Dual infection and recombination of Kaposi sarcoma herpesvirus revealed by whole-genome sequence analysis of effusion samples

Abstract

Kaposi sarcoma herpesvirus (KSHV) is the etiological agent of three malignancies, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and KSHV-associated multicentric Castelman disease. KSHV infected patients may also have an interleukin six-related KSHV-associated inflammatory cytokine syndrome. KSHV-associated diseases occur in only a minority of chronically KSHV-infected individuals and often in the setting of immunosuppression. Mechanisms by which KSHV genomic variations and systemic co-infections may affect the pathogenic pathways potentially leading to these diseases have not been well characterized in vivo. To date, the majority of comparative genetic analyses of KSHV have been focused on a few regions scattered across the viral genome. We used next-generation sequencing techniques to investigate the taxonomic groupings of viruses from malignant effusion samples from fourteen participants with advanced KSHV-related malignancies, including twelve with PEL and two with KS and elevated KSHV viral load in effusions. The genomic diversity and evolutionary characteristics of nine isolated, near full-length KSHV genomes revealed extensive evidence of mosaic patterns across all these genomes. Further, our comprehensive NGS analysis allowed the identification of two distinct KSHV genome sequences in one individual, consistent with a dual infection. Overall, our results provide significant evidence for the contribution of KSHV phylogenomics to the origin of KSHV subtypes. This report points to a wider scope of studies to establish genome-wide patterns of sequence diversity and define the possible pathogenic role of sequence variations in KSHV-infected individuals.

Keywords: KSHV; genetic diversity; recombination; virology; virus taxonomy.

Figure 1.

Graphical representation of Taxonomer results for reads not mapped to the human GRCh37 (GCF_000001405.25) or KSHV GK18 (AF148805) genome of fourteen effusion samples. (a) High level taxonomic classification of sequencing data is presented as the relative abundance of sequences within each effusion sample. (b) Viral sequencing reads classified at the species or subtype level for twelve of the fourteen effusion samples.

Figure 2.

Partial representation of the alignment of FNL001_US to GK18 within the K1 coding region (355-583 bp). Gray bar across the top shows the GK18 genome sequence. Only bases in which reads disagree in reference to the GK18 sequence are shown. Reads belonging to the minor frequency genome (K1 A subtype) are highlighted in pink, whereas reads belonging to the consensus variant (K1 B subtype) are highlighted in blue.

Figure 3.

Number and effect of KSHV SNVs detected within the nine genomes included in this study. (a) A total of 954 nucleotide variants were identified when KSHV sequences were compared with GK18 as a reference. Variants within coding regions constituted ∼72 per cent of all SNVs and were classified as synonymous and non-synonymous. In addition, non-protein coding variants, deletions and insertions were also identified. (b) Distance matrix of eighty-six coding regions of KSHV genomes compared with the GK18 reference sequence. Amino acid (aa) sequence identity (% similarity) is proportionally color-coded ranging from 100 per cent in white to ≤40 per cent in dark blue. ORF73 was excluded as the internal repeat region had to be masked due to low coverage. Highlighter tool plots displaying mismatches in the protein sequence of the most variable coding regions are shown for K1 (c), ORF4 (d), ORF8 (e), and K8 (f). Plots were generated using the Highlighter tool by the Los Alamos National Laboratory.

Figure 4.

Graphic overview of the KSHV genome depicting coding regions (CDS, dark blue), repeat regions (light blue) and RNA coding regions (orange). Bar graph indicates the number of variants of all genomes identified in comparison to the reference GK18. Variants were counted within a 500 bp sliding window with masked regions displayed as light gray boxes. Vertical black lines mark variants that have been found in at least two genomes of the K1 A and C subtypes and variants identified in the K1 A5 genome. The predicted functional relevance of the variants is indicated by different shapes (circle, synonymous; square, non-synonymous; rhombus, non-protein coding). A total of 752 variants are shown.

Figure 5.

Phylogenetic NeighborNet splits network analyses of KSHV genomes, K1 and K15 sequences. All networks were generated with 1,000 bootstrap replicates. (a) Alignment comprising twenty-five genome sequences, including representative sequences for available K1 subtypes. Sequences were trimmed and repeat regions were masked prior to analysis. The network presents two main partitions dividing the network into K15 P and M subtypes (blue edges). In addition, African subtypes are separated by another set of parallel edges (orange), also called splits. The Japanese and European groups are supported by uncontradicted splits (purple and pink splits). Phi-test P value=0.0 (b) K1 network of forty-seven sequences, with thirty-nine representative sequences of all known K1 subtypes shows the majority of genomes from this study to belong to the A and C subtype. Two distinct subtypes were identified for FNL001_US (A and B). Phi-test P value=1.0. (c) Splits network of twenty-six K15 sequences distinguishes the three known subtypes. All genomes identified in this study belonged to the P subtype. Phi-test P value=1.0.

Figure 6.

KSHV recombination analysis of three representative genomes compared with fifteen published KSHV genomes as well as VG-1 using BootScan analysis (2,000 bp window, 200 bp step). (a−c) BootScan analysis shows highly fragmented genomes. Similar patterns of fragmentation were observed for sequences with K1 A and K1 C subtypes as seen for (b) FNL005_US and (c) FNL004_US, respectively. Repeat regions were masked out (gray bars).