Human cytomegalovirus (HCMV) is the most common cause of congenital infection as well as the most common opportunistic viral infection in immunocompromised patients. As cytomegaloviruses exhibit species specificity, only human tissues may be infected by HCMV; therefore, direct laboratory study has been limited. A strain of severely immunodeficient mice that can support grafts of human tissue (SCID-hu mice) has recently been tested as a manipulable animal model for HCMV infection. Initial studies revealed that the ability to persistently replicate in tissue was not shared by all strains of HCMV tested. Using the SClD-hu mouse model the determine the genetic basis of the ability of HCMV to persistently replicate in human tissues and vital tissue tropism will be studied. A clinical isolate (Toledo) which is able to persistently replicate and laboratory-passaged stains AD169 and Towne which do not possess this ability will be studied In phase I, human fetal thymic epithelial cell cultures and a graft of fetal thymus/liver into the SCID mouse will be used to biologically characterize tissue tropism and the level of the block in AD169 replication. Specific murine monoclonal antibodies in immunofluorescence studies and lacZ+ recombinant viruses will be used to localize known viral proteins and determine the stage of viral infection at which the block occurs. Polyclonal antisera against Toledo and AD169 will be raised and antibody-viral protein reactivity profiles will be analyzed. In Phase II, the genetic determinants of persistent replication will be identified by a number of available approaches including the creation of Toledo-lacZ+ recombinant viruses possessing the growth phenotype, Cotransfection of AD169 DNA with cosmid clones of Toledo, restriction length and fragment polymorphism analysis, and genomic difference cloning. The gene product(s) will be further characterized by evaluation of sequence homology, RNA transcript analysis, and the use of indirect immunofluorescence with specific antibody for subcellular localization of the vital protein. The further characterizations of (l) the determinants of the ability of HCMV to persistently replicate and (2) the SClD-hu mouse as a model for natural human infection will add significant insight into the pathophysiology of this infection in the hopes of devising new therapeutic agents.
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