Epidemic measles recently resurfaced in the U.S., raising public health concerns about current vaccine strategy and design and doubts that eradication of this serious acute viral infection can be attained by the turn of the century. While measles resurgence largely can be ascribed to failure to achieve age-appropriate mass immunization, additional features of measles epidemics throughout the world point to deficiencies in the current vaccine's efficacy. Clearly, a new generation of measles vaccines that induce abundant, long-lived immunity even when administered to infants 6-9 months of age is internationally needed. Intelligent vaccine design, however, must rest on a far better understanding of measles virus (MV) and in particular, on the as yet unknown identity of the virally encoded determinants of virulence as well as of those genomic changes that lead to attenuation. This proposal seeks to address the hypothesis that important virulence/attenuation determinants reside in viral genomic non- protein coding regulatory regions: the 3' and 5' cis-acting putative promotor and/or regulatory sequence elements directing genomic transcription, genome and antigenome encapsidation, and replication; and the short regions encompassing internal intergenic boundaries specifying transcription termination and reinitiation. Thus, specific nucleotide changes in these regions may affect virulence by modulating transcriptional and/or replicative efficiency thereby determining the abundance of cytopathic viral gene products and/or virion progeny. To address this hypothesis, the nucleotide sequences of the non-protein coding regulatory regions of various epidemiologically distinct wild-type MV isolates as well as vaccine strains will be determined. Unique nucleotide changes found in the genomic regulatory regions of these MV strains will be tentatively assigned as virulence or attentuation determinants. Validity of that tentative assignment will be appraised b determining the influence of these nucleotides on transcription and replication when incorporated into a newly developed measles 'minireplicon', a chimeric genome sense RNA consisting of the MV 3' and 5' nonprotein coding sequences flanking an antisense firefly luciferase coding sequence from which luciferase mRNA is transcribed and new MV:luciferase minigenomes replicated when introduced into helper-cells. Evaluating the role of distinctive MV strain nucleotide changes in intercistronic regulatory domains will be approached by a similar strategy upon completion of construction of a bicistronic artificial MV: reporter gene minigenome now underway. Finally, the means must be developed by which such virulence/attenuation determinants can be introduced into a predefined measles sequence from which new genetically altered virion progeny can be propagated and tested for their efficacy as a live attenuated vaccine. To that end, continued effort will be directed towards refining the vector and expression system by which genetically modified infectious MV strains can be produced from full-length MV cDNA.
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