Human Herpesvirus 8 in Uganda: Seroprevalence in Blood Donors, Genome Variability and Evolution

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. HHV-8 is also associated with primary effusion lymphomas (PEL) and multicentric Castleman's disease. KS is a prominent disease in Uganda accounting for approximately 50% of reported cancer cases. 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 11-77%, 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%). Estimates of seroprevalence in the USA range from 0-20%. The mode by which HHV-8 is transmitted is not known, but transmission most probably occurs via sexual means in countries where KS is non-endemic, and via non- sexual means in countries where KS is endemic, such as Uganda and Zambia. 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 PEL cell line, BC-1 and one from an AIDS KS lesion. 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 highly variable type 1 membrane protein. Five HHV-8 K1 subtypes, generally called A, B, C, D and E have been identified. Subtype B appears to predominate in Africa, together with a variant (A5) of the A subtype, while subtypes A and C predominate elsewhere in the world. The K15 gene, at the right end of the genome, occurs as two highly diverged alleles, P (predominant) and M (minor), showing only 30% amino acid sequence identity. The P allele is more frequent among HHV-8 genomes, and the M allele is rarer. Evidence for recombination has been observed in 20-30% of HHV-8 strains with almost half of the African strains displaying mosaic genomes. Although KS is a prominent disease in Uganda, little is known about the prevalence of HHV-8 in the Ugandan population and the characteristics of HHV- 8 strains present in this country. The aims of this project were to determine the prevalence of HHV-8 in Ugandan blood donors and to characterise the genomes of HHV-8 strains in Ugandan KS patients. During the course of this work, one report on HHV-8 seroprevalence in Ugandan blood donors and three on K1 subtypes in samples from various parts of the world, including some from Uganda, were published. Using a combination of two serological tests, ELISA (against lytic (ORF65) and latent (ORF73/LANA) antigens) and LANA IF test, antibodies were detected in a high percentage (74%) of 114 HIV-negative blood donors, confirming previous results. Attempts to detect HHV-8 DNA by PCR in peripheral white blood cells of the blood donors were largely unsuccessful. PCR, sequence and phylogenetic analysis of the K1 gene in tumour samples from 17 KS patients indicated that the majority (11) were infected by the B subtype of HHV-8. Five patients were infected by the A5 subtype. A variant of the C subtype, showing characteristics of both A and C subtypes in addition to unique characteristics, was detected in a single patient. PCR and sequence analysis of other genome loci implied that at least five of nine (55%) HHV-8 strains are recombinants between subtypes. Three of the five strains with a K1 A5 gene were analysed and all were found to be subtype B in the rest of the genome. The single K1 subtype C strain was also a recombinant between subtypes C and B. PCR analysis for the K15 gene in tumour samples from 30 KS patients confirmed previous results that the P allele predominates. For the first time, a single strain bearing the M allele was identified in East African samples. Sequence analysis of the entire K15 gene in selected samples indicated that the P and M alleles occur in two distinct forms. Divergence between the M allele found in the Ugandan strain and that in the previously sequenced BC-1 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. The study increases our understanding of the characteristics of HHV-8 strains involved in KS in Uganda and provides new insights into the evolution of HHV-8