Inhibition of enterovirus 71 infection by small interfering RNA-mediated RNA interference

Autor: Meng -Tsai Wu, 吳孟采
Rok vydání: 2003
Druh dokumentu: 學位論文 ; thesis
Popis: 91
RNA interference is a sequence-specific posttranscriptional gene silencing mechanism. In mammalian cells, U6 and H1 promoters have been used to produce small interfering RNA. We have developed human and mouse RNA polymerase III H1, U6 promoters to drive shRNA, siRNA duplex, sense RNA and antisense RNA expression. Using pGL3-control as target vector, we examined the effect of firefly luciferase gene expression and compared the suppression efficiency by these small RNAs. We found that antisense RNA and sense RNA weakly inhibit gene expression, however siRNA duplex and shRNA efficiently suppress gene expression in SK-N-SH cell line. The transcriptional activities between these four Pol III promoters are not significantly different in SK-N-SH cell line. Since its discovery in 1969, EV71 has been recognized as a frequent cause of epidemics of hand-foot-mouth disease (HFMD) associated with severel neurological sequelae in a small proportion of cases. In 1998, the largest EV71 outbreak in Taiwan, more than 90000 children with HFMD have been reported, and at least 55 died, suggesting neurovirulence of the pathogen. We focus on prevention of EV71 infection, and use small interfering RNA to inhibit EV71 infection. We found two siRNA target site could inhibit the Taiwan/4643 EV71 isolate infection. The inhibition of viral infection was even more efficient when two siRNA expression cassettes were linked together. Single nucleotide changes within the poliovirus IRES have been found to result in large alterations in neurovirulence. We compared the sequence of 5´NTR from three EV71 isolates, and found that there are six nucleotides different. However, these differences have no significant effect on the different neurovirulence between EV 71 isolates.
Databáze: Networked Digital Library of Theses & Dissertations
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