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

<div><p><i>Dengue virus</i> (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.</p></div

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

<p>(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).</p

Description of DENV-2 isolates from Piauí, Brazil.

a<p>F is female and M is male.</p>b<p>Number of passages in cell culture (C6/36 cell line) are shown.</p

Age and sex distribution of IgM-ELISA dengue positive patients from the 2006/2007 outbreak in Piauí, Brazil.

<p>NI – not informed.</p

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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104516#pone.0104516.s003" target="_blank">Table S1</a>) 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.</p

Geographic location of Piauí and its meso-regions.

<p>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.</p

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

<p>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.</p

The small non-coding RNA response to virus infection in the <i>Leishmania</i> vector <i>Lutzomyia longipalpis</i>

<div><p>Sandflies are well known vectors for <i>Leishmania</i> but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to <i>Vesicular stomatitis virus</i> (VSV) infection in the sandfly <i>Lutzoymia longipalpis</i>. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in <i>Lutzomyia</i> Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between <i>L</i>. <i>longipalpis</i> and viruses and should also open the way for studies with other sandfly-borne pathogens.</p></div

RNAi pathway genes are not modulated by VSV infection in <i>L</i>. <i>longipalpis</i>.

<p><b>(A)</b> Expression of <i>L</i>. <i>longipalpis</i> genes encoding <i>Dicer-2</i>, <i>AGO2</i> and <i>r2d2</i> in control and VSV-infected LL5 cells at different time points. <b>(B)</b> Expression of <i>L</i>. <i>longipalpis</i> genes encoding <i>Dicer-2, AGO2 and r2d2</i> in adult sandflies fed with a blood meal containing VSV. Control sandflies (Mock) were fed with blood without virus. Black circles indicate individuals with detectable viral RNA levels. Numbers of infected individuals and the total are indicated at each time point. No significant differences were observed.</p

VSV replication in adult <i>L</i>. <i>longipalpis</i>.

<p><b>(A)</b> Adult <i>L</i>. <i>longipalpis</i> sandflies were fed with blood containing different concentrations of VSV and the prevalence of infection was analyzed by RT-qPCR at 2 and 4 dpf. The number of individuals tested is indicated at each time point. <b>(B)</b> Adult sandflies were given a blood meal containing 10⁸ PFU/mL of VSV and viral RNA levels in individual sandflies were determined at 1, 2, 4 and 6 dpf. The number of infected individuals is indicated at each time point. Control sandflies (Mock) were fed with blood without virus. Black circles indicate individuals with detectable viral RNA levels. Individuals used to prepare small RNA libraries shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006569#pntd.0006569.g003" target="_blank">Fig 3</a> are indicated in green. Scatter dot plot shows median with interquatile ranges. Statistical significance was determined using Dunn’s Multiple Comparison test for infected groups at different times after feeding. Significant <i>p</i> values are indicated in the figure. <b>(C)</b> <i>L</i>. <i>longipalpis</i> were given a blood meal with containing 10⁸ PFU/mL of VSV and infectious particles in each individual were assayed by PFU. The number of infected individuals and the total are indicated at each time point. Black circles indicate individuals with detectable infectious particles. Experiments are representative of at least 3 biological replicates.</p