Phylogenetic Tree for DENV-4 Samples from Manaus, Brazil.

<p>The tree was inferred with the maximum likelihood criterion implemented in the program GARLI v0.95 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000390#pntd.0000390-Zwickl1" target="_blank">[8]</a>. Node support was evaluated with 100 independent runs with GARLI (percent values above branches), 500 non-parametric bootstrap replicates with PhyML <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000390#pntd.0000390-Guindon1" target="_blank">[9]</a> (percent values in between brackets below branches), and the posterior probability from the maximum credibility tree among 2,000 trees obtained after running 20 million generations in 15 chains with the parallel implementation of MrBayes v3.0B4 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000390#pntd.0000390-Ronquist1" target="_blank">[10]</a> (in bold to the right of the critical nodes). Genotypes II (yellow) and III (green) lineages were collapsed for clarity. The high support values are indicative of the membership in genotype I, but have to be taken cautiously since only 390 bp of the prM-core junction were reported (for more details, see the open-access paper by Figueiredo et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000390#pntd.0000390-Figueiredo1" target="_blank">[4]</a>).</p

Comparison of clusters with lower and higher time trend increase.

<p>Comparison of clusters with lower and higher time trend increase.</p

Dengue incidence in the clusters in Vitória, September 2012 to June 2013.

<p>Dengue incidence in the clusters in Vitória, September 2012 to June 2013.</p

Dengue dispersion during an epidemic in Vitória, September 2012 to March 2013.

<p>N.B.: The color gradients vary from the minimum to the maximum number of dengue cases reported in the respective month per an area of 1 km of diameter.</p

Cases of dengue reported in Vitória per month, September 2012 to June 2013.

<p>Cases of dengue reported in Vitória per month, September 2012 to June 2013.</p

Clusters with lower or higher time trend increase in Vitória, September 2012 to June 2013.

<p>TTI—Time trend increase: incidence increase over the time.</p

Spatial variation in temporal trends for clusters with lower (cluster 1 to 5) and higher (cluster 6 to 11) time trend increase in Vitória, from September 2012 to June 2013.

<p>Spatial variation in temporal trends for clusters with lower (cluster 1 to 5) and higher (cluster 6 to 11) time trend increase in Vitória, from September 2012 to June 2013.</p

Inter- and Intra-Host Viral Diversity in a Large Seasonal DENV2 Outbreak

<div><p>Background</p><p>High genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections.</p><p>Methods and Principal Findings</p><p>We deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level.</p><p>Conclusions and Significance</p><p>Despite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability.</p></div

Blood-feeding efficiency of the Glytube feeder device compared with mice blood-fed females.

<p>The experiments were done in three biological replicates, two with three and another one with six independent experiments. To generate blood-feeding efficiency, the number of engorged females after 30 minutes of feeding was divided by total number of females allowed to feed. The numbers of females fed on Glytube or mice were not significantly different by Mann-Whitney test (p = 0.1189).</p

Materials used to assemble the Glytube blood feeder device.

<p><b>A.</b> A conical tube (50 mL) filled with 40 mL warmed 100% glycerol and top sealed with Dura Seal^™ heat-resistant sealing film. The sealing film is laterally held to the tube using a Parafilm-M^® thin strip (2.5 cm×5.0 cm). <b>B.</b> Screw cap of the conical tube. Dashed circular black line indicates the cap region where plastic is removed by cutting to generate the feeding element. <b>C.</b> Screw cap with 2.5 cm diameter hole. <b>D.</b> Screw cap covered externally with stretched Parafilm-M. A strip of Parafilm is fixing the feeding membrane to the cap. <b>E.</b> A piece of Parafilm-M (5 cm×5 cm) as a feeding membrane. Parafilm must be stretched to cover the screw cap. <b>F.</b> A piece of Dura Seal heat-resistant sealing film is used to sealing the conical tube filled with pre-heated 100% glycerol. <b>G.</b> Blood supplying the feeding element at internal side of the screw cap with the stretched Parafilm membrane. <b>H.</b> Heating and feeding elements assembled together to feed the <i>Ae. aegypti</i> females. <b>I.</b> Non blood-fed (black arrowhead) and artificially blood-fed females with dilated abdomens (black arrows).</p