Data collection

Sequence alignment and phylogenetic analysis

Multiple sequence alignment was performed with protein-coding sequences from dataset B using MUSCLE v3.8.425 [42] implemented in AliView v1.25 [43]. Alignments were manually checked for insertion and deletion errors. Maximum likelihood trees were generated using IQ-TREE v1.6.10 [44] with 1000 bootstraps. A general time-reversible substitution model with unequal base frequency and gamma distribution for rate heterogeneity (GTR+F+I+G4) was selected out of 88 models available using jModelTest [45] implemented in IQ-TREE v1.6.10 [44] based on the Bayesian information criterion. Sylvatic strains EF457905 (DENV-1), EF105379 (DENV-2) and JF262779-80 (DENV-4) were used as outgroups to root the respective trees. The root for DENV-3 was obtained using the best fitting root by the correlation method in TempEst [46]. The maximum likelihood phylogenetic trees were used to obtain the root-to-tip distances using TempEst. Trees were visualized using Figtree v1.4. We assigned new lineages within a genotype if the difference between the sequences from each phylogenetic branch is more than 3% at the nucleotide level and 1% at the amino acid level.

Bayesian analysis was performed with the E gene, NS5 gene and whole-genome sequences from dataset B.1 using the BEAST v1.8.3 [47] to get the substitution rates for each gene. Five sets were generated for each serotype by selecting 80% sequences randomly, the substitution rate was calculated for each run, and the average substitution rate was reported. The constant rate clock model was selected based on the AICM values of the Bayes factor and harmonic means (S2 Table). Markov Chain Monte Carlo (MCMC) was run for 10⁷ generations for each run, and the first 10% of samples were discarded as burn-in. The phylogeographic movement of the virus across the countries was obtained using the Bayesian stochastic search variable selection procedure implemented in BEAST v1.8.3 with whole genome sequences from dataset B.1 (MCMC chain length ~10⁸). SpreaD3 v0.9.7 [48] was used to visualize the spatio-temporal dynamics of the virus. Tracer v1.6 was used to check the convergence of the chains. Effective sample sizes for the parameters of interest were greater than 200.

Single-likelihood ancestral counting (SLAC) and fixed effects likelihood (FEL) methods were used to identify the positions that are undergoing selection using the HyPhy package (v2.5.1) [49]. Sites with a p-value <0.1 were considered significant only if they were detected by both methods.

Dynamics of amino acid variation in the envelope gene sequence

Temporal dynamics of E gene amino acid variation was examined in the sequences from South India due to the availability of a larger dataset (n = 164). Sequences with at least 50% coverage (mean coverage of 93%) of the E gene were selected for the analysis. The Hamming distance between the pair of sequences was divided by the length of the overlapping region between the pair. This was further converted to the z-score using the mean and standard deviation to obtain the normalized distance. To extract the dynamics within the serotype, we measured the normalized distance of new variants over the years with respect to the ancestral sequences (sequences from 2007/08 were used as ancestors due to a lack of sufficient sequences before that). The inter-serotype distance was calculated by selecting the sequences from two different serotypes during a particular year. Sample bootstrapping (n = 100) was used to determine the median and standard error of the normalized distances for each year. The time period of oscillations was calculated using an autocorrelation function with different lags for each trace. A lag with the maximum correlation coefficient was assigned as the period of oscillation for that trace. Bootstrap replicates (n = 100) were used to obtain the distribution of the time period. We performed the same analysis with the CprM gene sequences (position 55 to 155 of the coding sequence) from South Indian sequences (n = 411).

Pearson’s correlation coefficient was obtained between the serotype and inter-serotype distance dynamics. The robustness of the correlations was checked by three methods. First, by randomly deleting up to 3 data points from each dynamics (1000 bootstraps). Second, by estimating the cross-correlation between individual traces from two comparing groups randomly with 1000 bootstraps. As a control, we also checked correlations between the groups by randomly shuffling the time series of normalized hamming distances (before correlating) to ensure that the correlations do not arise merely from the yearly fluctuations (1000 bootstraps).

Dominant epitope selection from the database

Experimentally determined epitopes for all dengue serotypes were obtained from the Immunome Browser tool available on the Immune Epitope Database [50]. B cell and T cell epitopes were selected based on their response frequency (RF) score, calculated as reported earlier [51]. Epitopes having RF-score > 0.25 were selected as dominant epitopes for further analysis. For the epitopes examined in multiple studies, the RF-score was calculated by combining the number of subjects from all the studies. Apart from these known epitopes, we also included 77 sites in which residue variation conferred antigenic effects [52].

