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, New Guinea C strain (DEN2 NGC) such that a faithful, infectious copy of the genome can be made by in vitro transcription using the commercially available SP6 RNA polymerase. Creatio of an infectious clone will allow introduction of defined mutations into th 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. Stocks of DEN2 NGC have been grown and genomic RNA prepared. Primers for reverse transcription/polymerase chain reaction (RT PCR) have been designed and synthesized. 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. The best PCR results have been obtained using unprimed R reactions. The entire genome except for the right-hand 120 nucleotides has been successfully amplified. The left-hand 5.3 kb was amplified in one piece, but is not yet cloned. The right-hand 3 kb also has been amplified but not cloned. The next steps are to clone the remainder of the genome, chemically synthesize the right-hand 120 nucleotides, and then assemble a full-length clone from the pieces. Hopefully, in vitro transcription of this clone will produce infectious RNA.
