Herpes simplex virus (HSV) is the leading infectious cause of corneal blindness in the United States. Although there have been some advances in the development of drugs to treat ocular disease induced by this pathogen, current therapy for most forms of herpetic infection is inadequate. More basic research is need, particularly at the molecular level, to elucidate mechanisms involved in the infection process. It is well known that strains of HSV vary remarkably in their capacity to induce corneal disease, and recent work indicates that the attenuation or virulence of any given strain relates to its genetic makeup. Advances in biotechnology now make it possible to design studies to analyze the molecular basis for HSV virulence. The specific goal is to identify the gene(s) and gene product(s) that distinguishes a virulent from an avirulent HSV strain with respect to induction of corneal disease. Our preliminary studies have established that HSV-2 strains 333 and 186 replicate and induce pathology in the Balb/c cornea whereas HSV-1 strain 35 does neither. Intertypic recombinants will be constructed by transfecting restriction endonuclease digested HSV-2 DNA and intact strain 35 DNA into rabbit kidney cells. The recombinants generated will be selected for in vivo, or via marker rescue. The DNA from plaque purified progeny will be subjected to restriction endonuclease analysis to map the HSV-2 DNA fragment(s) incorporated into HSV-1 strain 35 DNA. Each recombinant will be inoculated onto the scarified murine cornea to determine its replicative and pathogenic potential. Fine mapping of the HSV-2 DNA fragment initially inserted into strain 35 DNA will be done in order to identify the smallest type 2 DNA fragment needed to bestow virulence. Finally, a corneal epithelium culture model will be used to probe for the function or product coded for by the HSV virulence gene(s). The experiments will produce a series of intertypic recombinants of defined genetic composition. Because of their varied genetic makeup, the recombinants will vary in their ability to replicate in corneal cells and produce disease. By correlating genetic structure with in vivo virulence, it will be possible to delineate the molecular basis for HSV virulence in the cornea.
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