Comparison with the vaccine strains

All Indian DENV envelope sequences post-2000 from dataset C were compared with three vaccines: CYD-TDV developed by Sanofi Pasteur, TV003 by NIH/Butantan and TAK-003 by Takeda. Multidimensional scaling based on the Hamming distances between the sequences was used to visualize and evaluate the differences between Indian and vaccine strains.

Homo-dimeric structures of the envelope proteins were obtained by homology modelling using the SWISS-MODEL server [53]. Template PDB structures were selected based on global model quality estimates and QMEAN statistics. Vaccine strain information and template selection for each serotype is shown in the S3 Table. Sites with differences in >10% of sequences were identified as significant differences and were mapped onto the envelope protein structure using PyMOL (v2.4.1) [54].

Dengue serotype dynamics in India

The reported dengue cases in 2018 have increased more than 25-fold (three year average) since 2002 in India (S1A Fig). All four geographical regions, namely–North, East, South and West-Central India, show periodic spikes in dengue cases as well as deaths over 2–4 years (S1B–S1D Fig). Comparing all published dengue sequences till 2018 from India (dataset A), we find all four dengue serotypes co-circulating in the country since 2000 (Fig 1). Although dengue sequence reporting from various parts of the country is sporadic and focused on the urban areas, the number of annual cases and deaths in the past two decades correlate well with the number of available sequences each year (S1E Fig, Pearson’s correlation coefficient = 0.67). In particular, we noted the increase in DENV-2 and DENV-4 sequences since 2011 and 2014, respectively (Fig 1A). Corresponding to the reported cases, we found periodic peaks in the number of sequences reported from North and South India. Genomes reported from North India show a pattern of serotype replacement in consecutive peaks (Fig 1B). Since most of the sequences from North India were collected from Delhi (~80.5%), the spikes in the dengue sequences in 2006, 2010 and 2013 also correspond well with the outbreaks in Delhi [31,55] (S1C–S1D Fig). Consistent with previous reports, we find DENV-3 dominated during the 2006 outbreak [56] while DENV-1 and DENV-2 serotypes dominated during the outbreaks in 2010 and 2013, respectively [31,57]. Although dengue outbreaks have been observed in East and West-Central India (S1C and S1D Fig), a relatively smaller number of sequences are available from these regions, possibly due to poor genomic surveillance (Fig 1B). Nevertheless, DENV-2 emerged as the dominant serotype in East India in 2016, while all serotypes were identified in West-Central India from 2016 to 2018. In South India, three peaks are evident in the number of sequences corresponding to 2009, 2013 and 2016 (Fig 1B) and correlated peaks in the number of cases/deaths were observed in 2009–10, 2012–13 and 2017 (S1C and S1D Fig). DENV-1 and DENV-2 have been the dominant serotypes over the last decade, but DENV-4 has recently emerged as a major serotype in South India. Similar periodic fluctuations in dengue incidence and replacement of dengue serotypes have been proposed previously to be driven by population-level immunity [18,19], but whether high endemicity and seropositivity are driving case incidences, serotype-specific dynamics and virus evolution in India is not known. In summary, DENV-1 and DENV-3 were the dominating serotypes in India till 2012. DENV-2 has become the dominant serotype in most regions in India since then, and DENV-4 is establishing itself in South India.

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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.

https://doi.org/10.1371/journal.ppat.1010862.g001

Prevalence and spatio-temporal dynamics of Indian dengue genotypes

Maximum likelihood trees and maximum clade credibility trees of complete genome coding sequences suggest that dengue genotypes are constrained by geography exemplified by the poor intermixing of the genotypes across continents (Figs 2 and S2–S4). Asian sequences consist of two dominant genotypes for all the serotypes, while the recent Indian dengue sequences represent only one of those genotypes (except for DENV-1). Genotype III for DENV-1, cosmopolitan genotype for DENV-2, genotype III for DENV-3, and genotype I for DENV-4 have been the dominating genotypes in the past two decades in India. The Indian genotypes are remarkably divergent from dengue in other regions of the world beyond Asia and merit further examination of their evolutionary dynamics (Fig 2A).

