in Rhesus Monkeys

The dengue type 4 virus (DEN4) genome contains a 384-nucleotide (nt) 3 * noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. Immedi-ately upstream of the secondary structure, short RNA sequences that are conserved among mosquito-borne flaviviruses have been identified. A series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length DEN4 cDNA to create viable deletion mutants, some of which might prove to be useful for inclusion in a live attenuated virus vaccine. When studied by an infectious-center assay, most full-length RNA transcripts of the deletion constructs exhibited reduced infectivity when transfected into simian LLC-MK2 cells compared with the full-length RNA transcripts of wild-type parental virus. Deletion mutations that extended as far as the 5 * boundary of the 3 * noncoding region and whose 3 * boundary did not extend beyond the last 113 nt of the 3 * end were viable. With the exception of mutant 3*d 303-183, which contained a deletion of nt 303 to 183 from the 3 * terminus, deletion mutants produced plaques that appeared late on simian LLC-MK2 cells or exhibited a small-plaque morphology on mosquito C6/36 cells compared with the wild-type virus. These mutants also replicated less efficiently and attained a lower titer in LLC-MK2 cells than parental wild-type virus. Significantly, mutant 3*d 303-183 grew to a high titer and was least restricted in growth. Mutant 3*d 303-183 and four other moderately to severely restricted mutants were selected fo