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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030566-04
Application #
2065725
Study Section
Virology Study Section (VR)
Project Start
1991-01-01
Project End
1995-12-31
Budget Start
1994-01-01
Budget End
1994-12-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Osorio, J E; Grossberg, S E; Palmenberg, A C (2000) Characterization of genetically engineered mengoviruses in mice. Viral Immunol 13:27-35
Martin, L R; Neal, Z C; McBride, M S et al. (2000) Mengovirus and encephalomyocarditis virus poly(C) tract lengths can affect virus growth in murine cell culture. J Virol 74:3074-81
Backues, K A; Hill, M; Palmenberg, A C et al. (1999) Genetically engineered Mengo virus vaccination of multiple captive wildlife species. J Wildl Dis 35:384-7
Martin, L R; Duke, G M; Osorio, J E et al. (1996) Mutational analysis of the mengovirus poly(C) tract and surrounding heteropolymeric sequences. J Virol 70:2027-31
Duque, H; Palmenberg, A C (1996) Epitope mapping of monoclonal antibodies raised to recombinant Mengo 3D polymerase. Virus Genes 13:159-68
Osorio, J E; Hubbard, G B; Soike, K F et al. (1996) Protection of non-murine mammals against encephalomyocarditis virus using a genetically engineered Mengo virus. Vaccine 14:155-61
Osorio, J E; Martin, L R; Palmenberg, A C (1996) The immunogenic and pathogenic potential of short poly(C) tract Mengo viruses. Virology 223:344-50
Martin, L R; Palmenberg, A C (1996) Tandem mengovirus 5' pseudoknots are linked to viral RNA synthesis, not poly(C)-mediated virulence. J Virol 70:8182-6
Hahn, H; Palmenberg, A C (1995) Encephalomyocarditis viruses with short poly(C) tracts are more virulent than their mengovirus counterparts. J Virol 69:2697-9
Palmenberg, A C; Osorio, J E (1994) Cardioviral poly(C) tracts and viral pathogenesis. Arch Virol Suppl 9:67-77

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