One of our major research objectives for the dengue viruses has been the construction of full-length dengue cloned DNA that could be used for the production in vitro of full-length RNA transcripts able to initiate dengue virus infection following transfection of permissive cells in culture. Our initial strategy was to use the pBR322 plasmid vector to clone full-length dengue DNA from two fragments, each containing approximately one-half of the dengue genome sequence. The full-length dengue virus cDNA was placed under the control of an SP6 polymerase promoter and a unique Asp 718 cleavage sequence was inserted at the 3'-end of the dengue sequence to be used for producing a run-off transcript. Asp 718 linearized DNA was then used as a template for in vitro transcription of RNA using SP6 polymerase. m7G capped GTP was added to the transcription reaction to produce full-length dengue RNA transcripts containing a cap structure at the 5' end. Transfection of LLCMK2 cells with the transcription products yielded infectious dengue virus as indicated by the appearance of dengue antigens in a majority of cells. Progeny dengue virus was recovered at a titer of 10(5)pfu/ml from the transfected cell lysate. Treatment of the transcription products with DNase did not affect infectivity, while RNase treatment completely abolished their infectivity. Formal proof for the infectivity of the RNA transcripts was provided by engineering two silent mutations into the dengue cDNA and demonstrating the presence of these mutations in dengue virus recovered from cells transfected with the mutant RNA transcripts. Experiments are underway using the full-length dengue cDNA to engineer mutations at strategic sites in the dengue viral genome. Subsequently, infectious dengue virus containing the mutations introduced into dengue cDNA will be evaluated for various biological and immunological properties in vitro as well as alteration in virulence for experimental animals. This strategy may yield attenuated mutants that could be used in a safe, effective, live virus vaccine.
