DENGUE TYPE-4 VIRUS MUTANTS CONTAINING DELETIONS IN THE 3'-NONCODING REGION OF THE RNA GENOME - ANALYSIS OF GROWTH RESTRICTION IN CELL-CULTURE AND ALTERED VIREMIA PATTERN AND IMMUNOGENICITY 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 seco ndary structure, are thought to be essential for virus replication, Im mediately upstream of the secondary structure, short RNA sequences tha t are conserved among mosquito-borne flaviviruses have been identified , A series of deletions that range from 30 to 262 nt were introduced i nto this upstream region of full-length DEN4 cDNA to create viable del etion 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 ex hibited reduced infectivity when transfected into simian LLC-MK(2) cel ls 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 th e last 113 nt of the 3' end mere viable, With the exception of mutant 3'd 303-183, which contained a deletion of nt 303 to 183 from the 3' t erminus, deletion mutants produced plaques that appeared late on simia n LLC-MK(2) cells or exhibited a small-plaque morphology on mosquito C 6/36 cells compared with the wild-type virus, These mutants also repli cated less efficiently and attained a lower titer in LLC-MK(2) cells t han parental wild-type virus, Significantly, mutant 3'd 303-183 grew t o a high titer and was least restricted in growth, Mutant 3'd 303-183 and four other moderately to severely restricted mutants were selected for evaluation of infectivity and immunogenicity in rhesus monkeys, T here was a suggestion that occurrence and duration of viremia were red uced for some of the deletion mutants compared with the wild-type viru s, However, more convincing evidence for attenuation of some of the mu tants was provided by an analysis of antibody response to infection. M utant 3'd 303-183 induced an antibody response equivalent to that stim ulated by wild-type virus, whereas other mutants induced low to modera te levels of antibodies, as measured by radioimmunoprecipitation and v irus neutralization. The immunogenicity of these 3' DEN4 deletion muta nts in monkeys appeared to correlate with their efficiency of growth i n simian LLC-MK(2) cells, One or more mutants described in this paper may prove to be useful for immunization of humans against disease caus ed by dengue virus.