Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain).

Dengue virus type 2 (DEN-2), a member of the Flaviviridae family, has a positive-strand RNA genome, 10,723 nucleotides (nt) in length and encoding a single polyprotein precursor consisting of 3391 amino acids (aa). In order to construct a full-length cDNA clone, the viral genome was cloned into 5' (nt 1-2203 under the control of the T7 promoter (pT7)) and 3' (nt 2203-10,723) constructs. A full-length DEN-2 cDNA under pT7 control was assembled in vitro after excising the two cDNA inserts from the 5' and 3' constructs, and joining them with T4 DNA ligase. The RNA produced by in vitro transcription of the cDNA using T7 RNA polymerase was infectious, as shown by transfection of permissive BHK-21 and Vero cells, and propagation of the virus particles released into the culture media. The virus particles stably maintained the conservative mutation introduced into the 5' construct, and the cells infected with the infectious RNA-derived virus synthesized virus-specific DEN-2 antigens, as shown by immunofluorescence and immunoprecipitations. The full-length infectious clone for DEN-2 should be useful for the study of molecular mechanisms involved in viral RNA replication and virus assembly.