Clinico-genomic study reveals association of dengue virus genome high frequency mutations with dengue disease severity

Abstract

Transmission of the dengue virus (DENV) places a huge burden on public health in several endemic regions. Like other RNA viruses, mutations in the DENV genome greatly governs its virulence, transmissibility, and interaction with the host immune system. Present study focuses on integrated analysis of mutation and clinical data accompanied at the onset of dengue fever. The findings from the associated clinical data with the variants of the DENV are critical for early detection of the disease and understanding the disease progression. RNA was isolated from the 1310 serum samples collected from the NS1-antigen positive dengue patients. Serotyping reveals that DENV-2 was predominant in circulation. The genome of 1305 DENV-2 was sequenced using Oxford Nanopore Technology and Illumina platforms. A total of 1023 DENV-2 demonstrated > 50% genome coverage. Mutation analysis across the 1023 DENV-2 genomes yielded a total of 2667 mutations including 627 non-synonymous and 2040 synonymous mutations. We observed a notable over-representation of synonymous mutations in prM and ancC genes while a higher occurrence of non-synonymous mutations was found in ancC, prM, and M proteins. Comparison of mutation frequency between mild and severe demonstrates higher mutation frequency in severe phenotype. Moreover, we observed a total of 56 significant mutations including 23 in severe, 17 in moderate and 16 in mild. The E-protein having non-synonymous mutations were docked with DC-SIGN with lower binding energy (ΔG = - 11.9 kcal/mol) for severe as compared to mild (ΔG = - 13.5 kcal/mol), suggesting lesser affinity of E-protein and DC-SIGN in case of severe as compared to the mild. We have identified the core set of high frequency mutations significantly associated with distinct dengue disease severity viz., mild, moderate and severe. Furthermore, in-silico protein modelling and docking studies demonstrate the potential functional role of the non-synonymous mutations identified across E-protein in severe dengue.

Keywords: Dengue; Disease severity; Leukopenia; Mutation analysis; Thrombocytopenia.

Fig. 1

DENV serotype distribution and mutational dynamics across the DENV-2 genomes. (A) Overview of the experimental design, including RNA isolation from serum samples, followed by qRT-PCR, whole genome sequencing, and variant analysis. (B) The sunburst plot represents the distribution of DENV serotypes in circulation from August 2022 to November 2022 in Delhi, India. (C) Bar graph showing the range of breadth of coverage across the number of DENV-2 genomes sequenced. (D) Line-range plot depicting the mutation spectrum across the DENV-2 genomes. The length of each line indicates the number of samples in which genomic mutations were detected at that particular position. (E–F) Bar chart representing means of gene length normalized count, (E) synonymous, and (F) non-synonymous mutation counts (× 1000) across the DENV-2 genomes.

Fig. 2

Clinical characteristics of dengue-positive individuals. (A) Stacked bar plot representing the percentage distribution of dengue patients across all the clinical parameters, categorized as low, normal, and high based on their clinical parameters derived from both complete blood count (CBC) and liver profile test (LFT) reports. (B–O) Sample-wise distribution of clinical parameters for (B) platelet count (10⁹/L), (C) total leucocyte count (TLC) (10⁹/L), (D) hemoglobin (g/dl), (E) packed cell volume or hematocrit (%), (F) lymphocytes (%), (G) eosinophils (%), (H) mean corpuscular hemoglobin or MCH (pg), (I) monocytes (%), (J) red blood cell or RBC count (10¹²/L), (K) mean platelet volume or MPV (fL), (L) neutrophils (%), (M) basophils (%), (N) aspartate aminotransferase or AST (U/L), and (O) alanine aminotransferase or ALT (U/L). The points above the bar represent samples that were categorized according to the range of each parameter: low: green, normal: orange, and high: purple.

Fig. 3

Phylogenetic analysis of 1023 DENV genomes and depiction of genotypes identified. The analysis revealed three clades of DENV-2 serotypes, where clade A and clade C represent cosmopolitan, while clade B is a mixture of both cosmopolitan and asian-I. The clades with different genotypes are labeled as mild (green), moderate (orange), and severe (purple).

Fig. 4

Association of high frequency mutation with distinct disease severities. (A) Graphical representation of dengue patients (n = 724), where mild (n = 317), moderate (n = 222), and severe (n = 185) were categorized based on the leukopenia and thrombocytopenia clinical parameters. The bars depict the average coverage and mutations per sample across mild, moderate, and severe. This figure was created using Biorender.com. (B) The upset plot shows the number of overlapping mutations across mild, moderate, and severe patients. (C) Scatter plot representing percentage of mutation frequency across the DENV-2 genomes. The dots within the circle depict mutation frequency within the cluster (clustered mutation), while only the dots represent mutation frequency outside the cluster (unclustered mutation). The color represents mild, moderate, and severe. (D–F) Line plots showing mutation frequency across the DENV-2 genome for Cluster A (80–100%) between three comparison groups: (D) mild vs. severe, (E) moderate vs. severe, and (F) mild vs moderate. (G) Violin plot showing significant differences between percentage of mutation and severity cohort (mild, moderate, and severe) for cluster A. One-way ANOVA test was used for the significant test (p value < 0.05). (H) Lollipop plot representing significant synonymous and non-synonymous mutations between 2 comparison groups, i.e., moderate versus severe (upper) and mild versus severe (lower), across the DENV-2 genomes.

Fig. 5

Binding and Interactions of E-protein and DC-SIGN. Binding interface of E-protein with DC-SIGN is depicted in (A) Wild-type, (B) Mild, (C) Moderate, and (D) Severe.