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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.

https://doi.org/10.1371/journal.ppat.1010862.g002

Interestingly, we find distinct temporally and spatially co-circulating lineages within the genotypes in India (Fig 2B–2E), yet it remains unclear what factors contribute to their co-prevalence. Among the two DENV-1 genotypes (I and III), genotype III has been dominant across India (S2 Fig). Within genotype III, two dominant lineages (lineage I and II) are circulating simultaneously within the country. These lineages differ at 39 positions across the genome (S5A Fig) and five positions in the immunologically dominant E gene (T272M, I337F, T339I, V358A and I/V461I). Residues 272 and 337 are on the exposed part of the E protein dimer, and position 272 is linked to having an antigenic effect suggesting differential antigenicity. Even though the phylogenetic clustering did not include the untranslated regions, all the sequences from lineage-I carry a deletion of 21 nucleotides in the hypervariable region of the 3’UTR (nucleotide positions 10294–10314 with respect to the reference sequence NC_001477, S6 Fig). A similar deletion has been reported previously from India [58], but the significance of such a large deletion is unclear. This region is not involved in any stem-loop structures or the genome cyclization region in the 3’UTR. In vitro studies have shown that a 19-nucleotide deletion (nucleotide positions 10289–10309) overlapping this region of 3’UTR did not affect the growth kinetics of the virus [59], while larger deletions (nucleotide positions 10274–10728) in the 3’UTR variable region show growth defects in human cell lines [60]. Therefore, it is likely that DENV-1-III lineage-I can tolerate the 3’UTR deletion without a significant fitness cost. On the other hand, a cluster of sequences in DENV-1-III lineage-II carry an insertion of two nucleotides in the hypervariable region of 3’UTR (C10274 and A10297 with respect to NC_001477, S6 Fig) and is restricted to South India. This shows how at least three sets of genotype III strains are co-circulating in the country. Spatio-temporal analysis reveals that DENV-1-III lineage-I is a relatively new lineage that emerged in 1992 (95% HPD: 1990–1995) and is limited to India, Singapore, and China (Figs 2B and S7A and S4 Table). Our analysis shows the early emergence of this genotype in India, with subsequent spread to South Korea, Comoros, and Singapore between 1990 and 2005 and multiple import-export events between India and Singapore post-2005 (S7A Fig). Apart from this, genotype I of DENV-1, the primary genotype in most Asian countries (Fig 2A), has also been reported exclusively from South India since 2012, suggesting a more recent introduction (S8 Fig).

The earliest DENV-2 sequences from India belong to the American genotype. This genotype was predominant in India before 1971 but was eventually replaced by the cosmopolitan genotype [61]. The DENV-2-cosmopolitan genotype was introduced to India and China in the early 1980s. From here, it has spread to Southeast Asia, Australia, and East Africa (S7B Fig). DENV-2 in India has two distinct co-circulating lineages (lineage-I and III) that emerged in the 80s with a common ancestor that dates back to 1979 (1977–1981) (Figs 2C and S3 and S4 Tables) [32,62]. While prevalent in the Indian subcontinent, cosmopolitan lineage-III has been reported in East Africa, and spatio-temporal analysis suggests that this lineage was most likely exported from India, in line with the study from Kenya [63]. These lineages differ at 41 positions across the genome, including three positions in the envelope gene (S5B Fig), all of which are in known epitopic regions or have antigenic effects (E protein residues 141, 162 and 322), suggesting immune selection pressure playing a role in their divergence. Otherwise, the E gene did not show significantly higher nonsynonymous mutation density compared to other genes (S5E Fig). Similarly, these lineages do not differ in their in-vitro virus growth kinetics and disease severity [32]. This could explain how these two lineages have been able to avoid replacement by the other since their emergence.

All the Indian DENV-3 genotype III sequences cluster along with other Asian countries (S4 Fig). DENV-3-III was first detected in Sri Lanka and disseminated to multiple countries over time (S7D Fig). After its initial spread, it evolved into two distinct geographical clusters: lineages IIIA (Asian) and IIIB (American). Most of the spread of genotype IIIB in the Americas was from 1990 to 2005 (S7D Fig). In contrast, diffusion of genotype IIIA in Asia took place after 2005, yet these two lineages have not intermixed (Figs S7D and 2A). In the E gene, these lineages differ at positions 158 and 380, with position 158 being a part of a known B cell epitope. Within lineage IIIA, we found two dominating sub-lineages co-circulating in India (IIIAa and IIIAb). These sub-lineages differ at 26 positions across the genome and eight positions in the E gene (S5C Fig). Although the E gene displays a high density of mutations (S5E Fig), most of these positions (7 of 8) carry the same amino acid in majority of sequences for both the sub-lineages (S5C Fig).

