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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060
EISSN: 1678-8060
Vol. 108, No. 8, 2013, pp. 983-991
Bioline Code: oc13164
Full paper language: English
Document type: Research Article
Document available free of charge

Memórias do Instituto Oswaldo Cruz, Vol. 108, No. 8, 2013, pp. 983-991

 en Construction and characterisation of a complete reverse genetics system of dengue virus type 3
da Silva Santos, Jefferson José; Cordeiro, Marli Tenório; Bertani, Giovani Rota; de Azevedo Marques, Ernesto Torres & Gil, Laura Helena Vega Gonzales

Abstract

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli check for this species in other resources has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast-E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli. RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Keywords
reverse genetics; dengue virus; molecular cloning

 
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