RESUMO
Viruses of the genus Flavivirus, which are arboviruses, of the Flaviviridae family, are amongst the most important agents of infectious disease in Brazil, causing human infections with a high morbility and mortality. In this work, the phylogeny of 14 virus amplicon sequences that were obtained by RT-PCR with universal primers for mosquito-borne Flavivirus were studied. The amplicons included a region of the Flavivirus genome of 129 nucleotides at the 3' terminus of the NS5 gene and the 145 initial nucleotides of the 3' non-coding region (NS5-3'NCR). Based on phylogenetic trees, most Brazilian Flaviviruses were grouped into two main branches, including a yellow-fever branch and a second main branch divided into a dengue branch that in its turn is subdivided into serotype 1, 2 and 4 branches, and another (Japanese Encephalitis Virus Complex) branch including SLE and Ilhéus. Rocio and Cacipacoré viruses were included in the Japanese Encephalitis Virus Complex branch in one of the two phylogenetic trees. Iguape virus appears in phylogenetic trees as a separate distant branch.
Assuntos
Culicidae/virologia , Flavivirus/classificação , Proteínas não Estruturais Virais/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Brasil , Células Cultivadas , Clonagem Molecular , Flavivirus/genética , Genoma Viral , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
We report a simplified reverse transcription-polymerase chain reaction (RT-PCR) method for identification of Brazilian flaviviruses based on the patterns of electrophoretic separation of the amplicons. The RT-PCR was done on the culture fluids of Aedes albopictus C6/36 cells infected with Brazilian flaviviruses, without previous extraction of viral RNA, using Flavivirus universal primers that anneal to highly conserved sequences within the nonstructural protein 5 and 3'- non translated region of the virus genome. Genomes of 13 Brazilian Flavivirus isolates were amplified. It was not possible to amplify the genome of Bussuquara virus. Analysis of the RT-PCR products gave reproducible results and three distinct amplicon patterns were observed. Cacipacoré (800-850 basepairs [bp]) and yellow fever viruses (600 bp) yielded a single amplicon; dengue virus types 1 and 2 (650 and 550 bp), dengue virus type 4 (550 and 450 bp), Iguape (650-600 bp and 750-700 bp), St. Louis encephalitis (700 and 650-600 bp), and Rocio viruses (600 and 500-550 bp) yielded two amplicons; and Ilheus virus yielded five amplicons, two larger than 1,000 bp, one 650-700 bp, one 550-600 bp, and one 450-500 bp. The analysis of amplicon DNA sequences of six viruses showed homology with the 3'- nontranslated region of Flavivirus genome. The use of the Flavivirus universal primers in this simple RT-PCR technique is suitable as a screening test for the genus Flavivirus, with the exception of Bussuquara virus, in Brazilian isolates in tissue culture fluid.
Assuntos
Primers do DNA , DNA Viral/análise , Flaviviridae/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Viral/genética , Aedes , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/química , DNA Viral/química , Eletroforese em Gel de Ágar , Flaviviridae/classificação , Flaviviridae/genética , Genoma Viral , Dados de Sequência Molecular , RNA Viral/química , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
We show here a simplified reverse transcription-polymerase chain 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 reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50 microliters 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, 7.5 U of reverse transcriptase, and IU of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37 degrees C for RT followed by a variable amount of cycles of two-step PCR amplification (92 degrees C for 60 sec, 53 degrees C for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% 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 10(2.8) 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.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Reação em Cadeia da Polimerase , Vírus da Dengue/genética , Humanos , DNA Polimerase Dirigida por RNARESUMO
We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue 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 and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/virologia , Reação em Cadeia da Polimerase/métodos , Brasil , DNA Viral/análise , Dengue/diagnóstico , Vírus da Dengue/genética , HumanosRESUMO
O objetivo deste estudo foi o de comaparar variáveis biomecânicas selecionadas do ato de sentar para crianças portadoras de paralisia cerebral moderada em dois sistema convencional. Dezenove crianças com idade variando de cinco a treze anos, portadoras de paralisia cerebral moderada espástica, participaram do estudo. Pontos de referência foram marcados e fotografias foram tiradas zero, cinco e dez minutos após as crianças atingirem posiçäo sentada a mais cômoda em ambos os sistemas. Após, os sujeitos foram instruídos para que se movessem para uma posiçäo funcional posterior e duas fotografias foram tiradas nesta posiçäo, após a qual os sujeitos retornaram à posiçäo relaxada e duas chapas finais foram feitas. Uma análise de variância 2 x 4 (sistema versus postura) para medidas repetidas foi realizada para cada uma das dez variáveis dependentes pré-selecionadas. Os resultados do estudo mostraram que o sistema de testes foi bem sucedido em distribuir a massa corporal superior reduzindo os momentos negativos lombares e aumentando os momentos positivos isquiáticos. Conclui-se, portanto, que o sistema de testes melhorou a postura sentada de crianças portadoras de paralisia cerebral moderada