Dengue Virus Type 3 Adaptive Changes during Epidemics in Sao Jose de Rio Preto, Brazil, 2006–2007

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of Sao Jose de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak

Maximum clade credibility (MMC) tree showing the phylogenetic relationships among SRJP clades (light gray boxes) and other Brazilian isolates.

<p>Posterior probability values are shown for main nodes only. Light grey text represents localities (sources) for other Brazilian isolates.</p

Non-neutral codons according to the assayed methods (SLAC and REL) at the specified significance levels (p = 0.05 and Bayes factor = 50, respectively).

<p>Non-neutral codons according to the assayed methods (SLAC and REL) at the specified significance levels (p = 0.05 and Bayes factor =  50, respectively).</p

Positively-selected codon transformations tracked over a clade credibility (MMC) tree.

<p>Light gray boxes represent SJRP clades. Numbers indicates codon position and one-letter symbols indicate amino-acids. Transformations for each character can be evaluated as follows: • Unique transformation all over the tree; ▪ character state fixed along that branch; ◂ same transformation occurs in another branch (homoplasy); ▸ transformation(s) in the same character occurs along that branch; ⧫ same transformation occurs in another branch (homoplasy) and transformation(s) in the same character occur along that branch.</p

GenBank and SJRP sequences used for phylogenetic inference.

<p>GenBank and SJRP sequences used for phylogenetic inference.</p

Genetic analysis per genomic region.

*<p>Total number of mutations.</p>**<p>Nucleotide diversity.</p>***<p>Average number of nucleotide differences.</p

Rank of immunogenicity of DENV-3 sequences.

<p>Thirty-three DENV-3 sequences were submitted to B and T epitope prediction using bioinformatics servers and further classified according to their immunogenic potential. Sequences previously clustered in B and C clades have epitopes, in envelope, NS1, NS2a and NS5, putatively less immunogenic when compared to the same epitopes of sequences clustered in clade A, except for sequence ACY70777.1. Substitutions in the capsid were irrelevant.</p

Per-site and per-gene d<i>N</i>/d<i>S</i> plot.

<p>The calculated SLAC d<i>N</i>/d<i>S</i> and REC posterior probability for positive selection (d<i>N</i>>d<i>S</i>) are plotted for each site in the upper and lower part of the polyprotein respectively.</p