Comparative genome analysis reveals driving forces behind Monkeypox virus evolution and sheds light on the role of ATC trinucleotide motif

Abstract

Monkeypox (MPOX), a zoonotic disease originating in Western and Central Africa in 1970, has seen a recent surge in outbreaks across 100+ countries. A comparative analysis of 404 Monkeypox virus (MPXV) genomes revealed notable changes in microsatellite abundance and density, especially within Clades I, IIa, and IIb. Each clade exhibited unique microsatellite motifs, with twenty-six conserved loci specific to MPXV, suggesting their potential as molecular markers in diagnostics. Additionally, nine genes in the MPXV genome featured ten variable hotspot microsatellite regions associated with surface protein synthesis and host control. Notably, gene OPG153, especially at the SSR locus '(ATC)n', exhibited the most pronounced variations among lineages over time and plays a role in virus pathogenesis within the host cell. These findings not only enhance our understanding of MPXV unique molecular profile but also offer valuable insights into potential pathogenic and evolutionary implications.

Keywords: human pathogen; microsatellite; monkeypox; motifs; repeats.

Figure 1.

The worldwide distribution of MPXV sequencing data utilized in the present study is visualized through a map, highlighting the geographical locations of 1,718 genomes, color-coded from highest (red) to lowest (yellow) intensity. The final dataset comprising 404 genomes is represented by dots, where larger dots indicate a higher volume of sequencing data, while smaller dots depict lower sequencing data. Additionally, the graph illustrates the percentage of sequencing data obtained from various countries for both the 1,718 genomes (left Y-axis) and the subset of 404 genomes (right Y-axis).

Figure 2.

(A) Reference genome of monkeypox. The genome is a substantial 197 Kbp in length, featuring two distinct variable regions flanking the central region. These variable regions are characterized by their unique sequences and exhibit a fascinating inverted terminal repeat structure at their respective ends. (B) Microsatellite mining in reference genome: Track 0 provides an overview of the reference genome, which spans 197 Kbp in length. Track 1 showcases 190 genes, displaying their respective sizes and precise locations along the genome. The functional categories and their corresponding colors are as follows: blue green for genes involved in host modulation, light steel blue color for genes not yet annotated, violet for surface proteins, and dark pink for genes associated with replication/transcription. Additionally, light pink indicates genes that play a role in assembly and budding processes. In Track 2, the length of microsatellites at specific loci is presented through a visually informative line graph. The coding regions are represented in slate blue, while non-coding regions are displayed in light pink. Track 3 enhances the visualization by presenting the microsatellite length, with color variations indicating different classes of microsatellites. Finally, Track 4 differentiates microsatellite numbers located in coding regions (shown in blue) from those in non-coding regions (displayed in pink). (C) Average microsatellite count in the 404 dataset. On an average, 300 microsatellites were found to be present in each genome. (D) Pearson correlation graph shows positive correlation between genome size and number of microsatellite (P-value <2.2e^–16).

Figure 3.

Lineage wise distribution of microsatellites. (A) RA and RD of microsatellites across sixteen lineages shown through ridgeline plot. A distinct contrast is easily discernible between ancestral and contemporary lineages. (B) Stacked plot showing the distribution of RA and RD among the different classes of microsatellites in all sixteen lineages. Within ancient lineages, pentamers were abundant, while monomers were found almost absent. Conversely, in recent lineages, the pentamer amount was reduced, and there was a notable increase in the number of monomers.

Figure 4.

Microsatellites lineage class comparison. The Kruskal-Wallis test is used to rigorously assess the differences among multiple groups of MPXV lineages. The observed discrepancies in microsatellite classes between the ancient lineages (Lineage I, I (likely IIb A.1), the reference lineage (IIb B.1), and the recently evolved lineage (IIb B.1.8) were exactly identified by (A) microsatellite length: Lineage I (likely IIb A.1) exhibited considerable variation in monomer, dimer, trimer, tetramer, pentamer, and hexamer (B) microsatellite frequency: Lineage I showed significant variation in monomer, dimer, and pentamer. Monomer, dimer, trimer, tetramer, and pentamer variation was substantial in Lineage I (probable IIb A.1).

Figure 5.

(A) Conservation of microsatellites in MPXV. Among the 318 distinct types of microsatellites discovered across 404 genomes, 39 per cent were conserved, 16 per cent were unique, and the remaining 45 per cent microsatellites were shared across MPOX genomes. (B) Shared microsatellites in various lineages: upset plot depicts that approximately 45 per cent of microsatellite types were identified as shared among diverse lineages. Notably, the highest (33) occurrence of shared microsatellites was observed between lineage I and I (probable IIb A.1). Additionally, fourteen microsatellites were found to be present across all lineages.

Figure 6.

Clades-specific microsatellite motifs. Phylogenetic tree displaying MPXV lineages up to the IIb B.1.8 variant. Clades 1, 2, and 3 are represented by red, yellow, and green boxes, respectively. Unique motifs from each clade are displayed on the right. Green triangles represent lineage groups, while red circles represent dead ends.

Figure 7.

Variable hotspot microsatellite loci. (A) Heatmap displaying microsatellite length variation in fifty-four genes across sixteen lineages. (B) A total of 10 microsatellites hotspot loci were detected in nine genes. The most substantial changes in microsatellite length were recorded in genes OPG153 and OPG197, where notably gene OPG153 featured two microsatellite hotspot loci. (C) A comprehensive heatmap depicting microsatellite hotspot genes across all sixteen lineages. In addition to the reference genome, this visualization only included genomes with variants. The heatmap discernibly reveals the gradual disappearance of microsatellite in OPG164 over successive generations. (D) The plot depicts the functional role of nine hotspot genes, as well as lineages which have differences from the reference.