Whole-Genome Sequencing of Kaposi's Sarcoma-Associated Herpesvirus from Zambian Kaposi's Sarcoma Biopsy Specimens Reveals Unique Viral Diversity

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countries-where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment protocol followed by Illumina deep-sequencing, we generated and analyzed 16 unique Zambian, KS-derived, KSHV genomes. We enriched KSHV DNA over cellular DNA 1,851 to 18,235-fold. Enrichment provided coverage levels up to 24,740-fold; therefore, supporting highly confident polymorphism analysis. Multiple alignment of the 16 newly sequenced KSHV genomes showed low level variability across the entire central conserved region. This variability resulted in distinct phylogenetic clustering between Zambian KSHV genomic sequences and those derived from Western countries. Importantly, the phylogenetic segregation of Zambian from Western sequences occurred irrespective of inclusion of the highly variable genes K1 and K15. We also show that four genes within the more conserved region of the KSHV genome contained polymorphisms that partially, but not fully, contributed to the unique Zambian KSHV whole-genome phylogenetic structure. Taken together, our data suggest that the whole KSHV genome should be taken into consideration for accurate viral characterization. IMPORTANCEOur results represent the largest number of KSHV whole-genomic sequences published to date and the first time that multiple genomes have been sequenced from sub-Saharan Africa, a geographic area where KS is highly endemic. Based on our new sequence data, it is apparent that whole-genome KSHV diversity is greater than previously appreciated and differential phylogenetic clustering exists between viral genomes of Zambia and Western countries. Furthermore, individual genes may be insufficient for KSHV genetic characterization. Continued investigation of the KSHV genetic landscape is necessary in order to effectively understand the role of viral evolution and sequence diversity on KSHV gene functions and pathogenesis. K aposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), is the etiologic agent for all forms of Kaposi's sarcoma (KS) (1). KS manifests as an endothelial tumor primarily on the skin but can also involve mucosal membranes and visceral organs. Among the HIV-uninfected population, KS is rare worldwide; however, HIV infection and immunosuppression greatly increase the risk of developing KS (2). In sub-Saharan Africa, HIV is epidemic, and KSHV is endemic. Accordingly, KS is one of the most common cancers in sub-Saharan A...

Genotypic distribution of HHV-8 in AIDS individuals without and with Kaposi sarcoma

“…Investigation of biomarkers potentially useful for KS prognosis based on different loci of genome [9,10,12,14,15,18] or whole-genome data [16] has advanced parallel to the molecular characterization of a large number of HHV-8 strains infecting patients from different clinical and epidemiological forms of KS. [9,10,12,14,15] For example, several short targets of HHV-8 from AIDS-KS lesions were selected to identify distinctive molecular pattern, but with restricted coverage of the repertoire of HHV-8 genotypes.…”

Conservation of the glycoprotein B homologs of the Kaposi׳s sarcoma-associated herpesvirus (KSHV/HHV8) and old world primate rhadinoviruses of chimpanzees and macaques

“…The gB genes of several very closely related variants of KSHV, the human RV1 rhadinovirus, have been sequenced from different PEL cell lines, including the KSHV strain GK18 of Caucasian origin representing the NCBI reference sequence (NP_009333), and VG1, a more distantly related African B-subtype virus. Recently, the gB sequences from KSHV variants from sixteen Zambian KS biopsy specimens were obtained from the complete genome sequences (Olp et al, 2015). Whereas human herpesviruses have been identified in the RV1 (KSHV) and LCV (EBV) lineages, a human RV2 lineage herpesvirus has not been identified to date.…”

“…For KSHV, we compared the gB sequence of the GK18 strain (NC_009333) from a patient with classical (non-HIV) KS with the VG1 strain (ADB08179), an African B-subtype. Additional KSHV gB sequences from 16 Zambian KSHV strains (Olp et al, 2015) and strain JSC-1 (ACY00399) were also compared. The only other KSHV gB sequences available were essentially identical to GK18.…”

Kaposi sarcoma–associated herpesvirus: immunobiology, oncogenesis, and therapy

“…Only recently have whole KSHV genome sequences become available from patients and primary biopsies (150,151). These sequences augment extensive studies that trace the origin and evolution of KSHV based on single-gene analyses (152).…”