Evolution of dengue virus type 3 genotype III in Venezuela: diversification, rates and population dynamics

Abstract

Background: Dengue virus (DENV) is a member of the genus Flavivirus of the family Flaviviridae. DENV are comprised of four distinct serotypes (DENV-1 through DENV-4) and each serotype can be divided in different genotypes. Currently, there is a dramatic emergence of DENV-3 genotype III in Latin America. Nevertheless, we still have an incomplete understanding of the evolutionary forces underlying the evolution of this genotype in this region of the world. In order to gain insight into the degree of genetic variability, rates and patterns of evolution of this genotype in Venezuela and the South American region, phylogenetic analysis, based on a large number (n = 119) of envelope gene sequences from DENV-3 genotype III strains isolated in Venezuela from 2001 to 2008, were performed.

Results: Phylogenetic analysis revealed an in situ evolution of DENV-3 genotype III following its introduction in the Latin American region, where three different genetic clusters (A to C) can be observed among the DENV-3 genotype III strains circulating in this region. Bayesian coalescent inference analyses revealed an evolutionary rate of 8.48 x 10⁻⁴ substitutions/site/year (s/s/y) for strains of cluster A, composed entirely of strains isolated in Venezuela. Amino acid substitution at position 329 of domain III of the E protein (A→V) was found in almost all E proteins from Cluster A strains.

Conclusions: A significant evolutionary change between DENV-3 genotype III strains that circulated in the initial years of the introduction in the continent and strains isolated in the Latin American region in recent years was observed. The presence of DENV-3 genotype III strains belonging to different clusters was observed in Venezuela, revealing several introduction events into this country. The evolutionary rate found for Cluster A strains circulating in Venezuela is similar to the others previously established for this genotype in other regions of the world. This suggests a lack of correlation among DENV genotype III substitution rate and ecological pattern of virus spread.

Figure 1

Maximum likelihood phylogenetic tree analysis of DENV-3 genotype III strains isolated in Venezuela. Strains in the trees are shown by the standardized terminology (which identifies their serotype, country, name and year of isolation) for strains previously described. Strains reported in these studies are shown by name. Venezuelan strains are shown highlighted in grey. Numbers at the branches show aLRT values. The scale bar indicates nucleotide substitutions per site. Cluster A branch is highlighted in red, clusters B and C branches are highlighted in green and light blue, respectively.

Figure 2

Alignment of E protein ectodomain amino acid sequences from DENV-3 genotype III strains isolated in Venezuela. Strains are listed by their names on the left side of the figure, and the cluster to which the strain is assigned is indicated between parentheses (see also Fig. 1). Only one strain of each cluster is shown (for detailed results of substitutions found in the rest of the Venezuelan strains see Additional File 2, Table S2). Identity to strain FJ639750 (DENV-3/VE/BID-V2179/2000) is indicated by a dash. Amino acid positions (relative to strain FJ639750) are shown by numbers at the top of the alignment. Sequences corresponding to E protein domains I, II and III are shown in red, yellow and light blue, respectively. Surface-exposed sequences previously identified on the dimeric DENV-3 protein are shown by a black diamond. Potential ELK/KLE-type and KELK/KLEK-type motifs are shown by a star. Asparagine at position 388 is shown by a white circle. Amino acid residues recently identified to be critical for neutralization by complex-reactive monoclonal antibodies (Mabs) on domain III of DENV-3 are indicated by a black circle [43].

Figure 3

Structure of the E protein dimer of DENV-3. E protein domains I through III are indicated in red, yellow and light blue, respectively. Residues recently identified to be critical for neutralization [43] are shown in space-filling representation. Substitution at position 329 (A→V), found in cluster A strains, is shown in blue in space-filling representation. Substitution at position 346 (N→S), found in a Venezuelan Cluster B strain is shown in magenta in space-filling representation. Amino acid substitution at position 132 (Y→H), present in Cluster B with respect to Clusters A and C strains is shown in green in space-filling representation. Two views of the protein dimer, rotated over the z-axis, are shown in A and B.