Genetic manipulation of the viral genome at the molecular level has been an attractive approach to development of stable live attenuated vaccines for prevention of viral infection. Attempts were undertaken to produce: (a) full-length cloned dengue DNA or (b) its RNA transcript synthesized in vitro that is infectious after introduction into permissive mosquito or primate cells. Initially, seven overlapping DNA segments spanning the entire dengue type 4 virus genome were used to construct a complete DNA copy by joining DNA fragments cleaved by endonucleases at shared sites. The unit-length dengue DNA of 10,644 nucleotides appeared to be stable in pBR322 plasmid as a molecule as shown by restriction enzyme digestion. The following sequences were added to the dengue recombinant DNA in an attempt to increase infectivity of dengue nucleic acid: (1) an SV40 promoter to increase transcription of RNA from dengue DNA following its transfection into primate cells; (2) an SP-6 promoter to increase in vitro synthesis of RNA from a dengue DNA template. Under assay conditions which were efficient for detection of infectivity of dengue virion RNA, we failed to detect invectivity of either dengue DNA RNA transcripts in cell culture. Presence of a short length of non-dengue genomic sequences at the 5' and 3' ends of the complete dengue cDNA may be responsible for its failure to be infectious. Experiments are currently in progress to remove these additional non-viral nucleotides so that only precise dengue RNA sequences are transcribed in vivo or in vitro including a 7mG cap at the 5'-terminus.
