Dengue viruses are members of the flavivirus group of togaviridae that contain a positive strand RNA genome of approximately 12 kilobases. We employed recombinant DNA techniques to investigate the molecular biology of dengue virus with the intent of developing immunoprophylactic measures against this virus group that is epidemic in many geographical areas. The 428 full-length RNA from dnegue virus type 4, produced in C6/36 mosquito cells, was isolated and tailed with poly(A) at the 3'-terminus using E. coli poly(A) polymerase. Complementary DNA was synthesized by reverse-transcription using olibo(dT) as a primer and subsequently converted to double stranded DNA in the presence of E. coli RNase H, polymerase I, and ligase. The dengue cDNA products were inserted into the Pst I site of pBR322 using the dG/dC joining technique. A library of E. coli transfomrants containing dengue specific DNA inserts ranging from 2,000 - 3,500 base pairs was obtained. From these inserts a restriction enzyme map of an almost full-length dengue DNA sequence has been constructed by """"""""genome walking"""""""". Nucleotide sequences at both termini will be determined and verified to facilitate the construction of a full-length cloned DNA for further giologic studies.
