]
Fouchard, N., Lacoste, V., Couppie, P., Develoux, M., Mauclere, P., Michel, P., Herve, V., Pradinaud, R., Bestetti, G., Huerre, M., Tekaia, F., de The, G. & Gessain, A. (2000). Detection and genetic polymorphism of human herpes virus type 8 in endemic or epidemic Kaposis sarcoma from West and Central Africa, and South America.International Journal of Cancer85, 166-170.
Hayward, G. S. (1999). KSHV strains: the origins and global spread of the virus.Seminars in Cancer Biology9, 187-199.[Medline]
Hermans, P., Lundgren, J., Sommereijns, B., Pedersen, C., Vella, S., Katlama, C., Luthy, R., Pinching, A. J., Gerstoft, J., Pehrson, P. & Clumeck, N. (1996). Epidemiology of AIDS-related Kaposis sarcoma in Europe over 10 years. AIDS in Europe Study Group.AIDS10, 911-917.[Medline]
Hillis, D. M. & Bull, J. J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis.Systematic Biology42, 182-192.
Kasolo, F. C., Monze, M., Obel, N., Anderson, R. A., French, C. & Gompels, U. A. (1998). Sequence analyses of human herpesvirus-8 strains from both African human immunodeficiency virus-negative and -positive childhood endemic Kaposis sarcoma show a close relationship with strains identified in febrile children and high variation in the K1 glycoprotein.Journal of General Virology79, 3055-3065.[Abstract]
Katz, M. H., Hessol, N. A., Buchbinder, S. P., Hirozawa, A., OMalley, P. & Holmberg, S. D. (1994). Temporal trends of opportunistic infections and malignancies in homosexual men with AIDS.Journal of Infectious Diseases170, 198-202.[Medline]
Kimura, M. (1980). A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.Journal of Molecular Evolution16, 111-120.[Medline]
Martin, R. W.III, Hood, A. F. & Farmer, E. R. (1993). Kaposis sarcoma.Medicine72, 245-261.[Medline]
Martin, J. N., Ganem, D. E., Osmond, D. H., Page-Shafer, K. A., Macrae, D. & Kedes, D. H. (1998). Sexual transmission and the natural history of human herpesvirus 8 infection.New England Journal of Medicine338, 948-954.
Monini, P., de Lellis, L., Fabris, M., Rigolin, P. & Cassai, E. (1996). Kaposis sarcoma-associated herpesvirus DNA sequences in prostate tissue and human semen.New England Journal of Medicine334, 1168-1172.
Moore, P. S., Gao, S. J., Dominguez, G., Cesarman, E., Lungu, O., Knowles, D. M., Garber, R., Pellett, P. E., McGeoch, D. J. & Chang, Y. (1996a). Primary characterization of a herpesvirus agent associated with Kaposis sarcomae.Journal of Virology70, 549-558.[Abstract]
Moore, P. S., Kingsley, L. A., Holmberg, S. D., Spira, T., Gupta, P., Hoover, D. R., Parry, J. P., Conley, L. J., Jaffe, H. W. & Chang, Y. (1996b). Kaposis sarcoma-associated herpesvirus infection prior to onset of Kaposis sarcoma.AIDS10, 175-180.[Medline]
Oettle, A. G. (1962). Geographical and racial differences in the frequency of Kaposis sarcoma as evidence of environmental or genetic cause.Acta Unio Internationalis Contra Cancrum 18, 330-363.[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 Kaposis 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 Virology73, 6646-6660.
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 the Kaposi sarcoma-associated herpesvirus (HHV8).Proceedings of the National Academy of Sciences, USA93, 14862-14867.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees.Molecular Biology and Evolution4, 406-425.[Abstract]
Sitas, F., Taylor, L., Madhoo, J., Cooper, K., Carrara, H., Boshoff, C. & Weiss, R. A. (1997). Occurrence of human herpes virus 8 in Kaposis sarcoma and other tumours in South Africa. South African Medical Journal 87, 1020, 1022.[Medline]
Sitas, F., Carrara, H., Beral, V., Newton, B., Reeves, G., Bull, D., Jentsch, U., Pacella-Norman, R., Bourboulia, D., Whitby, D., Boshoff, C. & Weiss, R. (1999). Antibodies against human herpesvirus 8 in black South African patients with cancer.New England Journal of Medicine340, 1863-1871.
Smith, M. S., Bloomer, C., Horvat, R., Goldstein, E., Casparian, J. M. & Chandran, B. (1997). Detection of human herpesvirus 8 DNA in Kaposis sarcoma lesions and peripheral blood of human immunodeficiency virus-positive patients and correlation with serologic measurements.Journal of Infectious Diseases176, 84-93.[Medline]
South African Department of Health (1999). Ninth Annual National HIV Sero-prevalence Survey of Women Attending Public Antenatal Clinics in South Africa. Health Systems Research and Epidemiology.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research22, 4673-4680.[Abstract]
Viviano, E., Vitale, F., Ajello, F., Perna, A. M., Villafrate, M. R., Bonura, F., Arico, M., Mazzola, G. & Romano, N. (1997). Human herpesvirus type 8 DNA sequences in biological samples of HIV-positive and negative individuals in Sicily.AIDS11, 607-612.[Medline]
Wahman, A., Melnick, S. L., Rhame, F. S. & Potter, J. D. (1991). The epidemiology of classic, African, and immunosuppressed Kaposis sarcoma.Epidemiologic Reviews13, 178-199.[Medline]
Whitby, D., Howard, M. R., Tenant-Flowers, M., Brink, N. S., Copas, A., Boshoff, C., Hatzioannou, T., Suggett, F. E., Aldam, D. M., Denton, A. S., Miller, R. F., Weller, I. V. D., Weiss, R. A. & Schulz, T. F. (1995). Detection of Kaposi sarcoma-associated herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposis sarcoma.Lancet346, 799-802.[Medline]
Wilkinson, D., Sheldon, J., Gilks, C. F. & Schulz, T. F. (1999). Prevalence of infection with human herpesvirus 8/Kaposis sarcoma herpesvirus in rural South Africa.South African Medical Journal89, 554-557.[Medline]
Zong, J. C., Metroka, C., Reitz, M. S., Nicholas, J. & Hayward, G. S. (1997). Strain variability among Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genomes: evidence that a large cohort of United States AIDS patients may have been infected by a single common isolate.Journal of Virology71, 2505-2511.[Abstract]
Received 14 January 2000;
accepted 12 April 2000.