Construction and characterisation of a complete reverse genetics system of dengue virus type 3

• Main Authors: Jefferson Jose da Silva Santos, Marli Tenorio Cordeiro, Giovani Rota Bertani, Ernesto Torres de Azevedo Marques, Laura Helena Vega Gonzales Gil
• Format: Article
• Language: English
• Published: Instituto Oswaldo Cruz, Ministério da Saúde 2013-12-01
• Series: Memórias do Instituto Oswaldo Cruz.
• Subjects: reverse genetics; dengue virus; molecular cloning
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000800983&lng=en&tlng=en

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 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.