Table 1.
Criteria based on clinical parameters for defining mild, moderate, and clinically severe dengue patients.
Fig 1.
Experimental workflow and SNVs spectrum across DENV-2 genomes.
(A) Study design includes NS1-Ag-positive serum sample collection, RNA isolation, and serotype detection, followed by whole genome sequencing, variant calling, and downstream data interpretation. This figure was created using licensed version of Biorender.com. (B) SNV spectrum with distribution of synonymous and non-synonymous SNVs across DENV-2 genomes whose coverage are > 50% (n = 3254). (C and D) Bar chart representing means of number of unique SNVs divided by gene length, a normalized count in the y-axis, with mean from bootstrap standard error estimation. (C) synonymous, and (D) non-synonymous SNV counts (x1000) across the DENV-2 genomes. (E) Horizontal stacked bar chart depicts the percentage proportion of synonymous and various types of non-synonymous SNVs—including missense, frameshift, and stop-gained—across different genes of DENV-2 genome.
Fig 2.
Dengue severity subgrouping and comparative account of clinical manifestations.
(A) Schematic representation of the dengue patient categorization based on two primary CBC parameters: total leukocyte count (TLC) and platelet counts (thrombocytes) into mild, moderate, and clinically severe, and their association with fluctuations in CBC and LFT parameters during dengue virus infection. This figure was created using licensed version of Biorender.com. (B-F) Statistical comparison across the patient groups associated with mild, moderate, and clinically severe sub phenotypes for the major CBC parameters, including (B) total leukocyte count (109/L); (C) platelets (109/L); (D) hemoglobin (g/dl); (E) neutrophils (%); and (F) lymphocytes (%). (G-K) Statistical comparison across the patient groups associated with mild, moderate, and clinically severe dengue sub phenotypes for major LFT parameters, including (G) bilirubin (direct) (mg/dl); (H) bilirubin (total) (mg/dl); (I) ALT (U/L); (J) AST (U/L); and (K) GGTP (U/L). Kruskal-Wallis with Dunn’s comparison was performed for comparison of each clinical parameter across mild, moderate, and clinically severe. The significance value is denoted as *, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, and **** indicates p ≤ 0.0001.
Fig 3.
Phylogenetic analysis of 10164 DENV-2 genome sequences.
The distribution of genomes of different continents from various outbreaks, along with the in-house sequences from a single outbreak, is represented using distinct colors. The color codes are as follows: mild (green), moderate (orange), clinically severe (magenta), cohort data (sea green), India (salmon red), Asia (pink), Africa (cyan), North America (yellow), and South America (yellow-green).
Fig 4.
Phylogenetic analysis for the DENV-2 genomes associated with different disease sub-phenotypes.
A magnified view of phylogenetic tree for 929 genomes comprising clinical sub-phenotypes mild (light green), moderate (orange), and clinically severe (magenta).
Fig 5.
Association of SNVs with differential disease severity.
(A) The analytical workflow of the SNV association studies, showing a representation of clinico-genomic integration of mild, moderate, and clinically severe subgroups with differential genomic features, that is depicted through differential SNV burden in the genes for different categories. This figure was created using licensed version of Biorender.com. (B) Joint-grid plot depicting genome coverage and SNVs count across mild, moderate, and clinically severe sub phenotypes depicting a positive correlation for higher number of SNVs for higher coverage. (C) Venn diagram demonstrating the unique and common SNVs across the mild, moderate, and clinically severe patients. (D) Frequency of SNVs in mild, moderate, and clinically severe groups across the DENV-2 genomes has been smoothed by moving average in intervals of 10, with the average represented by square markers. (E) Bar plot depicting significant burden of SNVs across the gene of DENV-2, with the significance shown in the asterisk symbol obtained from the chi-square test of independence.
Fig 6.
Distribution of significant SNVs across disease severity.
Lollipop plot depicting significant SNVs across mild and clinically severe phenotypes. SNV is represented with different colors, signifying individual groups. Significance was calculated using fisher’s exact test (p < 0.05). The directionality and strength of association of significant SNVs with respective groups were confirmed with the phi coefficient correlation test.
Fig 7.
Comparison of statistically significant SNVs associated with mild and clinically severe phenotype with global frequency (A-B).
The matrix plot depicts the comparison of significant SNVs identified in the of DENV-2 genomes from the study with the global frequencies reported worldwide A) Mild, and B) Clinically severe.
Fig 8.
Summary of the identification of SNV hotspots in the DENV-2 genome.
It highlights E, NS4B and NS5 regions in the mild and clinically severe dengue sub-phenotypes through an integrative clinico-genomic approach. This figure was created using licensed version of Biorender.com.