Análise das seqüências de nucleotídios da regiäo 5' näo codificadora e dos genes das proteínas estruturais dos Flavivirus / Nucleotide sequence alignement of the 5' non coding and structural protein regions of the genome of Flaviviruses

Neste trabalho, analisaram-se comparativamente as seqüências de nucleotídios dos genes das proteínas estruturais C, prM e E de todos os Flavivirus, incluindo, também, a regiäo 5' näo codificadora, de 21 Flavivirus. Utilizou-se para a análise o programa de microcomputador DNAsis (Hitachi, Japäo) e construiu-se uma árvore filogenética, incluindo os vinte e um (21) vírus, após alinhamento de suas seqüências de nucleotídios. Na árvore filogenética obtida, observou-se uma ramificaçäo inicial, separando os vírus transmitidos por carrapatos daqueles transmitidos por mosquitos. Também, agruparam-se, em diferentes ramos, os vírus do dengue, os da febre amarela, e os da encefalite japonesa. Observou-se uma evidente relaçäo entre a árvore filogenética e os subgrupos e tipos virais, reconhecidos com base em relacionamento antigênico.

A simple reverse transcription-polymerase chain reaction for dengue type 2 virus identification

We show here a simplified reverse transcription-polymerase reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reation vessel was carried out following a digestion of virus with 1 per cent Nondet P-40. The 50 µl assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37ºC for RT followed by a variable amount of cycles of two-step PCR amplification (92ºC for 60 sec, 53ºC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7 per cent agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 10².8 TCID 50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription: polymerase chain reaction (RT-PCR) method

Apresentamos neste trabalho uma tecnica simplificada de RT-PCR para identificacao de virus de dengue. Cinco estirpes brasileiras de virus de dengue dos tipos 1 e 2, isoladas de pacientes e o virus da vacina de febre amarela como controle negativo, foram utilizadas nos testes. Celulas C6/36 foram infectadas e os fluidos de cultura coletados apos 7 dias. A RT-PCR, era efetuada em um unico tubo contendo 1 ul de fluidos infectados e diluidos 1/10 em agua destilada ou em mistura detergente contendo Nonidet P40. A mistura de reacao, num volume de 50 ul, continha 50 pmol de primers especificos, que amplificam sequencia com 482 pares de bases para virus do dengue tipo 1 e 210 pares de bases para dengue tipo 2, ou, ainda, primers para grupo dengue que amplificam sequencia com 511 pares de bases...