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Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level., (Published by Elsevier B.V.)

A phylogenetic approach was used to identify genetic variants of DENV-3 subtype III that may have emerged during or after its expansion throughout South America. We sequenced the capsid, premembrane/membrane and envelope genes from 22 DENV-3 strains isolated from Venezuela, Bolivia, Ecuador and Peru between 2000 and 2005. Phylogenetic analysis showed that the isolates sequenced in this study formed three clades within subtype III: one with the isolates from Venezuela, one with the Bolivian isolates and one with the isolates from Ecuador and Peru.

Virological surveillance of dengue viruses in Aedes aegypti populations constitutes a powerful tool for early prediction of dengue outbreaks. We have standardized a protocol for viral RNA extraction from individual and pools of mosquitoes that permits a sensitive detection of dengue virus without RNA degradation or PCR inhibition when we apply a semi-nested RT-PCR. The limit of detection for each dengue serotype was 0.1 PFU. In a prospective field study conducted from November 2000 to December 2001, adult female A. aegypti mosquitoes from several municipalities with high dengue transmission in Maracay, Aragua State, Venezuela were collected and screened for dengue viruses using RT-PCR. We analyzed a total of 296 A. aegypti pools (1,632 mosquitoes); of these, 154 pools (469 mosquitoes) were collected from houses with persons with clinical diagnosis of dengue (dengue houses), and 142 pools (1,163 mosquitoes) from adjacent residences (neighbour houses). From the dengue houses, eight mosquito pools (5.2%) were positive for DENV-1 (0.7%), DENV-3 (3.2%) and DENV-4 (1.3%) viruses. From the neighbour houses, 18 mosquito pools (12.7%) were positive for DENV-3 (12%) and DENV-4 (0.7%) viruses. From these 26 RT-PCR positive mosquito pools (containing 1-25 mosquitoes each), 22 pools (84.6%) were positive for DENV-3. The most prevalent serotype in the 2001 dengue outbreak was also DENV-3. The minimum infection rate in both A. aegypti collections, from dengue houses and neighbour houses was 17 and 15 per 1,000, respectively. The relevance of these results for dengue surveillance is discussed.