INFECTIOUS RNA TRANSCRIPTS FROM FULL-LENGTH DENGUE VIRUS TYPE-2 CDNA CLONES MADE IN YEAST

The dengue virus type 2 genomic RNA was amplified by reverse transcrip tion-PCR and cloned as four cDNA fragments, We could not assemble thes e four fragments into full-length cDNA in Escherichia coli. The full-l ength dengue virus cDNA was constructed by homologous recombination in yeast, tither as part of a yeast artificial chromosome or in a yeast- E. coli shuttle vector, Full-length cDNA clones were propagated once i n E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection , as judged by cytopathic effects and indirect immunofluorescence. Inf ectivity was sensitive to RNase digestion and was dependent on the pre sence of cap analog in the transcription reaction mixture. Virus in th e medium was passaged on C6-36 cells to produce stocks, and these stor ks had titers and plaque morphologies similar to those of the parental dengue virus type 2, Intracellular dengue virus RNA from cells infect ed with transcripts virus contained an introduced BstEII site, proving that infectivity was derived ft om RNA transcripts and not from conta mination with parental dengue virus, Transcript-derived virus was comp arable Co dengue virus type 2 for growth and protein expression in tis sue culture cells, Sequence analysis of the dengue virus cDNA in one f all-length clone revealed only one unexpected silent mutation. By usin g yeast technology, it will be easy to introduce specific mutations in to the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts.