The dengue virus genome is a positive-strand RNA molecule of approximately 11 kb. Purified genomic RNA is infectious upon transfection into appropriate cells. The goal of this project is to clone a cDNA copy of the genome of dengue virus type 2 such that a faithful, infectious copy of the genomic RNA can be made by in vitro transcription. Creation of an infectious clone will allow introduction of defined mutations into the dengue genome. Such a capability would be useful in many types of studies, including mapping mutations responsible for altered host range in neurotropic strains of DEN2, and attempting to attenuate DEN2 for possible use as a vaccine. The strategy we have been using is to prepare genomic RNA and then use reverse transcription/polymerase chain reaction to make large amounts of cDNA. We have tried to amplify relatively large segments of the genome, so that only a few cloned pieces have to be assembled to make a full-length copy. We can routinely amplify 3-4 kb segments, but we can not reliably amplify larger pieces. Thus, the entire genome has been successfully amplified and cloned as four pieces. Two of the pieces were easily combined to make the right half-genome. Efforts to obtain the left half-genome have so far proved unsuccessful; all such clones isolated to date have been rearranged. We are currently pursuing several alternative strategies to solve our problems: we are attempting to make the right three-quarter-genome, in order to produce a full-length cDNA by two-piece in vitro ligation without having to clone the entire genome; we are trying to clone the left half-genome in a low copy-number vector in E. coli, and then assemble a full-length clone in the same vector; we are experimenting with E. coli strains designed to propagate """"""""difficult"""""""" pieces of DNA; we are beginning to try cloning in yeast. Hopefully, one or more of these approaches will soon succeed.
