DENV-3 was introduced into the Americas via Panama and Nicaragua in 1994 , spreading rapidly to neighboring countries and reaching Brazil through Rio de Janeiro in December 2000 . Interestingly, DENV-1 and DENV-2 were also introduced into Brazil through Rio de Janeiro . Thus, it seems that the main route of entrance for DENV in Brazil was always Rio de Janeiro. Analyzing the E gene and the 3′UTR sequences, we have previously found that isolates BR/BV4/02 isolated in Boa Vista, Roraima, and BR8/04 isolated Belem, Para, in the Northern Region of the country, were phylogenetically more closely related to viruses isolated in the Caribbean islands rather than to those isolated in Rio de Janeiro, suggesting that DENV-3 was also introduced into Brazil by the northern region . In the present study, now analyzing the entire ORF, the isolates BR/BV4/02 and BR/BR8/04, as well as new viruses isolated between 2006 and 2007 by other groups, were phylogenetically more closely related to viruses isolated in the Caribbean islands than to viruses circulating in Brazil, supporting the hypothesis that DENV-3 was introduced by the Norther Region of the country in addition to the well documented introduction by Rio de Janeiro. According to our phylogenetic analysis, at least two groups of DENV-3 genotype III are circulating in Brazil (Figure 2, sub-lineages A and B). A single virus (BR_V2387_03, FJ850079) isolated in the Northern Region of Brazil was more closely related to viruses isolated in Venezuela, suggesting that was an imported case. In addition to the DENV-3 genotype III, recent studies have shown that genotype I viruses are also circulating in Brazil [20–22]. The Paraguayan isolates were closely related to viruses isolated in Brazil within sub-lineage B of lineage III, suggesting that these viruses were introduced into Paraguay from Brazil as previously described .
A previous phylogenetic analysis of DENV-3 genotype III isolated in Sri Lanka, based on a 966 nt fragment spanning part of capsid, preM/M and part of E genes, identified the emergence, after 1989, of a new sub-type, which was correlated with severe disease epidemics that spread to Africa and then to the Americas . Our phylogenetic analysis showed a similar distribution of DENV-3 genotype III isolates (Figure 2). Thus, our lineage I corresponds to DENV-3 genotype III circulating in SriLanka before 1989, which was called as sub-lineage A by Messer and colleagues (2003). On the other hand, LK_V2411_89 and LK_V2409_97 viruses, located basally in the branch of lineage III, could correspond to the new sub-type described by Messer and colleagues (2003) that they called Group B. The topology of our phylogenetic tree suggests that this last sub-type migrated to East Africa and Indian subcontinent and later to the Americas in agreement with observations made by Messer and colleagues (2003). Interestingly, phylogentic analysis of genotype III showed also that NI/V2420/1994 virus isolated in Nicaragua is located at the base of the branch containing the American isolates (Lineage III), suggesting that the introduction of genotype III in the Americas occurred through Nicaragua in 1994, in agreement with the epidemiological data [4, 23].
Previous studies analyzing the C, PrM, E and NS3 genes have shown that DENV-3 segregated into four genotypes [13, 14, 24, 25]. In this study, we have found that DENV-3 segregated in the same genotypes I, II and III as shown in the previous studies mentioned above, analyzing either the entire ORF or each protein gene sequence. Thus, DENV-3 genotype can be determined by sequencing any part of the ORF. The genotype IV was not observed in our analysis because no complete genome sequence of any of these viruses is available in the GenBank. Klungthong and colleagues (2008) have also suggested that any genomic region can be sequenced to determine the genotype ; however, these authors used the entire genomic sequences of only 12 isolates, while our results were supported by the analysis of more than 500 isolates.
Similar to other RNA viruses, DENV exhibit a high degree of genetic variation due to the non-proofreading activity of its RNA polymerase, the high rates of mutation, the immense population size, and the immunological pressure, leading to the emergence of new subtypes of DENV . Recently, we have described the existence of various taxa or viral sub-types within DENV-3 genotypes by analyzing of the E protein gene . In this study, we have found similar, and even new, internal groups within each genotype. The segregation of the viruses into genotypes, lineage, sub-lineage and groups as suggested in this study were supported by high posterior probability, by nucleotide divergence, and by the presence of characteristic amino acid motifs.
The first studies that identify different genotypes within each DENV serotypes were based on the topology of the trees, supported by bootstrap values [13, 24, 29–32]. The nucleotide divergence among the genotypes of DENV-1 and DENV-2 was in average 6% when the genomic sequence corresponding to the junction E/NS1 (240 bp) was analyzed , and 7% in mean when the E protein gene (1,485 bp) was analyzed for DENV-1 and DENV-2 [31, 32]. In this study, we carried out a more detailed analysis of the nucleotide divergence among the different genetic groups of DENV-3. We have observed that nucleotide divergence varied in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages when the complete ORF of genotypes II and III was analyzed. For genotype I, a higher nucleotide divergence rate was observed among lineages and sub-lineages.
In addition, our analysis showed that the nucleotide divergence among genotypes varied depending on the genomic region, ranging from 4.1% for C protein gene to 8.8% for NS2a protein gene. These comparisons showed also that C protein gene sequence is the most conserved, and NS2a, NS4a, E protein genes the more variables. However, when the amino acid divergence was analyzed, a different picture was observed; NS2b, NS4b and NS3 were the more conserved proteins, while NS2a, NS4a, E proteins were the more variables. Knowledge of the rates of divergence among the different taxonomic levels, are an important tool for the detection of new viral groups, as well as, information about the variability of each of the genes among different viral groups, could be used to select targets for: the design of probes for diagnosis, antiviral therapies and the construction of candidate vaccines.
Recently, Wittke and colleagues have suggested the existence of an additional genotype (genotype V) within DENV-3 . Our previous phylogenetic analysis based on the E protein, however, has suggested that the genotype V corresponds to a lineage within the genotype I . In this study, the nucleotide divergence among lineage I (called genotype V by Wittke and colleagues) and lineage II of genotype I was 4.9% in average, lower than the 6% observed among genotypes. Therefore, we suggest the maintenance of the classification of DENV-3 into four genotypes as previously proposed [14, 24].
In this work, we performed phylogenetic analysis and evolutionary divergence dynamics of DENV-3 and provided data related to the processes that control the viral evolution. These data will be useful to better characterize the DENV-3 epidemics in future and might even be used for selection of vaccine candidates.