Human herpesviruses cause a variety of diseases which have a significant impact on the health of the nation. Varicella-zoster virus causes chickenpox in nearly all Americans, and when it reactivates from latency, causes shingles in approximately 15% of the population. Herpes simplex virus causes oral and genital herpes, and can cause severe, fatal infections, especially in neonates and the immunocompromised. Currently, over 100,000 Cesarean sections are performed annually in the U.S. to prevent neonatal herpes. Products which influence the course of these viral infections will have a dramatic impact on the health of the nation, and in particular, on women's health. This project addresses specific manufacturing, purity, and safety-related problems which are unique to herpesvirus based biological products. 1) These viruses are the only ones to establish latency and reactivate, thus presenting safety concerns with respect to reactivation of live vaccines (as has been shown to occur with live varicella vaccine). Molecular biological assays to evaluate the influence of vaccines and drugs used in herpesvirus infections on the establishment and maintenance of viral latency are being developed, including polymerase chain reaction based assays and the construction of mutant viruses which express marker genes. Using herpes simplex virus as a model, this project is using molecular virology techniques to identify the genes involved in latency and reactivation, and appropriate methods for attenuating these viruses for use as vaccines and gene delivery vectors are being studied. The latency-associated transcript has been identified as the critical determinant of site-specific viral reactivation. 2) Herpesviruses are capable of providing helper function to other viruses (e.g. adeno-associated viruses [AAVs]) which are not detected by currently used cell substrate testing techniques. As no well standardized test is in place to test for potential contamination of vaccines, another goal of this project is to identify means by which vaccines can be tested for such agents. A superinfection assay to detect AAVs in cell substrates and biological products is being developed. Varicella-zoster virus was shown to provide full helper function to AAV. 3) Herpesviruses may play a role as vectors in neuronal gene therapy or intracellular immunization. Techniques for attenuation of these vectors to eliminate potential rescue of infectious virus or damage to cells are being studied. Attenuated HSV strains are being further mutated, and their ability to deliver foreign genes and antisense RNAs to cells in tissue culture and animals are being tested.
