1) Varicella-zoster virus (VZV) is the etiologic agent of chickenpox and herpes zoster. We have developed a system to mutate individual genes or to insert foreign DNAs in the VZV genome. These mutant viruses are being assayed for their ability to grow in cell culture or in VZV animals models. 2) Mutations have been engineered into the VZV genome that result in virus that is unable to express the viral glycoprotein V, ORF1 protein (expressed on the surface of infected cells), ORF47 protein kinase, or ORF10 protein (a transactivator). Viruses that are unable to express each of these genes grow at similar rates as the parental virus in cell culture. VZV ORF10 is the functional homolog of herpes simplex virus VP16. While VP16 is essential for replication of herpes simplex in vitro, deletion of ORF10 did not alter the growth of VZV in vitro. Comparison of phosphorylation products in cells infected with parental or ORF47 mutant VZV indicates that the ORF47 protein kinase is responsible for phosphorylation of several proteins in infected cells. Guinea pigs will be inoculated with selected VZV mutants to determine if the viruses have lost the ability to establish latency or reactivate from the central nervous system. Such viruses might be useful and candidate vaccines. 3) The E. coli beta-galactosidase gene, has been inserted into the VZV genome, and the resultant virus produces plaques that stain blue with X gal. Intraocular inoculation of guinea pigs with VZV expressing beta- galactosidase results in a chronic uveitis with mononuclear inflammatory cells in the vitreous and VZV present in the ciliary body and retina. Virus was detected in the eye at least two months after inoculation. This model should be useful for studying the role of individual viral genes during infection in vivo. 4) The herpes simplex virus (HSV) glycoprotein D (gD) gene, encoding a major neutralizing antigen, has been inserted into VZV. The resulting virus expresses HSV gD on its surface, and cells infected with the virus express gD on their cytoplasmic membrane. Inoculation of guinea pigs with VZV expressing HSV gD, followed by challenge with intravaginal HSV, resulted in production of neutralizing antibodies to HSV and in reduced severity of genital herpes. A second generation vaccine has been constructed that expresses HSV gD and glycoprotein B and will be tested in animals.
