Abstract
BackgroundDengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The current lack of an approved tetravalent vaccine and ineffective insecticide control measures warrant a search for alternatives to effectively combat DENV. The trans-splicing variant of the Tetrahymena thermophila group I intron catalytic RNA, or ribozyme, is a powerful tool for post-transcriptional RNA modification. The nature of the ribozyme and the predictability with which it can be directed makes it a powerful tool for modifying RNA in nearly any cell type without the need for genome-altering gene therapy techniques or dependence on native cofactors.ResultsSeveral anti-DENV Group I trans-splicing introns (αDENV-GrpIs) were designed and tested for their ability to target DENV-2 NGC genomes in situ. We have successfully targeted two different uracil bases on the positive sense genomic strand within the highly conserved 5'-3' cyclization sequence (CS) region common to all serotypes of DENV with our αDENV-GrpIs. Our ribozymes have demonstrated ability to specifically trans-splice a new RNA sequence downstream of the targeted site in vitro and in transfected insect cells as analyzed by firefly luciferase and RT-PCR assays. The effectiveness of these αDENV-GrpIs to target infecting DENV genomes is also validated in transfected or transformed Aedes mosquito cell lines upon infection with unattenuated DENV-2 NGC.ConclusionsAnalysis shows that our αDENV-GrpIs have the ability to effectively trans-splice the DENV genome in situ. Notably, these results show that the αDENV-GrpI 9v1, designed to be active against all forms of Dengue virus, effectively targeted the DENV-2 NGC genome in a sequence specific manner. These novel αDENV-GrpI introns provide a striking alternative to other RNA based approaches for the transgenic suppression of DENV in transformed mosquito cells and tissues.
Highlights
Dengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year
While we identified several ribozymes that are effective in significantly reducing DENV 2-NGC infection of Ae. albopictus C6/ 36 cells, our inability to target sequences that are conserved among all serotypes require investigation of additional ribozymes with potential for wider specificity
Analysis of highly conserved elements in the Dengue genome All nucleotide position designations used throughout the study are relative to the published DENV- 2 New Guinea strain C genome (DENV-2 NGC; GenBank Accession: M29095)
Summary
Dengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The mosquito-borne Dengue viruses (DENV) are responsible for approximately 100 million infections and 200,000 deaths each year with 2.5 billion people remaining at risk for DENV infection, making DENV one of the most important viral diseases in the world (1). Newer approaches that have received considerable attention include interference with the extrinsic incubation cycle of DENV replication within the arthropod vector [2,3] One such approach envisions population replacement of vector competent mosquitoes with those refractory for infection and/or transmission of the virus, which could theoretically halt disease transmission [2,3]. This approach has distinct advantages for environmental safety, cost effectiveness, and long term disease suppression
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