A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2A⌬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⌬30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (⌬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⌬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 ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n ‫؍‬ 13) or only in HuH-7 cells (n ‫؍‬ 7). Each of the 20 ts 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 ts 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 ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the ⌬30 deletion, thereby creating rDEN4⌬30-4995, a recombinant virus which is ts and more attenuated than rDEN4⌬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.The mosquito-borne dengue (DEN) viruses (serotypes 1 to 4 [DEN1 to -4]) are members of the Flavivirus genus and contain a single-stranded positive-sense RNA genome of approximately 10,600 nucleotides (nt) (43). The genome organization of DEN viruses is 5Ј-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3Ј (where UTR is untranslated region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural) (10, 48). A single viral polypeptide is cotranslationally processed by viral and cellular proteases, generating three structural proteins (C, M, and E) and seven NS proteins. The disease burden associated with DEN virus infection has increased over the past several decades in tropical and semitropical countries. Annually, there are an estimated 50 to 100 million cases of DEN fever (DF) and 500,000 cases of the more severe and potentially lethal DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS) (19).The site of viral replication in DEN virus-infected humans and the pathogeneses of DF and DHF/DSS are still incompletely understood (26). In humans, DEN virus infects lymphocytes (32, 55), macrophages (21, 52), dendritic cells (34, 63), and hepatocytes (36, 39). The liver is clearly involved in DEN virus infection of humans, as indicated by the occurrence of transient elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the ...