NS1 antigen capture tests applied to tissues (<i>n = </i>74) from confirmed dengue fatal cases (<i>n = </i>23).

<p>*Total positive /total tested (%), ND: not done, NA: not available.</p

Dengue fatal case confirmation by any of the NS1 capture tests per tissues per case.

<p>*Positive/total analyzed (%), +: positive sample, −: negative sample, NA: not available.</p

NS1 antigen capture tests analysis in tissues from dengue fatal cases (<i>n = </i>23).

<p>NS1 antigen capture tests analysis in tissues from dengue fatal cases (<i>n = </i>23).</p

Twenty Years of DENV-2 Activity in Brazil: Molecular Characterization and Phylogeny of Strains Isolated from 1990 to 2010

<div><p>In Brazil, dengue has been a major public health problem since its introduction in the 1980s. Phylogenetic studies constitute a valuable tool to monitor the introduction and spread of viruses as well as to predict the potential epidemiological consequences of such events. Aiming to perform the molecular characterization and phylogenetic analysis of DENV-2 during twenty years of viral activity in the country, viral strains isolated from patients presenting different disease manifestations (<i>n</i> = 34), representing six states of the country, from 1990 to 2010, were sequenced. Partial genome sequencing (genes C/prM/M/E) was performed in 25 DENV-2 strains and full-length genome sequencing (coding region) was performed in 9 strains. The percentage of similarity among the DENV-2 strains in this study and reference strains available in Genbank identified two groups epidemiologically distinct: one represented by strains isolated from 1990 to 2003 and one from strains isolated from 2007 to 2010. No consistent differences were observed on the E gene from strains isolated from cases with different clinical manifestations analyzed, suggesting that if the disease severity has a genetic origin, it is not only due to the differences observed on the E gene. The results obtained by the DENV-2 full-length genome sequencing did not point out consistent differences related to a more severe disease either. The analysis based on the partial and/or complete genome sequencing has characterized the Brazilian DENV-2 strains as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. It was established that strains circulating prior DENV-2 emergence (1990–2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II. Furthermore, all DENV-2 strains analyzed presented an asparagine (N) in E₃₉₀, previously identified as a probable genetic marker of virulence observed in DHF strains from Asian origin. The percentage of identity of the latter with the Dominican Republic strain isolated in 2001 combined to the percentage of divergence with the strains first introduced in the country in the 1990s suggests that those viruses did not evolve locally but were due to a new viral Lineage introduction in the country from the Caribbean.</p> </div

Primers used for amplification of the partial and complete genes (coding region) from Brazilian DENV-2.

<p>Primers used for amplification of the partial and complete genes (coding region) from Brazilian DENV-2.</p

Maximum likelihood phylogeny based on the C/prM/M/E genes of 25 Brazilian DENV-2, 1991–2010.

<p>Black circles represent DENV-2 sequences generated in this study. Strains representative from the four genotypes available in Genbank (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) were used for the comparison, DENV-1, DENV-3 and DENV-4 strains were used as outgroup to root the trees. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. DENV strains used were named as follows: Country/strain number/state/year. RJ: Rio de Janeiro, ES: Espirito Santo, CE: Ceará, BA: Bahia, RS: Rio Grande do Sul, RN: Rio Grande do Norte.</p

Strains representative of the different DENV-2 genotypes and strains used as outgroup for comparison purposes.

<p>Strains representative of the different DENV-2 genotypes and strains used as outgroup for comparison purposes.</p

Maximum likelihood phylogeny based on the complete coding region sequencing of 9 Brazilian DENV-2, 1990–2008.

<p>Black circles represent DENV-2 sequences generated in this study. Strains representative from the four genotypes available in Genbank (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) were used for the comparison, DENV-1, DENV-3 and DENV-4 strains were used as outgroup to root the trees. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. DENV strains used were named as follows: Country/strain number/state/year. RJ: Rio de Janeiro and CE: Ceará.</p

DENV-2 used in this study for partial (<i>n</i> = 25) and complete coding region (<i>n</i> = 9) sequencing.

<p>BA: Bahia, CE: Ceará, RJ: Rio de Janeiro, RS: Rio Grande do Sul, RN: Rio Grande do Norte, ES: Espírito Santo; DF: Dengue Fever; DHF: Dengue Hemorrhagic Fever; DSS: Dengue Shock Syndrome; Fem: Female; Male; C/prM/M/E: Capsid/pré-membrane/Membrane/Envelope; Complete CR: Complete coding region;</p>*<p>Imported case; NA: Not available; ND: Not done; P: primary infection; S: secondary infection.</p