Fig 1.
Year-wise dengue sequences reported from India.
(A) Dengue serotype sequences available from India till 2018. (B) Distribution of dengue serotypes in different regions of India. DENV-1: blue, DENV-2: orange, DENV-3: green, DENV-4: red. Arrows indicate the peaks in the number of sequences.
Fig 2.
Dengue genotype distribution and phylogeny.
(A) Heatmap of the number of whole-genome sequences from different geographical regions and India. (B-E) Time-dated phylogenetic trees for all serotypes with the circulating genotypes in India (DENV-1-III, DENV-2-cosmopolitan, DENV-3-III, DENV-4-I). The colour of the rectangle at the tip of the branches represents the region of the sample collection. The estimated time of the common ancestors is denoted for the important nodes along with the 95% highest posterior density (HPD) intervals. Asterisk (*) denotes posterior probability support of ≥ 0.95.
Fig 3.
Substitution rates for Indian genotypes and DENV-4-I lineage.
(A) Comparison of the substitution rates (substitutions per site per year) for different segments of dengue virus for Indian genotypes. The p-values were obtained using Welch’s t-test (unpaired, two-tailed, unequal variance). (B) Root-to-tip-distance of global dengue whole-genome nucleotide sequences. The Indian sequences are highlighted in red, and the R2 value for the linear fit is shown. (C) The maximum-likelihood phylogenetic tree for complete coding sequences of DENV-4. Distinct branches (corresponding to Ia/b/c/d, IIAa/b and IIBa/b) used for the dN/dS analysis are colour coded. Indian DENV-4 cluster is shown in the inset. (D) dN/dS values for the consensus sequences generated from the branches in (C) are represented as a box plot for each gene. Whiskers denote the 5th and 95th percentile. The dN/dS values for Ic/d pair are marked in red. The generalized extreme Studentized deviate test was performed to find the outliers, and p-values are reported. (E) Sequence logo showing the amino acid variations between pairs Ic/d and Ia/b. The amino acids at these locations for DENV-1-3 are shown at the bottom. Highlighted colour depicts the DENV-4 branch with the same amino acid residue.
Fig 4.
Dynamics of E gene amino acid variation in South India between 2007 and 2016.
(A) Normalized amino acid distances with respect to the 2007/2008 dengue strains is plotted in black. Hamming distances were normalized with the sequence length and converted to a z-score. Distances were bootstrap sampled (n = 100) to calculate the reported median. Error bars represent standard error. The blue line represents a linear regression fit to the data with 80% (gray shade) confidence interval. Di indicates the distance dynamics between DENV-i from its (2007/2008) ancestral sequence. (B) Year-wise normalised interserotype distances (as z-score) calculated by randomly selecting the sequences from each serotype every year (100 bootstraps) is plotted. The inset heatmap depicts the time period of oscillation binned yearly (range of 0 to 5 years). The mean and standard deviation (in brackets) of the time period distribution is denoted next to the heatmap. Dij indicates the interserotype distance dynamics between i-th and j-th serotype. (C) Median of the Pearson’s correlation coefficients between the distance dynamics Di and Dij is shown as a heatmap with corresponding colormap. A schematic depicting the relative evolution of dengue serotypes in two-dimensional sequence space with positive and negative correlation between Dij and Di or Dj is shown at the bottom. Central point represents the ancestral strain, and the arrow represents the direction of the virus evolution.
Fig 5.
Comparison of Indian envelope protein sequences with vaccine strains.
(A) Relative amino acid differences between Indian envelope sequences (post-2000) and vaccine strains (CYD-TDV, TV003 and TAK-003). The multidimensional scaling was used to reduce the dimensions of the data using the hamming distance matrix. Genotype cluster boundaries are indicated with dotted lines for visual clarity. Indian sequences are shown in grey; vaccine strains are shown with empty circles. (B) Amino acid differences at a frequency >10% with respect to the CYD-TDV vaccine are shown on the envelope protein dimer structure. Different positions in the known epitopic regions are shown in red. Variable residues within the predicted antigenic effect positions are shown in black. Residues present in epitopic regions with predicted antigenic effects are highlighted with a box. Venn diagram shows the total number of differences in the full E protein (including the stem and transmembrane region), epitopic sites and antigenic sites. The circle size represents the number of differences category-wise. (C) The density of differences (number of differences/ length of the domain) is shown for different domains of the envelope protein (envelope domains EDI-III, stem and transmembrane (TM) region). The error bars represent the standard deviation across the three vaccine strains.
Fig 6.
Schematic for antibody-mediated protection from secondary dengue infection.
(A) Antibody decay dynamics and (B) corresponding level of protection during the secondary infection. Homotypic antibodies and protection from homotypic infection wane slowly (green). Cross-reactive antibodies to heterotypic dengue and protection wane faster (purple). Sequence similarity between the primary and secondary infecting serotypes is shown by shades of purple. The red shaded area represents the window of the antibody titer in which ADE is in effect. The dotted purple line represents protection response in the absence of ADE.