The spatio-temporal reconstruction identifies the earliest sequences of DENV-4-I of Philippines origin (S7C Fig) consistent with the E gene analysis reported earlier [64]. Most of the subsequent spread of DENV-4-I was restricted to Southeast Asian countries. Although it was introduced in India around 1945, there has been a significant increase in reported DENV-4-I sequences from 2015 onwards, especially in South India (Fig 1) [33,65]. It is possible that it remained undetected due to underreporting of mild infections since the primary cases of DENV-4 have been reported to cause mild disease [66,67]. All Indian DENV-4-I sequences cluster separately from the other prevalent genotype I groups (Fig 2E). We identify two lineages in Indian DENV-4 sequences (Ic and Id); DENV-4-Id lineage seems to be replacing DENV-4-Ic since 2016 (Figs 2E and 3C). This novel lineage of DENV-4 (Id) was reported in Pune in 2016 using the CprM sequences [68]. We noticed remarkable differences in the E and NS2A genes between the two lineages (S5D Fig), as discussed in detail in the next section. Overall, all prevalent DENV genotype lineages carry signatures of immune selection contributing to their divergence.

Emergence of DENV-4-Id suggests dominant immune selection pressure

To examine the selection pressure on the dengue virus in India, we employed FEL (Fixed Effect Likelihood) and SLAC (Single Likelihood Ancestral Counting) methods. Apart from DENV-3, there was no substantial evidence of positive selection, and most amino acid changes were deleterious and negatively selected (S5 and S6 Tables). In DENV-3-III genotype, we found a single position in NS5 protein (50I/T) under positive selection, while this site is highly conserved in all other serotypes. Significant negative selection in DENV is consistent with the requirement for horizontal transmission between taxonomically diverse host species, which imposes a strong purifying immune pressure [10,12,13].

Further, the substitution rates of dengue genotypes in India are comparable (7.59E-4, 6.31E-4, 7.83E-4, 6.50E-4 and 6.26E-4 substitutions/site/year for DENV-1-I, DENV-1-III, DENV-2-cosmopolitan, DENV-3-III and DENV-4-I, respectively) (S4 Table) and similar to earlier reported rates [7,62,69]. Although 95% highest posterior density (HPD) intervals for all the genotypes overlap, the largest substitution rate is observed for the DENV-2-cosmopolitan genotype. However, the substitution rates obtained for the E gene were about 26% (11.5% to 44.2%) higher than those for the whole genome (Fig 3A). Interestingly, the substitution rate was 44% larger for the DENV-4-I E gene compared to the whole genome, suggesting high immunological pressure driving the divergence of the DENV-4 E gene. This is consistent with previous reports for the HIV envelope gene and spike protein of the SARS-CoV-2 virus [70,71].

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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 5^th and 95^th 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.

https://doi.org/10.1371/journal.ppat.1010862.g003

Root-to-tip distance analysis showed that most Indian dengue viruses follow the molecular clock similar to that observed in other parts of the world (Fig 3B). However, Indian DENV-4 lineage Id is highly divergent with residuals larger than three times the interquartile range and displays the most extended branch in the phylogenetic tree (Fig 3B and 3C). Indeed, when we compare dN/dS ratios between branches within the DENV-4 phylogenetic tree (Fig 3C), we found significantly higher dN/dS values for the E gene for the DENV-4-Ic/d pair compared to other DENV-4 clade pairs (Fig 3D). Besides the E gene, NS2A also showed a relatively higher dN/dS ratio and nonsynonymous mutation density (Figs 3D, S5D and S5E). This suggests a role of immune selection pressure in DENV-4-Id evolution consistent with co-evolution of E and NS2A genes correlating with virus antigenicity [52]. On the other hand, NS1 and NS5 genes showed significantly lower dN/dS ratios for the same pair. In fact, 10 out of 34 mutations in DENV-4-Id (with respect to DENV-4-Ic) are present in the E gene (S5D Fig). The E gene variant positions 132, 174, 200, 202 and 351 are also in the known epitopic regions and/or have antigenic effects pointing to the immune escape-driven divergence of DENV-4-Id. Intriguingly, five of the acquired E gene mutations (I130V, K200T, N202K/E, A221T, H494Q) in DENV-4-Id are similar to the DENV-1 and DENV-3 E genes (Fig 3E). This was unexpected since the prior prevalence of DENV-1 and DENV-3 in the country should have forced the DENV-4 to drift away from them. On the other hand, antibody-dependent enhancement may confer some fitness advantage to the DENV-4-Id clade due to shared antigenic features with DENV-1 and DENV-3. Such movement towards other serotypes is not evident in the phylogenetically related DENV-4-Ia/b pair (Fig 3E), suggesting a unique signature of the Indian DENV-4-Id lineage.

