Phylogenetic analysis of DENV-2 based on the complete genome sequence.

<p>The evolutionary history was inferred using the Maximum Likelihood method, using the General Time Reversible model for nucleotide substitution with discrete Gamma distribution to model evolutionary rate differences among sites [4 categories (+<i>G</i>, parameter = 0.45)]. The tree with the highest log likelihood (−46330.60) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 91 nucleotide sequences with a total of 10,167 positions in the final dataset. A total of 1000 bootstrap replicates were run and values ≥99 are represented as percentage in respective nodes. The Brazilian DENV-2 lineages are shown in grey. For clarity purposes some branches were collapsed. VE/CO/GU/BZ/MX 1999–2009 contains isolates from Venezuela (VE61095/2007, DENV-2/VE/BID-V2944/2005, -V2424/2004, -V2492/200, -V2216/2003, -V2262/2006), Colombia (DENV-2/CO/BID-V3370/2004, -V3369/1999, -V1603/2004), Guatemala (FDA-GUA09/2009), Belize (BZ/BID-V2952/2002) and Mexico (DENV-2MX/BID-V3661, -V3714 and –V3768); PR 1986–1995 contains isolates from Puerto Rico (DENV-2/US/BID-V1356/1993, -V855/1992, -V1182/1989, -V1175/1988, -V1183/1990, -V1171/1987, -V1164/1986, DENV-2/PR/17DN/1995 and DENV-2/PR/6780DN/1994 ) and PR 1994–2007 also contains isolates from Puerto Rico (DENV-2/US/BID-V37DN/1994, -V1424/1996, -V1427/1999, -V1398/1997, -V1038/1998, -V1367/1995, -V1463/2000, V1472/2001, -V593/2005 and –V1412/2007). Evolutionary analyses were conducted in MEGA5.0.</p

Amino acid differences in the envelope protein of Brazilian DENV-2 lineages.

1<p>- except HQ012515.BR59382/RN/1997 and HQ012518.BR66985/RJ/2000 (K).</p>2<p>- except DENV-2/BR/BID-V2386/2003, DENV-2/BR/BID-V2396/2006, JF804028.BR/DB015/2006 (V).</p

Bayesian coalescent and discrete phylogeography analyses of Brazilian DENV-2 based on envelope nucleotide sequence.

<p>Subtree of the maximum clade credibility tree was inferred using 144 DENV-2 envelope sequences (1,485 nt). The subtree containing isolates of the American/Asian genotype is displayed here. Time of the most recent common ancestor (MRCA) was estimated using the year of isolation as the calibration point, under the relaxed molecular clock, with the Tamura Nei Model, with discrete Gamma distribution and an estimated nucleotide substitution rate of 7.5⁻⁴. The posterior probabilities values ≥0.96 are represented by (*) and values ≥0.99 are represented by (**), inside the nodes. The years that the MRCA was estimated to exist are shown for some nodes with upper and lower intervals in parenthesis. The origin value of the reverse scale axis corresponds to year 2010. Using different colors, (legend shown on the left side), terminal branches were annotated based on geographic location of DENV-2 isolates. Internal nodes of the tree which presented modal state posterior probability ≥0.60 were also colored according to their most probable location states, inferred by discrete phylogeographical analysis. Brazilian lineages are delimited by square brackets. For clarity purposes some branches were collapsed. SJRP/2008 contains 11 isolates from São José do Rio Preto/São Paulo/Brazil (DENV-2/BR/BID-V3653/2008, -V3638/2008, -V3640/2008, -V3483/2008, -V3637/2008, -V3495/2008, -V3645/2008, -V3481/2008, -V3486/2008, -V3650/2008 and -V3648/2008); CU/US/KN 1997–2001 contains isolates from Cuba (Cuba115/97 and Cuba165/1997), Puerto Rico (US/BIC-V1387/1998) and Saint Kitts and Nevis (KN/BID-V2951/2001); VE/CO/GU/BZ/MX 1999–2009; PR 1986–1995 and PR 1994–2007 contain the same isolates as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059422#pone-0059422-g001" target="_blank">Figure 1</a> caption. Analyses were performed using programs from BEAST package v.1.6.1, BEAUTi, Tracer v.1.5.0, TreeAnotator v.1.6.1 and FigTree v.1.3.1 (B) Map of Brazil showing the macro-regions and states. The black pin indicates the approximate location of São José do Rio Preto/São Paulo/Brazil.</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

GenBank and SJRP sequences used for phylogenetic inference.

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

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

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