Five years ago, West Nile virus (WN) entered and spread rapidly throughout North America. During the 1999-2003 outbreaks of WN in the USA, there were 13,693 WN illnesses reported that included 548 deaths. Importantly, severe neurologic disease, which required hospitalization and long-term rehabilitation, was observed in over 30% of WN cases, most common in the elderly. As a result, WN illness is considered to be an emerging disease in the USA and presents a significant public health threat since a human vaccine is not available and since the magnitude of epidemic disease continues to increase. We have extended our flavivirus chimeric vaccine strategy to include WN and its closely related St. Louis encephalitis virus (SLE). Goals of this project are to construct and evaluate WN/DEN4 and SLE/DEN4 chimeric viruses in humans as possible vaccines against disease caused by WN or SLE. We constructed a viable WN/DEN4 chimera in which the structural prM and E protein genes of WN were substituted for the corresponding genes of DEN4 that differed in amino acid sequence by 61.5% for prM and 55.9% for E. In order to generate a further attenuated derivative of WN/DEN4, we introduced a 30-nucleotide deletion (D30) in the 3' non-coding region of genome that is a genetically stable mutation and has attenuated DEN4 for monkeys and humans. Both chimeric WN/DEN4 and WN/DEN4-3?D30 viruses have been evaluated for safety, infectivity, and immunogenicity in mice, geese, horses, and rhesus monkeys and for infectivity in mosquitoes. Chimeras: (1) exhibited more than 108-fold restriction of replication in mouse brain compared to wild-type WN when inoculated IC; (2) failed to induce fatal encephalitis in adult Swiss or SCID mice inoculated IP; (3) were highly attenuated in geese, horses, and monkeys; (4) were immunogenic in mice, horses, and nonhuman primates; (5) provided complete protection of mice and monkeys against WN challenge, and (6) were restricted in their ability to replicate in mosquitoes. The safety evaluation of a clinical vaccine lot of the WN/DEN4-3?D30 chimera in mice and nonhuman primates was recently performed under Good Laboratory Practice conditions at Bioqual, Inc. A Phase I clinical testing will be initiated as soon as we receive approval for our Clinical Protocol and IND submissions.