Generating Cloned Attenuated Dengue 1 Viral cDNA Sequences

Wild type (wt) dengue 1 viral RNA will be used as substrate to generate a complementary DNA (cDNA) library of clones that contain identified and sequenced DNA segments spanning the entire dengue 1 genome. Point mutations designed to generate amino acid alterations will be introduced at the nucleotide level in the cloned dengue 1 DNA sequences by polymerase chain reaction amplification. These mutations will be based upon sequence alterations that have previously been identified by Naval medical researachers in non-virulent or attenuated dengue 1 strains as potentially useful for development of an attenuated dengue 1 vaccine. In Phase II, mutagenized cDNA clones will be combined with appropriate cloned parental wt dengue 1 sequences to produce full-length chimeric dengue 1 cDNAs. These full-length viral cDNAs will subsequently be transcribed into infectious dengue 1 viral RNAs containing mutations of interest for development of a live attenuated dengue 1 viral vaccine and for analysis of dengue 1 pathogenicity and immunogenicity.