Immunization with live, attenuated vaccines has been the most effective strategy employed for the control of virus disease. However, undesirable complications can result from reversion of the attenuated vaccine strain to a strain which is virulent, in some cases inducing the disease syndrome that the vaccine was designed to prevent. The goal of the proposed research is to introduce multiple, well-defined attenuating mutations into the genome of the alphavirus, venezuelan eguine encephalitis (VEE), thus producing a live virus vaccine candidate strain wit an extremely low rate of reversion to virulence. First, we propose to identify the loci which control VEE virulence by sequencing a series of closely related attenuated and virulent VEE mutants which we have isolated previously. Second, the RNA genome of VEE will be converted to a form ammenable to genetic manipulation by construction of a full-length cDNA clone of VEE placed downstream from a T7 promoter. Infectious virus may be recovered from such cDNA clones by in vitro synthesis of infectious VEE RNA and transfection of cultured cells. Third, multiple, well- defined, attenuating mutations will be introduced into the full- length VEE construct by allelic replacement and/or site-directed mutagenesis. If each of these mutations independently renders the virus attenuated, then virus derived from such a construct will be much less likely to revert to virulence. Finally, the derived virus will be tested for pathogenicity, immunogenicity and reversion to virulence in animal models. We feel that successful completion of this study will make a significant contribution in four major areas. One, the availability of a full-length molecular clone of VEE will greatly facilitate the study of this important pathogen. Two, specific domains controlling virulence of VEE will be identified. Three, the potential usefulness of this approach in the design of live attenuated vaccines for positive-strand RNA viruses will be evaluated. And four, if the genetically engineered strains developed in this study prove superior to the existing investigational vaccine in animal models, they can be considered as candidates for use in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS026681-03
Application #
3412667
Study Section
Experimental Virology Study Section (EVR)
Project Start
1989-03-01
Project End
1991-03-31
Budget Start
1990-03-01
Budget End
1991-03-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Ryman, Kate D; White, Laura J; Johnston, Robert E et al. (2002) Effects of PKR/RNase L-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis. Viral Immunol 15:53-76
Charles, P C; Trgovcich, J; Davis, N L et al. (2001) Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse. Virology 284:190-202
White, L J; Wang, J G; Davis, N L et al. (2001) Role of alpha/beta interferon in Venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region. J Virol 75:3706-18
Schultz-Cherry, S; Dybing, J K; Davis, N L et al. (2000) Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses. Virology 278:55-9
Bernard, K A; Klimstra, W B; Johnston, R E (2000) Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice. Virology 276:93-103
Davis, N L; Caley, I J; Brown, K W et al. (2000) Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles. J Virol 74:371-8
Aronson, J F; Grieder, F B; Davis, N L et al. (2000) A single-site mutant and revertants arising in vivo define early steps in the pathogenesis of Venezuelan equine encephalitis virus. Virology 270:111-23
MacDonald, G H; Johnston, R E (2000) Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis. J Virol 74:914-22
Charles, P C; Brown, K W; Davis, N L et al. (1997) Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate. Virology 228:153-60
Davis, N L; Brown, K W; Johnston, R E (1996) A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge. J Virol 70:3781-7

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