VIRAL DETERMINANTS OF HSV DISEASE IN MICE

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The long-range goals of this project are to define cell and viral requirements for entry of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) into cells of the genital tract and for spread of infection from the portal of entry to other tissues, including the nervous system. The immediate aims are to identify HSV-1 and HSV-2 glycoproteins that mediate the entry of virus into cells of the vaginal epithelium and that are required for the cell-to-cell spread of virus from the epithelium to cells of the peripheral and central nervous system. The limited information available about requirements for infection of cultured cells by HSV-1 may not be fully generalizable to HSV-2 or to the infection of differentiated cells of the intact host. Mutant viral strains deleted for individual glycoprotein genes, or expressing altered forms of the glycoprotein, will be derived from the parental strains HSV-1 (F) and HSV-2(G). The parental and mutant strains will be used to inoculate mice by intravaginal instillation. The cells of the genital tract and nervous system that become infected will then be identified and pathological changes, including CNS lesions and demyelination, correlated with the-spread of infection. The parental and mutant viral strains will express beta- galactosidase under control of the strong IE cytomegalovirus promoter, from a single intergenic region, so that infected cells can readily be identified in mouse tissues by the colored product resulting from cleavage of X-gal by beta-galactosidase. The glycoproteins to be deleted include, in order of priority, gB, gC, gD, gE, gI, gG, gH, and gL (some of the desired deletion mutants may be available from another project). Each of these glycoproteins has been implicated in viral binding to or entry into cultured cells of various types, or in mediating cell-to-cell spread of infection. Viral mutants with deletions of the gB, gD, gH and gL genes must be propagated on complementing cells that provide the missing gene product, which is required for replication in cultured cells. Both complemented and noncomplemented forms of virus can be tested for ability to infect mice. We expect to determine which viral glycoproteins are required for entry of virus into the apical surfaces of epithelial cells that line the vaginal cavity, which regions of the epithelium are preferentially infected, which viral glycoproteins are required for the spread of infection from the portal-of-entry cells to other cells of the epithelium and also to neurons in dorsal root ganglia and to other cells in the spinal cord and brain. These results, considered with results obtained in other planned studies in guinea pigs and in cells cultured from the human female genital tract, should allow determination of specific requirements for infection of the female genital tract; exploration of factors that can govern differences in natural history of disease and pathology caused by HSV-1 and HSV-2; and identification of mutations that would be desirable for the engineering of infectious non- pathogenic vaccine strains.