Viral Latency and Virus Detection

Investigators

Abstract

A major focus of this program is the development of methods to detect viruses in biological samples. This includes specific methods to detect individual viruses, non-specific methods that could be used to detect a broad array of viruses, and more sensitive methods that could be used to screen large quantities of vaccine, cell substrate, or raw materials. This challenging project has numerous important public health implications, ranging from assurance of vaccine safety (i.e., absence of adventitious agents) to identifying pathogenic viruses in clinical samples. We also are investigating the pathogenesis of viral latency, in particular as it relates to alpha-herpesviruses. Our goal is to understand molecular mechanisms in viral latency and reactivation, with particular reference to relevant vaccine safety and efficacy issues. Most of our studies have involved attempting to understand the role of the latency-associated transcripts (LAT) in HSV-2 latency. Our primary laboratory-based approach is to test our hypotheses by constructing and evaluating mutant viruses. Analysis of vaccines for the presence of adventitious viral agents. Vaccines are among the most cost-effective public health measures available today. Because many vaccines are administered to all healthy children, and development of herd immunity is an important contributor to their effectiveness, it is important for the public to have confidence that vaccines are safe. It is thus very important from a public health perspective to ensure that administered vaccines are free of known adventitious agents. The goal of this project is to develop new tests that may be reliably used to test vaccines for potential adventitious agents. Most current vaccine testing relies on highly sensitive tissue culture assays. Newer molecular techniques have not in general been adapted for this kind of testing, in part because of the high reliability and sensitivity of the tissue culture assays. However, these molecular techniques have the potential to detect agents that theoretically could yield negative tissue culture results. Thus, new molecular techniques are being used to develop assays that could detect potential viral adventitious agents. Polymerase chain reactions were used to screen vaccine lots for the presence of simian cytomegalovirus (SCMV) DNA. SCMV DNA was found in some lots of oral poliovirus vaccine. However, viable SCMV could not be cultured from any vaccine lot. Improved tissue culture methods for detection of SCMV were investigated. A framework for using the results of laboratory studies to assess adventitious agent risk in a regulatory setting was developed. This project also focused on development of non-specific assays, that detect adventitious viral nucleic acid based on specific features of contaminating viruses, without a clear need to know the DNA or RNA sequence of the virus being detected. The current approach is to purify viral nucleic acids from biological samples and to use non-specific PCR methods to amplify those viral nucleic acids. These approaches are shown to be sensitive and very non-specific in nature. Continuing work will address improvement in sensitivity and application of these assays to relevant samples, as well as investigation of additional approaches to non-specific detection of viruses. A third approach is to develop nanotechnology approaches for detecting very small quantities of viruses or viral nucleic acids. We are working on developing devices that could potentially detect single copies of analytes of interest. Safety of live vaccines that establish life-long, persistent infections. This project addresses the safety of herpesvirus vaccines as follows: 1) study of the mechanisms by which herpesviruses establish latency and reactivate. Studies of the herpes simplex virus LAT regions are defining the specific viral genetic elements that direct the establishment and maintenance of, and reactivation from, latency. Information from these studies is relevant to development of safe herpesvirus vaccines and vectors. In addition, methods are developed to assess the ability of a vaccine or other intervention to influence the establishment of viral latency. These methods are based on quantitative polymerase chain reaction and on the construction of viral mutants that express reporter genes during latency, enabling the easy detection of latent virus. 2) development of assays to clearly differentiate vaccine strain and wild-type chickenpox strains. An assay was developed by sequencing of the vaccine strain genome, and identification of specific differences. This assay correctly identifies VZV strains as vaccine or non-vaccine in 100% of test cases. Availability of improved assays will enable more reliable assessment of potential human to human transmission of vaccine strain, and of vaccine strain reactivation in humans. Identification of these differences may also yield important information regarding the basis for attenuation of the vaccine strain, providing a basis for the performance of additional important studies. As a component of these studies, we also have been examining rapid methods to evaluate the antiviral susceptibility or resistance of vaccine strain viruses. 3) Study and analysis of data related to the chickenpox vaccine to identify safety concerns. New methods to analyze clinical data related to duration of vaccine response and to the ability of the vaccine strain to reactivate are being developed and used to predict the long term efficacy and safety of the vaccine. This project incorporates FY2002 projects 1Z01BK005011-05 and 1Z01BK005012-05.