Dengue fever, dengue hemorrhagic fever and dengue shock syndrome are considered to be the most important arthropod-borne viral diseases due to its high prevalence, and significant morbidity and mortality. The global incidences of dengue has now increased enormously and about 50 - 100 million dengue infections are now reported annually from more than 100 tropical and subtropical countries of the world [1]. Infection with any of the dengue viruses can be manifested in a range of symptoms from a relatively mild flu-like syndrome with rash (dengue fever, DF) to a severe and potentially fatal disease characterized by capillary leakage, thrombocytopenia, and sometimes hypovolemic shock (dengue hemorrhagic fever, DHF/dengue shock syndrome, DSS). Immune enhancement mechanisms following a second heterologous dengue virus infection have been associated with a higher risk of developing DHF/DSS [2], however, cases of DHF/DSS in confirmed primary infections have been observed [3]. Different studies have shown that changes in the viral genetic structure may contribute to shifts in the epidemic potential or pathogenicity of dengue virus serotypes [4,5]. These observations point to the importance of viral factors in determining disease severity. In addition, some strains have been associated with increased pathogenic potential [6].

DENV-3 re-appeared in Latin American in 1994 after an absence of 17 years [7]. Before, serotypes DENV-1 and DENV-2 were circulating for more than a decade in Brazil. In 2002, the introduction of DENV-3 in the state of Bahia produced the first cases of DHF. This event transformed Bahia into a hiperendemic area with three serotypes circulating at the same time [8]. DENV-3, after its introduction, was the serotype responsible for the majority of infections in both Brazil and in Bahia; isolates from different parts of Brazil have been analyzed with respect to their genetic variability. Molecular techniques including sequencing of the viral genomic RNA and direct sequencing of reverse transcriptase/polymerase chain reaction (RT/PCR)-amplified genes have provided many information on geographic distribution, origin and evolution among dengue viruses. Many genes have been used as 5′3′UTR regions, E gene and E/NS1 junction [9,10].

Here we report the analysis of a genomic interval spanning the E/NS1 junction of the dengue genome used for molecular surveillance of this flavivirus and to determine which strains of dengue virus for DENV-2 and DENV-3 were circulating in Bahia state during the 2006- 2007 transmission seasons.

The regions, in the dengue virus genome, encoding the envelope protein (E) and the nonstructural protein 1 (NS1) seem to be the most appropriate to characterize genotypes within serotypes [14,15]. In this way, for some flaviviruses, as yellow fever viruses, most molecular markers for pathogenicity have been found in the E gene. However for dengue, several studies of cloned or cell culture-passaged wild and attenuated viruses have been performed, but each report described different sites of nucleotide and amino acid changes possibly associated with virulence [16,17].

DENV-3 introduction was responsible, in 2002, for almost 80,000 cases notified in all Bahia State [8], and being the first time that DHF cases occurred in this region. Between 2006 and 2007, about 25,000 cases were notified. Dengue circulation in Bahia (Brazil) from 2006 to 2007 has consisted mainly of a mixture of DENV serotypes. Between two serotypes (DENV-2 and DENV-3), the former has been consistently detected in outbreaks/ epidemics since its introduction 2002. Before this study, dengue virus genotypes in Bahia state were not available or few samples have been analyzed. In this report, we analyzed 31 local DENV isolates collected in a span of two years to further understand the ecology and dynamics of this virus in the region. As known, the potential for causing severe disease has been described for all four serotypes of DENV, and the main factors considered to explain its pathogenicity are host genetic susceptibility [18], antibody dependent enhancement and differences in virulence among strains [19], in this way determinants of virulence have been located in three genomic regions [5] and have been tested in vitro [20], so the genotypes have been more or less related with potential to cause DHF. Different studies concluded that the spread of genotype III, of DENV-3, from the Indian subcontinent to Africa, and then consequently to Americas, was correlated with an increase in severe cases of dengue disease in this region [21,22].

The routine laboratory diagnosis of dengue virus infection is primarily achieved by the isolation of virus, detection of IgM/IgG antibodies by serodiagnosis and/or molecular detection by the demonstration of viral RNA by RT-PCR [23,24]. RT-PCR is one of the most important confirmatory tests to confirm dengue infection. But, its positivity is highly dependent of the time in the clinical diagnosis and conditions in the sample maintenance (better when is collected within first five days of onset of the symptoms). The lower rate of the virus isolation may be attributed to the absence of live virus in the sample. This must be due to misclassification of the cases by epidemiological staff and others factors as improper maintenance or storage system. All positive cell culture isolations were confirmed and identified by RT-PCR and nested PCR (31 available samples). Still, we have carried out the molecular epidemiology and genotyping study of the detected DENV-2 and DENV-3 viruses, during 2006 and 2007 circulation seasons. We studied the sequence of these dengue viruses, directly from patient serum sample, as recently advocated by several researchers [5,25]. Various genomic regions of dengue viruses have been selected

for molecular phylogenetic analysis [26]. However, we have selected the E/NS1 gene junction as it also harbours epidemiologically important sequence information and sensitive for surveillance application [9].

The results obtained for DENV-3 in this study bring some interesting findings how can samples from two years, 2006 and 2007, display a high identity with isolated from Peru in 2002? And in the same time samples from 2007 showed a high similarity with samples differrent countries in all American Continent. Other important finding was that almost all grouping were with samples from the same area in Bahia state. In some way, the microregion where the virus circulate in any level determine how they become grouped. During 2006 and 2007, the main serotype circulating in Bahia state was DENV-3, in agreement with the displacement of serotypes 1 and 2 of DENV after its introduction in Brazil in 2002 [27]. A study developed in Rondônia and Acre found both genotype I and V [10], while we found just genotype III after two years of surveillance. This confirm the epidemic potential of this genotype by the displacement of serotypes 1 and 2 of DENV when a highly virulent genotype of DENV-3 was introduced in Brazil in 2002 [27]. Since its introduction this genotype have been related with severe disease in Bahia and Brazil [8,28].

In conclusion, the effective worldwide control of dengue virus into Bahia and consequently in all Brazil, requires the definition of the epidemical viral sources, and the precise identification of virus genotypes. A key objective of DF and DHF surveillance programmes is early detection of outbreaks to permit the implementation of control measures by the public health authorities. DHF outbreaks can be anticipated by monitoring the emergence of new genotypes in a region like Bahia State.

This project was funded by FAPESB PPSUS, CNPq and FIOCRUZ.