Updated Phylogeny of Chikungunya Virus Suggests Lineage-Specific RNA Architecture

Abstract

Chikungunya virus (CHIKV), a mosquito-borne alphavirus of the family Togaviridae, has recently emerged in the Americas from lineages from two continents: Asia and Africa. Historically, CHIKV circulated as at least four lineages worldwide with both enzootic and epidemic transmission cycles. To understand the recent patterns of emergence and the current status of the CHIKV spread, updated analyses of the viral genetic data and metadata are needed. Here, we performed phylogenetic and comparative genomics screens of CHIKV genomes, taking advantage of the public availability of many recently sequenced isolates. Based on these new data and analyses, we derive a revised phylogeny from nucleotide sequences in coding regions. Using this phylogeny, we uncover the presence of several distinct lineages in Africa that were previously considered a single one. In parallel, we performed thermodynamic modeling of CHIKV untranslated regions (UTRs), which revealed evolutionarily conserved structured and unstructured RNA elements in the 3'UTR. We provide evidence for duplication events in recently emerged American isolates of the Asian CHIKV lineage and propose the existence of a flexible 3'UTR architecture among different CHIKV lineages.

Keywords: RNA virus; epidemics; evolution; phylogeography; secondary structure.

Figure 1

Maximum-likelihood phylogenetic tree of 590 Chikungunya virus genomic sequences highlighting the geographic locations (tree branches) and lineages (tree leaves). Tree was computed using MAFFT alignment of individual ORFs and partitioned tree search with model test using IQ-Tree. Leaves are colored by lineage association. Branches of the tree are colored by geographic location of a sample. Colors of ancestral nodes and branches do not imply geographic origin and therefore are depicted in gray.

Figure 2

Worldwide spread of major epidemic Chikungunya clades: Asian Urban American (AUL-Am), ECSA Indian Ocean Lineage (ECSA-IOL) and ECSA Middle Africa South America (ECSA-MASA). This figure is rendered using the R package Leaflet with the underlying data based on parsimony ancestral reconstruction of metadata for place of isolation on the tree resulting from neighbor-joining tree search with collapsed branches under 80% bootstrap threshold (see Materials and Methods for more details). The circles on the map represent locations from which a transmission event originates. The color transparency represents the number of times a transition between characters occur on a location (node) or between locations (edge) (darker lines = more transitions). Boxes labeled “I” and “II” indicate that line crossed the International Date Line (e.g., AUL) or the shortest physical path between the two points (for mapping purposes only) was through the North Pole (e.g., ECSA-IOL).

Figure 3

(a–d) Secondary structure cartoons, structural alignments and base-pair probability plots (dot plots) of the structured CHIKV 3’UTR elements SL-Y, CSE, SL-a, and SL-b. Colored squares in the dot plot report a pairing of two bases, with full squares corresponding to 100% pairing probability. The base pairings pattern of the corresponding most stable, minimum free energy structures are shown in the lower triangles, while the upper triangles show the base pairing probabilities considering all possible alternative, suboptimal structures. Colors in structure drawings, alignments and dot plots indicate how many different types of base combinations are found for a particular base pair (red: 1; yellow: 2). All structure plots show the consensus structures as calculated by RNAalifold [57]. For each structure, the free energy (E) and the RNAz [65] z-score (z) are provided. Negative z-scores indicate a structure is more stable than it would be expected for other RNAs of the same dinucleotide content. (e) Sequence logo of the intrinsically unstructured repeat element UR, computed from all HMM-derived homologous sequences within the data set. Structure cartoons are not shown since the overall base pairing probability is essentially zero for this sequence.

Figure 4

Schematic of the 3’UTR architecture of all major CHIKV strains. The phylogeny on the left is derived from a maximum-likelihood tree based on a genomic alignment of six representative sequences. For each strain, the sequence with the longest UTR was selected as a representative (WA: AY726732.1, ECSA-IOL: JF274082.1, ECSA-MASA: KY038946.1, AUL-Cbn: KT327163.2 (Mexico), AUL-Am: MF001517.1 (USA), AUL-Asia: KF318729.1 (China). All boxes correspond to exact coordinates of annotated structural/nonstructural elements. A scale bar is provided for the nucleotide coordinates.