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 has been to clone a cDNA copy of the genome of dengue virus type 2 (DEN2) such that a faithful, infectious copy of the genomic RNA can be made by in vitro transcription. This has now been accomplished, as will be outlined in the next section. Creation of this infectious clone now allows introduction of defined mutations into the dengue genome. Such a capability will 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. Work has been initiated to engineer selected mutations into the infectious clone. The strategy we used was to prepare genomic RNA and then use reverse transcription/polymerase chain reaction (RT-PCR) to make large amounts of cDNA. The entire genome was amplified, and cloned in E. coli, as four pieces. Two of the pieces were easily combined to make the right half-genome. Efforts to obtain the left half-genome failed. The right half-genome was combined with the left quarter-genome in a yeast shuttle plasmid. An attempt to directly add the missing quarter-genome to this clone in E. coli was unsuccessful. The full-length genomic clone was obtained by homologous recombination between the """"""""three-quarter clone"""""""" and the missing quarter-genome upon cotransfection in yeast. The full-length clone was successfully grown in E. coli, and large amounts of DNA were prepared. RNA transcripts made from this DNA were transfected into susceptible cells using lipofectin, and a typical dengue virus infection resulted after about 3 weeks. Conditions have since been worked out to electroporate the RNA into cells, which results in a full-blown dengue infection after only 4 days. Virus recovered from transfected cells can be passaged and plaqued just as wild-type virus. Virus derived from transcripts behaves as wild-type in a one step growth curve on a mammalian and an insect cell line. cDNA made by RT-PCR of RNA from cells infected by transcript-derived virus was shown to carry an introduced restriction enzyme site, thus proving that the virus was derived from RNA transcripts and not from contaminating wild-type virus. The infectivity of the transcripts was sensitive to RNAse but resistant to DNAse, and depended on the presence of CAP analog in the transcription reaction. Efforts are currently underway to directly plaque after electroporation of RNA into cells, to allow a comparison of the specific infectivity of full-length transcripts and DEN2 RNA. We anticipate that we will determine the DNA sequence of the infectious clone, in order to compare it to the published DEN2 genomic sequence.
