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

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Abstract
A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2AΔ30, 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. 2AΔ30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (Δ30) 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 2AΔ30 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. Twentytsmutant viruses were identified that weretsin both simian Vero and human liver HuH-7 cells (n= 13) or only in HuH-7 cells (n= 7). Each of the 20tsmutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20tsmutant 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. Atsmutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the Δ30 deletion, thereby creating rDEN4Δ30-4995, a recombinant virus which istsand more attenuated than rDEN4Δ30 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.