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Title: A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate
Authors: Santos, Jefferson J S
Cordeiro, Marli T
Bertani, Giovani R
Marques, Ernesto T A
Gil, Laura H V G
Affilliation: Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.
Abstract: Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.
Keywords: Dengue Virus
Transcription, Genetic
Virus Replication
Clone Cells
DNA, Complementary
RNA, Viral
keywords: Vírus da Dengue
Replicação Viral
Células Clonais
Transcrição Genética
RNA Viral
Issue Date: 2014
Citation: SANTOS, J. J. S. et al. A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate. Anais Da Academia Brasileira De Ciencias, v. 86, n. 4, p. 1749–1759, dez. 2014.
DOI: 10.1590/0001-3765201420130332
ISSN: 1678-2690
Copyright: open access
Appears in Collections:PE - IAM - Artigos de Periódicos

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