Whole-genome sequencing of KSHV from Zambian Kaposi’s sarcoma biopsies reveals unique viral diversity

24 Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi’s sarcoma 25 (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of 26 global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been 27 completed using isolates from Western countries—where KS is not endemic. The lack of whole28 genome KSHV sequence data from the most clinically important geographical region, sub29 Saharan Africa, represents an important gap as it remains unclear whether genomic diversity has 30 a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present 31 in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment 32 protocol followed by Illumina deep-sequencing, we generated and analyzed sixteen unique 33 Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 34 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting 35 highly confident polymorphism analysis. Multiple alignment of the sixteen newly sequenced 36 KSHV genomes showed low level variability across the entire central conserved region. This 37 variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic 38 sequences and those derived from Western countries. Importantly, the phylogenetic segregation 39 of Zambian from Western sequences occurred irrespective of inclusion of the highly variable 40 genes K1 and K15. We also show that four genes within the more conserved region of the KSHV 41 genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian 42 KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole 43 KSHV genome should be taken into consideration for accurate viral characterization. 44