Genetic divesity of Human Herpesvirus Type 8 in Northern South Africa

M.Sc. (Microbiology)

Department of Biochemistry and Microbiology

Background: Human herpesvirus type 8 (HHV-8), is an oncogenic virus responsible for causing all forms of Kaposi`s sarcoma (KS). HHV-8 prevalence varies globally, however, it is more prevalent in African countries, with South Africa having over 50% of HHV-8 infections. HHV-8 encodes a highly diverse open reading frame (ORF) K1 gene, which has led to the identification of seven major genotypes (A-F and Z) that are heterogeneously distributed across the world. The viral genetic landscape of any geographical area is of paramount importance in vaccine development and diagnostics. However, data on HHV-8 genotypes is scarce in northern South Africa. Therefore, this study will provide genetic diversity of HHV-8 in northern South Africa, and this may aid in the selection of genes for vaccine development. Objective: The main objective of the study was to describe the genetic diversity of human herpesvirus type 8 in northern South Africa. Methodology: Deoxyribonucleic acid (DNA) was extracted from 115 archived mouthwash samples collected from five healthcare facilities in northern South Africa. The partial open reading frame (ORF) K1 gene (~840bp) was amplified in a two round conventional PCR using JumpStart REDTaq master mix. The band of interest was extracted by phenol-freeze protocol and enriched using conventional PCR. Enriched amplicons were purified and sequenced in an Illumina MiniSeq platform. K1 genotypes were inferred using an online BioAfrica HHV-8 subtyping tool and confirmed by computing a phylogenetic tree. Intra-genetic diversity among HHV-8 genotypes was described by aligning study sequences with their respective prototype strains. Synonymous and nonsynonymous mutation rates were computed by the online SNAP tool. Results: K1 gene was successfully amplified in 61.7% (71/115) samples, along with unspecified DNA bands. The band of interest was successfully recovered in 67 amplicons (94.4%). Sixty-five gel extracted products (65/67; 97%) were successfully enriched and purified using magnetic beads. Of the 65 purified samples, 63 were sequenced using Illumina MiniSeq platform. Thirty-seven sequences had an acceptable nucleotide base call. The prevalence of HHV-8 in the study sequences was 94% (35/37) and majority of the sequences (24/35;68%) had sequence reads that span partial or complete K1 gene. Two major genotypes were detected (A and B); genotype B (19/24;79%) had a higher prevalence than genotype A (5/24; 21%). All sequences which grouped with genotype A were further classified as subtype A5. Interestingly, all sequences that were classified as genotype B did not cluster to any of the B subtypes. A higher genetic drift was observed among the study sequences reaching up to 33.7% at the amino acid level. Genotypes A and B exhibited 16.67% and 7.41% intra-genetic diversity at the amino acid level, respectively. Several amino acid polymorphisms were observed at the ITAM region of genotype A sequences (OUHC 013 and ODF 029), while the ITAM region of the B sequence was conserved. Conclusion: In this study, a predominance of HHV-8 genotype B was observed in northern South Africa. Additionally, there was a high degree of evolutionary divergence among the studied sequences. A higher frequency of nonsynonymous mutations was detected at the ITAM region of A5 sequences and these mutations may potentially affect the functionality of ITAM.

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