Dynamics of E gene evolution displays recurring variation

Taking a cue from the divergence of DENV-4-Id, we examined whether the high seroprevalence can play a role in the evolution of dengue in India. Dengue cross-reactive immunity has been shown to shape the antigenic evolution of dengue for the E gene at a city level [11]. Since serotype replacements in the different regions of the country display temporal fluctuations (Fig 1C), we asked whether there are signatures of immunity-driven evolution of dengue serotypes across large endemic areas. In the absence of antigenicity data, we evaluated the variation in amino acid sequences of the E gene longitudinally. To validate this approach, we rely on neutralization antibody titers of sera collected from humans (post-monovalent vaccination) and African green monkeys (post-infection) tested against a panel of viruses [72]. We found general correlation between the antibody titers and the similarity between the E protein sequences with the two datasets of antibody titer data (Pearson’s correlation coefficient: 0.530, S9 Fig). In South India, we find that, in general, the E gene diverges from the ancestral sequence for all serotypes, but this divergence fluctuates over time (Fig 4A). Overall, in our dataset, the E gene sequences drift away from their respective ancestral sequences, evolve to be similar and then diverge repeatedly. This behaviour was pronounced in DENV-2-4 with an estimated time period (peak-to-peak) of about three years (2.92 ± 0.58 years for DENV-2, 3.55 ± 0.9 years for DENV-3 and 2.99 ± 0.64 years for DENV-4). While we could not estimate a time period for a similar E gene dynamic in the case of DENV-1, we did note a peak in divergence in 2012–13 arising from a singular genotype I outbreak during a period of genotype III prevalence.

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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. D_i 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. D_ij indicates the interserotype distance dynamics between i-th and j-th serotype. (C) Median of the Pearson’s correlation coefficients between the distance dynamics D_i and D_ij 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 D_ij and D_i or D_j is shown at the bottom. Central point represents the ancestral strain, and the arrow represents the direction of the virus evolution.

https://doi.org/10.1371/journal.ppat.1010862.g004

Interestingly, the observed dynamics of the E gene amino acid variations were also correlated among the serotypes (Figs 4C and S10). The high correlation suggests that the divergence in each serotype was synchronous, i.e., when DENV-1 drift away (or converges) from its ancestral sequences, a similar dynamics is observed for DENV-2-4 with respect to their ancestral sequences (Figs 4C and S10). To understand whether the evolution of the dengue virus is shaped by the interserotype cross-reactive immunity in the population, we examined the dynamics of interserotype distance D_ij (year-wise hamming distance between DENV-i and DENV-j) (Fig 4B). We observe that the interserotype distances also displayed fluctuations over a similar time period of 2–4 years (2.54 ± 0.5 years for D₁₂, 4.13 ± 1.18 years for D₁₃ and 3.11 ± 0.58 years for D₂₃), suggesting an interplay between the serotypes at the population level. We also checked whether the serotype sequences became similar to each other or drifted apart based on the correlations between the serotype and interserotype evolutionary dynamics. For instance, when DENV-1 and DENV-2 display divergence (or convergence) with respect to their ancestral sequences (Fig 4A), the distance between the DENV-1 and DENV-2 (D₁₂) also increase (or decrease). While DENV-1 dynamics did not correlate with D₁₃ and D₁₄ dynamics, DENV-2 strongly correlated with all other interserotypic dynamics. In contrast, DENV-3 showed a negative correlation with D₁₃ and D₃₄ dynamics suggesting that as DENV-3 diverges, it moves closer to the circulating DENV-1 and DENV-4 strains (Figs 4C and S10). These signatures are specific to the immunologically dominant E gene. For example, when we compare the CprM gene sequences from South India, the fluctuations were not significant. When CprM Hamming distance fluctuations are observed as in the case of D₁ dynamics, it is linked to the transient introduction of genotype I in 2012 (S11A Fig). Consistent with this, we found no significant correlations between the within or inter-serotypic viral dynamics, except for DENV-4 (S11B and S11C Fig). Due to the availability of only 21 sequences for DENV-4 (and n = 3 between 2008–2015), we do not consider these correlations as significant.

