Overlapping cDNA clones were initially identified which spanned the entire dengue 4 sequence of 10,644 nucleotides in length. These separately cloned DNA segments were used to construct a full-length DNA copy. SV40 sequences that contained an early promoter and origin of SV40 DNA replication of COS-1 cells. We also constructed recombinant dengue DNA containing an SP6 promoter in order to produce RNA transcripts for assay of infectivity in transfected LLCMK2 cells. Special emphasis has been given to removing non- viral sequences from both termini of full-length dengue DNA. The modified SP6-engue 4 DNA produced precise transcripts in an in vitro system including a cap structure at the 5'-end. Thus far, non of these transcripts has proved to be infectious following the standard transfection assay. Direct transfection of COS-1 cells with SV-D4 DNA also failed to demonstrate infectivity. It is possible that our failure to detect infectivity with our cloned DNA resulted from deletion or substitution mutations present in the virus which was cloned or such alterations might have been introduced as an artifact during cloning. A second full-length dengue cDNA copy has been constructed from a new set of separate clones. Although the RNA transcripts made from the new clone also failed to show infectivity, these new full-length dengue sequences should prove to be valuable for identifying mutations in this and other full length addition, fragments in both non-infectious clones will be replaced with the corresponding fragments derived from other cloned cDNAs in a continuing attempt to construct a full length cDNA that is infectious.
