A cloned full-length DNA copy of the dengue type 4 virus genome was obtained and its complete sequence analyzed. Experiments were then undertaken to determine if cloned dengue DNA, or an RNA transcript derived from it, is infectious after introduction into permissive cells. First, we introduced SV40 DNA sequences which represent strong signals for replication and transcription into the plasmid vector in an attempt to increase the infectivity of cloned dengue DNA. Second, because dengue viral RNA is infectious we have initiated efforts to introduce dengue RNA transcripts from cloned DNA directly into cultured cells. In these efforts the versatile in vitro transcription system of SP6 and T7 phages is being utilized. The full-length dengue DNA will be inserted into such a plasmid vector (Gemini 3) at the unique Pst I site. The dengue DNA recombinant will then be linearized with Kpn I thus making it possible to obtain """"""""run-off"""""""" transcripts which can be assayed for infectivity. If we are successful, site-specific mutagenesis will be performed to construct dengue virus mutants containing mutations in strategic regions essential for viral replication and other gene functions. Dengue virus mutants constructed in this manner may exhibit an altered phenotype such as temperature-sensitivity of virus replication or reduced virulence for lower primates and humans.
