Mapeamento de arbovíroses no estado de Rondônia.

Abstract

The arboviruses represent serious problems of public health in Brazil, in particular in the Amazon area. Four arboviruses have shown to be particularly important, namely dengue, yellow, Oropouche and Mayaro, since they cause severe human disease with high mortalities and are involved in periodic outbreaks. The mapping of the main arboviruses in the Rondonia State is necessary, as well as the knowledge of their biological cycles, in order to define control measures with the understanding of their period and mechanisms of transmission. It is also necessary to improve diagnostic procedures by viral isolation and molecular characterization by RT-PCR, with the primers selected for identification of these viruses, directly from the serum or tissue fluids of patients, or indirectly from cell cultures inoculated with patient s materials. In the present manuscript are presented results of arbovirus isolation and characterization procedures from two hundred and sixty six samples of blood colleted from patients with suspected of arboviruses infections in the following Rondonia State s cities: Ariquemes, Cacoal, Colorado D´Oeste, Jarú, Ouro Preto D´Oeste, Porto Velho, Theobroma and Vilhena. The samples were inoculated in C6/36 monolayers and after seven days the cellular supernatants were collected for RNA extraction by Trizol® method. The RNA extracted was used for RT-PCR with primers selected from the specialized literature s data. The universal primer was selected for identify Flavivirus, Alphavirus genera and Simbu serogroup. Specific primers were also used to identify the four serotypes of dengue virus and yellow fever virus. The amplicons were submitted to electrophoresis in agarose gel 1,7% and visualized in ultra violet light. For genetic sequencing was used the interruption of extention of chain of nucleotide by dideoxynucleotide technique. The ABI PRISM 310 Genetic Analyser (PE Biosyntesis) was used for sequencing the dengue virus serotype 1, the Personal Seq 4X4 (Amersham Pharmacia Biotech, USA), for sequencing the dengue virus serotype 3 and the Mega BACE1000 (Amersham Pharmacia Biotech, USA) for sequencing the Cacipacore virus. One hundred fourteen samples were indentifyed by RT-PCR. The results of identification of positive collected samples from different periods of 2003 to 2005 was the following: 70 patients with dengue serotype 1, one patient with dengue serotype 2, 42 patients with dengue serotype 3 and one patient com Cacipacore virus. The genetic sequencing performed of dengue virus serotypes 1 and 3 samples analyzed by software Clustal W showed 98% of similarity with others dengue virus isolated and sequenced in southeastern areas of Brazil. The Cacipacore virus sample isolated fom the Theobroma patient was the first sample isolated from human infections and presented 91% of similarity with others virus samples isolated in Brazil from migratory birds. We concluded from, our studies, that the epidemiologic surveillance, using molecular methods associated to serological ones is more accurate and precise than serological methods alone. It must be added the easiness of the procedure and the speed for getting the result in contrast with complicated methodologies like hemaglutination inhibition or complement fixation methodologies that need double samples and eventually give cross-reacting results with others arboviruses. The selected primers, for Multiplex-RT-PCR and for nested-RT-PCR used in this work, identify the main arboviruses circulating not only in the Rondonia State that can be used in other centers of research in the region north region of Brazil. Our results also emphasize the agility of the method consisting in the direct use of patient s serum for the RT-PCR procedure which reduces the time for getting a diagnosis.