Dengue, the predominant arthropod-borne viral disease affecting humans, is caused by one of four distinct serotypes (DENV-1, -2, -3 or -4). A literature analysis and review was undertaken to describe the molecular epidemiological trends in dengue disease and the knowledge generated in specific molecular topics in Latin America, including the Caribbean islands, from 2000 to 2013 in the context of regional trends in order to identify gaps in molecular epidemiological knowledge and future research needs. Searches of literature published between 1 January 2000 and 30 November 2013 were conducted using specific search strategies for each electronic database that was reviewed. A total of 396 relevant citations were identified, 57 of which fulfilled the inclusion criteria. All four dengue virus serotypes were present and co-circulated in many countries over the review period (with the predominance of individual serotypes varying by country and year). The number of countries in which more than one serotype circulated steadily increased during the period under review. Molecular epidemiology data were found for Argentina, Bolivia, Brazil, the Caribbean region, Colombia, Ecuador, Mexico and Central America, Paraguay, Peru and Venezuela. Distinct lineages with different dynamics were found in each country, with co-existence, extinction and replacement of lineages occurring over the review period. Despite some gaps in the literature limiting the possibility for comparison, our review has described the molecular epidemiological trends of dengue infection. However, several gaps in molecular epidemiological information across Latin America and the Caribbean were identified that provide avenues for future research; in particular, sequence determination of the dengue virus genome is important for more precise phylogenetic classification and correlation with clinical outcome and disease severity.

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Dengue in Latin America: Systematic Review of Molecular Epidemiological Trends

Phenomena such as incomplete lineage sorting, horizontal gene transfer, gene duplication and subsequent sub- and neo-functionalisation can result in distinct local phylogenetic relationships that are discordant with species phylogeny. In order to assess the possible biological roles for these subdivisions, they must first be identified and characterised, preferably on a large scale and in an automated fashion. We developed Saguaro, a combination of a Hidden Markov Model (HMM) and a Self Organising Map (SOM), to characterise local phylogenetic relationships among aligned sequences using cacti, matrices of pair-wise distance measures. While the HMM determines the genomic boundaries from aligned sequences, the SOM hypothesises new cacti in an unsupervised and iterative fashion based on the regions that were modelled least well by existing cacti. After testing the software on simulated data, we demonstrate the utility of Saguaro by testing two different data sets: (i) 181 Dengue virus strains, and (ii) 5 primate genomes. Saguaro identifies regions under lineage-specific constraint for the first set, and genomic segments that we attribute to incomplete lineage sorting in the second dataset. Intriguingly for the primate data, Saguaro also classified an additional ~3% of the genome as most incompatible with the expected species phylogeny. A substantial fraction of these regions was found to overlap genes associated with both the innate and adaptive immune systems. Saguaro detects distinct cacti describing local phylogenetic relationships without requiring any a priori hypotheses. We have successfully demonstrated Saguaro’s utility with two contrasting data sets, one containing many members with short sequences (Dengue viral strains: n = 181, genome size = 10,700 nt), and the other with few members but complex genomes (related primate species: n = 5, genome size = 3 Gb), suggesting that the software is applicable to a wide variety of experimental populations. Saguaro is written in C++, runs on the Linux operating system, and can be downloaded from http://saguarogw.sourceforge.net/
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Unsupervised genome-wide recognition of local relationship patterns

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in Sao Jose do Rio Preto, Sao Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998–2000) and BR3 (2003–05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue.

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Circulation of different lineages of Dengue virus 2, genotype American/Asian in Brazil: dynamics and molecular and phylogenetic characterization.

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of Sao Jose de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

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Dengue Virus Type 3 Adaptive Changes during Epidemics in Sao Jose de Rio Preto, Brazil, 2006–2007

Following successive outbreaks of dengue fever caused predominantly by dengue virus (DENV) 2 and 3, DENV-1 is now the primary serotype circulating in Brazil. We sequenced and analyzed Brazilian DENV-1 genomes and found that all isolates belong to genotype V and are subdivided into three lineages, which were introduced during four different events. The first introduction occurred in 1984-85, the second in 1997-99, and the third and fourth occurred from 2004 to 2007. These events were associated with an increase in genetic diversity but not with positive selection. Moreover, a potential new recombinant strain derived from two distinct lineages was detected. We demonstrate that the dynamics of DENV-1 in Brazil is characterized by introduction, movement, local evolution, and lineage replacement. This study strengthens the relevance of genotype surveillance in order to identify, trace, and control virus populations circulating in Brazil and Latin America.

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Population dynamics of DENV-1 genotype V in Brazil is characterized by co-circulation and strain/lineage replacement.

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A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

P.F. Vasconcelos

Papers

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.