We interpret these coupled fluctuations between dengue serotypes in light of population-level cross-reactive immunity and antibody-dependent enhancement. When homotypic immunity is present in the population, the serotypes drift apart, manifesting in positive correlations between serotype divergence and interserotype dynamics, as observed for DENV-1 and DENV-2 (Fig 4C). However, when population immunity is poor against a particular serotype (as with a new introduction or waning levels), similarity to that serotype confers an advantage due to the presence of cross-reactive antibodies and associated ADE [14,15]. This is consistent with the negative correlation observed between DENV-3 and interserotype dynamics D₁₃ and D₃₄ with dropping DENV-3 prevalence and its replacement by DENV-1 and DENV-4 in South India (Figs 4C and 1B). Indeed, recent reports have argued the interserotypic convergence of dengue antigenicity to be correlated to the outbreaks [11].

Divergence of Indian dengue virus from vaccine strains

All the vaccine strains used by tetravalent vaccines are based on the strains isolated between 1964 and 1988. As the prevalent Indian dengue viruses are evolving under high population seropositivity, they might be diverging antigenically from the vaccine strains as well. Additionally, genotypes used in the vaccine strains are not observed in India (S3 Table). Therefore, we examined the E protein differences between the vaccine and the circulating dengue virus in India. Dimensionality reduction analysis of amino acid differences between the virus strains shows that dengue E gene sequences from India cluster together but are removed from the vaccine strains for all serotypes (Fig 5A). Only the DENV-1 and DENV-2 strains of CYD-TDV are close to the DENV-1-I and DENV-2-cosmopolitan clusters present in India. More than half of the significant amino acid differences (50% DENV-1, 68% DENV-2, 92% DENV-3 and 50% DENV-4) lie on the exposed surface of the envelope protein on the virus, which is accessible to the majority of the antibodies (Figs 5B and S12 and S7 Table). Overall, ~ 6% (2.7–13.5%) of all known epitopic regions are different in Indian dengue sequences compared to the vaccine strains (S8 Table). Further, almost half (34.6–66.7%) of all the E protein variations lie either in known epitopic regions selected from the IEDB database or have a positive antigenic effect [52] (S7 Table). This is in line with our assertion that the dengue virus in India has evolved under immunological selection pressures. Further investigation is needed to assess the impact of these mutations on vaccine efficacy in India.

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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.

https://doi.org/10.1371/journal.ppat.1010862.g005

Indian variants of DENV-1 and DENV-4 are distinct from all the vaccines compared to DENV-2 and DENV-3 (Fig 5C). Among the E protein domains, EDIII, a known antigenic domain and target of recent Indian dengue vaccine efforts [39,73], carry the largest density of variations, further confirming the immune escape-driven evolution of the protein. Apart from EDIII, the transmembrane region of the vaccines (especially for TAK-003) is distinctly different from the Indian variants (S7 Table). This region has been reported to be highly antigenic in DENV-2 [74,75]. Consistent with the role of the stem region in the viral membrane fusion process, it is highly conserved. Therefore, antigenic differences of Indian dengue viruses with respect to the vaccines define the majority of E protein differences. This can have important implications for vaccine efficacy, where vaccines will likely evoke poor neutralization against dengue virus in India. For example, monoclonal antibodies targeting the EDIII region of dengue display a 100-fold variation in neutralization titer across different genotypes [76,77]. Similarly, challenge studies (in humans, ClinicalTrials.gov Identifier: NCT03416036 and Cynomolgus Macaques) show complete protection against vaccine genotypes but confer only partial protection against other genotypes [78,79]. For the DENV-4-II based CYD-TDV vaccine, efficacy in young children against DENV-4-I is only 23.9%, which has been attributed to eight specific residues in the E/prM gene [38]. Four of these (T46I, L120S, F461L, and T478S) dramatically reduce vaccine efficacy (from as high as ~75% to less than 20%) [38]. DENV-4 in TV003 and TAK-003 vaccines also share three of these residue variations, implying potentially lower vaccine efficacy against the DENV-4-I genotype prevalent in India.