Dengue virus 2 American-Asian genotype identified during the 2006/2007 outbreak in Piauí, Brazil reveals a Caribbean route of introduction and dissemination of dengue virus in Brazil

Abstract

Dengue virus (DENV) is the most widespread arthropod-borne virus, and the number and severity of outbreaks has increased worldwide in recent decades. Dengue is caused by DENV-1, DENV- 2, DENV-3 and DENV-4 which are genetically distant. The species has been subdivided into genotypes based on phylogenetic studies. DENV-2, which was isolated from dengue fever patients during an outbreak in Piaui, Brazil in 2006/2007 was analyzed by sequencing the envelope (E) gene. The results indicated a high similarity among the isolated viruses, as well as to other DENV-2 from Brazil, Central America and South America. A phylogenetic and phylogeographic analysis based on DENV-2E gene sequences revealed that these viruses are grouped together with viruses of the American-Asian genotype in two distinct lineages. Our results demonstrate the co-circulation of two American-Asian genotype lineages in northeast Brazil. Moreover, we reveal that DENV-2 lineage 2 was detected in Piauí before it disseminated to other Brazilian states and South American countries, indicating the existence of a new dissemination route that has not been previously described.

Figure 1. Geographic location of Piauí and its meso-regions.

The state of Piauí is located in northeastern Brazil and is divided into the Mid-North, North Central, Southeast and Southwest regions. The state capital, Teresina, is indicated with an arrow on the map.

Figure 2. Evolutionary relationship between DENV-2 isolates from Piauí and the five genotypes of DENV-2.

A maximum clade credibility (MCC) tree was selected after Bayesian inference analysis (strict molecular clock; TN93+G; 400,000,000 iterations) of 72 DENV-2 envelope sequences. The five DENV-2 genotypes (American-Asian, American, Asian I, Asian II and Cosmopolitan) are highlighted in different colors. Brazilian isolates (bold blue letters) clustered within the American-Asian genotype and could be divided into two groups: lineage 1(dark green block) and lineage 2 (light green block). Each node is represented by colours black and red (), which presented posterior probability value>0.9 and >0.75, respectively. Blue bars represent the extent of the 95% highest probability density (95% HPD) for each divergence time. The most probable geographic state for each internal node was inferred by discrete phylogeographic analysis. Different colors in the branch represent distinct geographical states according to the legend on the left side of the figure. Branch width is proportional to the probability value of the inferred ancestral geographical state.

Figure 3. Amino acid polymorphisms in the envelope protein of the American/Asian DENV-2 genotype.

(Top) A diagram of the DENV-2 envelope protein showing its three protein domains (I, II and III). (Bottom) A partial alignment of the envelope protein showing sites of amino acid polymorphism within the American-Asian genotype. Other DENV-2 genotypes (Asian I, Asian II, Cosmopolitan and American) are shown for comparison. Amino acid sites in blue boxes are those that most likely had underwent a non-synonymous mutation after the divergence of the two lineages because they differ from the two ancient American-Asian DENV-2 samples (Jamaica/M20558/1983 and Puerto Rico/AY484607/1988). Amino acids are colored according to their side chain charge (hydrophobic: yellow; polar: green; negatively charged: red; and positively charged: blue).

Figure 4. Bayesian coalescent and discrete phylogeographic analyses of Brazilian DENV-2 lineage 1 based on envelope nucleotide sequences.

A maximum clade credibility tree was inferred by Bayesian inference analysis (strict molecular clock; TN93+G; 200,000,000 iterations) using 63 DENV-2 envelope sequences (summarized in Table S1) retrieved by a BLAST search against the entire GenBank database and using PI-111/2006 (indicated by a red arrow) as a query.PI-111/2006 is an isolate from the state of Piauí (Brazil) that clustered in Brazilian DENV-2 lineage 1. Nodes that presented posterior probability value of >0.9 and >0.75 are represented by black and red circle (), respectively. Blue bars in each node represent the extent of the 95% highest probability density (95% HPD) for each divergence time. The most probable geographic state for each internal node was inferred by discrete phylogeographic analysis. Different colors in the branch represent distinct geographical states according to the legend on the left side of the figure. The branch width is proportional to the probability value of the inferred ancestral geographical state.

Figure 5. Bayesian coalescent and discrete phylogeographic analyses of Brazilian DENV-2 lineage 2 based on envelope nucleotide sequences.

A maximum clade credibility tree was inferred by Bayesian inference analysis (strict molecular clock; TN93+G; 200,000,000 iterations). The 57 DENV-2 envelope sequences (summarized in Table S1) in the tree were retrieved using the BLASTn algorithm; envelope sequences from Piauí isolates (Brazil, indicated by red arrow) clustered in the Brazilian DENV-2 lineage 2 were used as a query. Brazilian isolates (in bold blue letters) weresubdivided into two subgroups that referred as lineage 2a and 2b, respectively. Nodes that presented posterior probability value of >0.9 and >0.75 are represented by black and red circle (), respectively. Different colors in the branch represent the different geographical states according to the legend on the left side of the figure. The branch width is proportional to the probability value of the inferred ancestral geographical state.