Rheumatology Research Laboratory

Basic Science Research
Herpes is familiar because of cold sores and genital lesions caused by herpes simplex virus. But herpes is more than that. Herpesviridae is a large family of viruses, including Epstein-Barr virus, cytomegalovirus and kaposi’s sarcoma-associated herpesvirus that cause diseases ranging from a mild rash to cancer.

All humans are infected with multiple herpesviruses by adulthood. However, unlike most viruses, herpesviruses are never cleared—that is, the immune system never eliminates these viruses from the body. Rather, they persist in certain cells for the life of the host. Most of the time there are no symptoms to reveal the underlying infection. During this period the virus is in a state of relative dormancy called latency. Latency, however, is frequently punctuated later in life by viral reactivation. Reactivation, unfortunately, is frequently associated with severe disease.

All humans are infected with multiple herpesviruses by adulthood. Unlike most viruses, herpesviruses are never cleared—that is, the immune system never eliminates these viruses from the body. They persist in certain cells for the life of the host. Most of the time there are no symptoms to reveal the underlying infection. During this period the virus is in a state of latency, however, later in life it can be punctuated by viral reactivation which is frequently associated with severe disease.

For decades, it was assumed that latency was detrimental to the health of the host because it puts the host at risk for reactivation. Interestingly, we’ve discovered that herpesvirus latency in mice activates the host innate immune system (macrophages and natural killer cells) and thereby provides a potential benefit by increasing the host's resistance against bacteria and tumors.

Our current research aims to understand how, at a molecular level, herpesviruses interact with the host immune system during latency. This work involves the generation and testing of new viral mutants for their ability to alter the host immune system. These studies are pursued in collaboration with Drs. Linda van Dyk and Eric Clambley at the University of Colorado, Laurie Krug at Stony Brook University and Craig Forrest at the University of Arkansas.

Specializing in bacterial artificial chromosome (BAC) mutagenesis
The ability to clone herpesvirus genomes into a bacterial artificial chromosome (BAC) has improved the efficiency and fidelity of targeted mutations in these genomes. Over the past several years, new technologies have helped to streamline and improve our mutagenesis strategies. These new strategies were pioneered by Darby Oldenburg, PhD, at Gundersen Medical Foundation. She has more than 5 years’ experience creating and characterizing various herpesvirus mutants for use in tissue culture and in vivo studies.

We use a combination of methods to create site-specific mutations (e.g. base-change, insertions, deletions, tagging and others) for DNA contained in BACs. We have successfully inserted up to 10kb of heterologous DNA into the MHV68 genome.

The turnaround time (TAT) for a basic mutation such as a small insertion/deletion (less than 1kb) or DNA base substitution is about three weeks. This includes sequence analysis of the mutated locus/loci and a three-enzyme restriction fragment length polymorphism (RFLP). The mutated virus is then transferred to a stable E. coli strain for long-term storage.

Our method provides robust, rapid and reliable mutations. Our improved techniques have been used by several MHV68 researchers including our collaborators.

The types of mutants that we have successfully created include:

  • Specific base pair changes
  • Deletion of open reading frames (ORFs)
  • Insertion of various TAGs, reporter genes (e.g. green fluorescent proteins, luciferase)
  • Deletion and insertion of ORFs into specific sites within the genome

We are always interested in new collaborative projects with researchers in the herpesvirus community who would like to have virus mutants created and characterized using our improved techniques.

Human Studies
In addition to the basic science studies described above, Gundersen Medical Foundation researchers participate in human studies through the CORRONA database and an investigator-initiated study measuring the prognostic value of a novel blood test (called the interferon signature) in patients with autoantibodies in their blood (antinuclear antibodies or ANA).

Some patients with a positive ANA go on to develop systemic lupus erythematosus or another autoimmune disease, yet many remain healthy. Our hope is that this new blood test will help clinicians distinguish between these two possibilities. This study, which enrolled its first subjects from Gundersen Health System in August of 2010, is in collaboration with Dr. Erik Peterson at the University of Minnesota.


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