Molecular Biology/animal models of herpesviruses Varicella zoster virus and Herpes simplex virus type 1

Description of projects available to graduate students:
The VZV projects
VZV causes severe pain (post herpetic neuralgia) when it reactivates from a neuronal latent state established during primary infection, chickenpox. Even though there are vaccines, VZV induced pain will remain a major human problem in need of better treatment. Our group is one of only a few that study the biology of VZV (since it is difficult to work with). We are currenty developing a model of VZV induced pain in the rat, aiming to shed light on why VZV induces pain and find better ways to treat it. The VZV projects are done in collaboration with Dr Bill Goins.

Project 1 is discovery driven and aims to determine why VZV causes pain in the rat. Does the virus go latent in the DRG ? Does it express specific genes/proteins in the dorsal root ganglia (DRG) showing pain? Is there an inflammatory response that correlates with pain and recovery from it? This project involves optimizing real time quantitative PCR detection methods, and using immunological/histochemical detection and flow cytometry of (DRG) of rats with VZV induced pain to study how VZV gene expression/immunity correlates with pain and recovery.

Project 2 is to identify if specific VZV genes are required to induce the pain in rats. Can a VZV mutant be created that does not induce pain? Can expression of one or more VZV genes alone induce pain? This requires development VZV mutants lacking viral genes, using recombineering methods, and the development of HSV expressing VZV genes: followed by their assessment to induce pain in the rat. Viral protein functions of specific viral genes associated with pain development are examined.

The HSV-1 projects
When HSV -1 reactivates from latency, it triggers a blinding disease known as stromal keratitis. The overarching goal is to seek to block reactivation through the manipulation of the CD8 T cell immune response at the trigeminal ganglia (TG), the site of latency, in the C57Bl6 mouse model of ocular infection. The CD8 T cells are dominant to a single epitope on the viral protein gB. The HSV projects are done in collaboration with an immunologist, Dr Robert Hendricks.

Project 1 will determine the nature of the TG associated CD8 T cell infiltrate in the absence of the major immunodominant gB epitope. What new viral antigens are CD8 T cells in the TG directed to? Can they still block reactivation? This will involve recombinant virus construction, characterizeration in the mouse model, and CD8 T cell immunology by flow cytometry and antigen peptide library screening.

Project 2 is to determine how the kinetics of viral gene expression influence the phenotype and specificity of CD8 T cell infiltrate in the TG. Does expressing a viral antigen late change the ability of CD8 T cells to infiltrate the TG? Does expressing the antigen continuously during latency boost the CD8 T cell infiltrate and afford better protection? Does expressing more of the epitope increase the infiltrate? This involves virus development in which the epitope is expressed from different viral promoters.

Techniques graduate student will learn:
The lab uses molecular biology/immunology methods in conjunction with animal models of human disease. The VZV projects are molecular biology/detection based, and involve recombineering and virus mutant construction, cloning, protein expression, cell culture and rat animal studies, in conjunction with PCR detection methods and immuno detection.
The HSV-1 projects are more immunology/flow cytometry based with molecular characterization of recombinant viruses and their assessment in mice. Cell and virus culture, recombinant virus generation and manipulation, cloning, protein expression, PCR, immunodetection, virus characterization. Animal studies in mice and/or rats.