Molecular characterization of the 5' and 3' ends of the dengue virus genome and its usefulness in pathogenesis studies
Rosales Munar, Alicia Alejandra
Dengue is one of the mosquito-borne diseases of major importance in public health, which has spread worldwide, especially in tropical and subtropical regions, as a result of globalization, precarious urbanization, and expansion of the mosquito vector. Although it has been extensively studied at the molecular level, most of the dengue virus (DENV) genomes at public databases lack or have uncorroborated ends of the untranslated regions (UTRs), becoming a limitation when conducting studies of association between the genetic background of the virus and the clinical outcome of the disease. In an attempt to solve this problem, the present study aims to characterize the 5' and 3' ends of dengue virus, to provide complete information for future studies of viral pathogenesis. The 5´ UTRs of twenty-seven Colombian DENV isolates (from patients with or without warning signs or severe disease) were readily amplified through a standard RACE approach while the 3´ UTRs required the development of a modified RACE protocol, For the cDNA synthesis of the 5' UTR, a specific anti-sense primer for each serotype was used, which were located at the coding region of the C or prM/M proteins. The cDNAs were purified and subsequently polyadenylated using the terminal transferase. By using an oligodT and an internal reverse primer located upstream of the first primer used for cDNA synthesis, the 5' UTRs were successfully PCR amplified and sequenced. For the molecular characterization of the 3' UTR, an adenine tail was directly added to the positive-sense viral RNA using the E coli poly (A) polymerase, which allowed the hybridization of the oligo-dT for the synthesis of cDNA, subsequent PCR amplification and Sanger sequencing. The results allow to demonstrate the successful characterization of the whole dengue virus UTRs and to assess the potential impact of point mutations at the RNA secondary structure level
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