Chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in Vero cells or human liver cells and attenuated in mice

A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2A Delta 30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2A Delta 30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Delta 30) in the 3 ' untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2A Delta 30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty is mutant viruses were identified that were is in both simian Vero and human liver HuH-7 cells (n = 13) or only in HuH-7 cells (n = 7). Each of the 20 is mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 is mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5 ' and 3 ' UTRs, with more than one change occurring, in general, per mutant virus. A is mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Delta 30 deletion, thereby creating rDEN4 Delta 30-4995, a recombinant virus which is is and more attenuated than rDEN4 Delta 30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.