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, 10723 nucleotides (nt) in length and enc oding a single polyprotein precursor consisting of 3391 amino acids (a a), In order to construct a full-length cDNB clone, the viral genome w as cloned into 5' (nt 1-2203 under the control of the T7 promoter (P-T 7)) 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 insert s 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 po lymerase 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 conservat ive 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. Th e full-length infectious clone for DEN-2 should be useful for the stud y of molecular mechanisms involved in viral RNA replication and virus assembly.