BACKGROUND: Antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rDEN4) have been replaced with those derived from DEN2 or DEN3. Two vaccine candidates were identified, rDEN2/4Delta30(ME) and rDEN3/4Delta30(ME), which contain the membrane (M) precursor and envelope (E) genes of DEN2 and DEN3, respectively, and a 30 nucleotide deletion (Delta30) in the 3' untranslated region of the DEN4 backbone. Based on the promising preclinical phenotypes of these viruses and the safety and immunogenicity of rDEN2/4Delta30(ME) in humans, we now describe the generation of a panel of four antigenic chimeric DEN4 viruses using either the capsid (C), M, and E (CME) or ME structural genes of DEN1 Puerto Rico/94 strain. RESULTS: Four antigenic chimeric viruses were generated and found to replicate efficiently in Vero cells: rDEN1/4(CME), rDEN1/4Delta30(CME), rDEN1/4(ME), and rDEN1/4Delta30(ME). With the exception of rDEN1/4(ME), each chimeric virus was significantly attenuated in a SCID-HuH-7 mouse xenograft model with a 25-fold or greater reduction in replication compared to wild type DEN1. In rhesus monkeys, only chimeric viruses with the Delta30 mutation appeared to be attenuated as measured by duration and magnitude of viremia. rDEN1/4Delta30(CME) appeared over-attenuated since it failed to induce detectable neutralizing antibody and did not confer protection from wild type DEN1 challenge. In contrast, rDEN1/4Delta30(ME) induced 66% seroconversion and protection from DEN1 challenge. Presence of the Delta30 mutation conferred a significant restriction in mosquito infectivity upon rDEN1/4Delta30(ME) which was shown to be non-infectious for Aedes aegypti fed an infectious bloodmeal. CONCLUSION: The attenuation phenotype in SCID-HuH-7 mice, rhesus monkeys, and mosquitoes and the protective immunity observed in rhesus monkeys suggest that rDEN1/4Delta30(ME) should be considered for evaluation in a clinical trial.