RESUMO
Dengue virus (DENV) and its four serotypes (DENV1-4) belong to the Flavivirus genus of the Flaviviridae family. DENV infection is a life-threatening disease, which results in up to 20,000 deaths each year. Viruses have been shown to encode trans-regulatory small RNAs, or microRNAs (miRNAs), which bind to messenger RNA and negatively regulate host or viral gene expression. During DENV infections, miRNAs interact with proteins in the RNAi pathway, and are processed by ribonucleases such as Dicer and Drosha. This study aims to investigate Drosha, DGCR8, and Dicer expression levels in human A-549 cells following DENV4 infection. DENV4 infected A-549 cells were collected daily for 5 days, and RNA was extracted to quantify viral load. Gene expression of Drosha, Dicer, and DGCR8 was determined using quantitative PCR (RT-qPCR). We found that DENV4 infection exhibited the highest viral load 3 days post-infection. Dicer, Drosha, and DGCR8 showed reduced expression following DENV4 infection as compared with negative controls. In addition, we hypothesize that reduced expression of DGCR8 may not only be related to miRNA biogenesis, but also other small RNAs. This study may change our understanding regarding the relationship between host cells and the dengue virus.
Assuntos
RNA Helicases DEAD-box/biossíntese , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Dengue/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/biossíntese , Ribonuclease III/biossíntese , Células A549 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Dengue/genética , Dengue/virologia , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Carga ViralRESUMO
Currently assembling genomes without reference is one of the most important challenges for bioinformaticists all over the world in an attempt to characterize new organisms. The current study has used two dengue virus type 4 (DENV-4) strains recently isolated in Brazil, which have its genomes sequenced using the GSFLX 454 sequencer (Roche, Life Science) by the pyrosequencing method. The GSFLX 454 data were used for testing different genome assembling strategies. We described a pipeline that was able to recover more than 96% of the sequenced genome in a single run and could be helpful for further assembly attempts of other DENV genomes, as well as other RNA virus-like genomes.