The dengue virus genome is a positive-strand RNA molecule of approximately 11 kb, which is capped but not polyadenylated. 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 (DEN2), New Guinea C (NGC) strain. This cDNA will be under control of the prokaryotic SP6 promoter, such that in vitro transcription using commercially available SP6 RNA polymerase will generate a faithful copy of the dengue genome. Our specific goal is to produce transcripts that are infectious when transfected into tissue culture cells. Creation of an infectious transcript will allow introduction of defined mutations into the dengue virus genome, which will be useful for basic studies on the life cycle of the virus. Also, there are neurotropic strains of DEN2 NGC that are adapted to growth in mouse brain, as well as strains that are not. By substituting portions of an infectious transcript with cDNA derived from the neurotropic virus, it should be possible to map the mutations responsible for adaptation to growth in mouse brain. Finally, it may be possible to introduce mutations into the genome to attenuate the virus for possible use as a vaccine. Initially, we attempted to plaque purify DEN2 NGC on LLC-MK2 cells. Although we were able to successfully obtain plaques, our efforts to grow stocks from selected isolated plaques have failed. We have elected to proceed without plaque purification. Stocks of DEN2 NGC have been grown. We are currently designing primers for reverse transcription/polymerase chain reaction. We intend to amplify relatively large pieces of the genome, so that only a few pieces have to be assembled to make a full-length copy.
