Analysis of the ORFK1 hypervariable regions reveal distinct HHV-8 clustering in Kaposi's sarcoma and non-Kaposi's cases

Abstract

Background: Classical Kaposi's Sarcoma (cKS) is a rare vascular tumor, which develops in subjects infected with Human Herpesvirus-8 (HHV-8). Beside the host predisposing factors, viral genetic variants might possibly be related to disease development. The aim of this study was to identify HHV-8 variants in patients with cKS or in HHV-8 infected subjects either asymptomatic or with cKS-unrelated cutaneous lymphoproliferative disorders.

Methods: The VR1 and VR2 regions of the ORF K1 sequence were analyzed in samples (peripheral blood and/or lesional tissue) collected between 2000 and 2010 from 27 subjects with HHV-8 infection, established by the presence of anti-HHV-8 antibodies. On the basis of viral genotyping, a phylogenetic analysis and a time-scaled evaluation were performed.

Results: Two main clades of HHV-8, corresponding to A and C subtypes, were identified. Moreover, for each subtype, two main clusters were found distinctively associated to cKS or non-cKS subjects. Selective pressure analysis showed twelve sites of the K1 coding gene (VR1 and VR2 regions) under positive selective pressure and one site under negative pressure.

Conclusion: Thus, present data suggest that HHV-8 genetic variants may influence the susceptibility to cKS in individuals with HHV-8 infection.

Figure 1

Likelihood mapping analysis of K1 gene (VR1 and VR2 regions) sequences of HHV-8. Each dot represents the likelihoods of the three possible unrooted trees for a set of four sequences (quartets) selected randomly from the data set: dots close to the corners or the sides represent, respectively, tree-like, or network-like phylogenetic signal in the data. The central area of the likelihood map, represents star-like signal (phylogenetic noise). Panel A and B represent the first and second data set, respectively.

Figure 2

ML phylogenetic tree for the first subtyping data set. The tree was rooted by the midpoint rooting. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test, and were drawn to scale with the bar at the bottom indicating 0.04 nucleotide substitutions per site. Sequences corresponding to HHV-8 isolates from different diseases are evidentiated in black (KS), red (MF), orange (eMF), green (no clinical symptoms). The asterisk (*) along the branches represents significant statistical significance for the clade subtending that branch (bootstrap value > 70%).

Figure 3

Bayesian time-scaled tree of the 27 K1 HHV-8 gene sequences. The numbers at the root and at the internal nodes represent the estimated date of the origin and the uncertainty indicated by 95% highest posterior density (95% HPD) intervals. Sequences corresponding to isolates from different diseases are evidentiated in black (KS), red (MF), orange (eMF), green (no clinical symptoms) The asterisks (*) along a branch represent significant statistical significance for the clade subtending that branch (posterior probability >97%). The line at the bottom represents time (in years).