A series of cDNA plasmids containing full-length copies of Mengovirus and MengoEMC (encephalomyocarditis) virus chimeric genomes have been developed and characterized. The isolates differ from each other and from the wildtype Mengo and EMC virus sequences only in the lengths of their 5' non- coding poly(C) tracts. RNA transcripts derived from these constructions are completely infectious to HeLa cells and the resulting genetically engineered progeny viruses. However, upon inoculation of mice, the natural host for cardioviruses, the engineered viruses carrying shortened versions of the poly(C) tract (eg: C8, C12 or C13UC10) exhibit dramatically attenuated pathogenicity relative to the original wildtype viruses or relative to a newly engineered progeny virus with a wildtype-Mengo length poly(C) tract (C50UC10). The attenuation is manifest in a 106-1010 fold increase in the virus LD50. Moreover, animals receiving sublethal doses from any of the short poly(C) strains characteristically develop high level of neutralizing antibodies and acquired lifelong protective immunity against (lethal) challenge with wildtype virus. It is the goal of this proposal to explore and characterize the molecular basis for poly(C)- mediated attenuation with the eventual objective of exploiting this phenomena and the principles to be learned from it, for the development of new and effective, live picornavirus vaccines.
The specific aims are: (1) To genetically engineer a spectrum of full-length cardiovirus plasmids containing altered poly(c) tracts, discontinuities, viral chimeric sequences, rearrangements and other variations within the 5' non-coding regions; (2) To test the relative effects of such constructions on transcript infectivity and progeny virus viability in several relevant tissue culture cell lines; (3) To assess the pathogenic effects of infectious genetically engineered progeny viruses in mice, in order to evaluate the host protective responses, viral etiology, replication, clearance and sequence reversion potential; (4) To formulate and test hypotheses to explain at the molecular level, the role of picornaviral 5' non-coding sequences, and in particular poly(C) tracts, in the restrictive determination of virus-host interactions. The potential role of cytokine induction as a contributing element to the enhancement of wildtype viral infections will be examined.
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