Miles Murri and Dr. Brian D. Poole, Microbiology and Molecular Biology
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For the last year I have been working on a project that would test the efficacy of a potential drug on a common human virus called Epstein-Barr Virus (EBV). Though the virus often resides in a latent or dormant state, it is commonly found in nearly all adults worldwide (1). When it is active, its upregulated gene expression leads to conditions ranging from ear infections and tonsillitis to highly serious conditions like lymphomas (1). It has also been linked substantially with multiple autoimmune diseases such as Lupus (2). A certain immunosuppressive plant extract called Kalanchoe has been used traditionally as an herbal medicine and is seen to have an anti-proliferative effect on cancer cells in the body (3). My aim has been to show that Kalanchoe pinnata, the extract used in the experiments, does inhibit EBV gene expression.
The results have been interesting. What I’ve found so far is that on a certain type of cell line that we have been using, a lymphoblastoid cell line, viral gene expression was actually increased due to the Kalanchoe extract. In fact, in the viral genes, BZLF-1, EBER-1, LMP-1, LMP-2, and EBNA-1, all of which are normally expressed as a result of EBV infection, the average change in gene expression from our control values was 91.2 times more. So some of the genes that are normally expressed in EBV-transformed B cells were found to be expressing at levels over 100 times more than the normal values obtained for those transfected B cells.
While this finding was somewhat striking to me, it does not necessarily mean that our finding is invaluable or incorrect. In fact, the opposite may be true. While it is apparent that EBV gene expression is actually increased instead of being downregulated, this might be the way that it suppresses Epstein-Barr Virus flare-ups. EBV has a way of hiding in the host and is difficult to detect by the immune system. By activating gene transcription to a significantly higher level, it may be that the extract is able expose the virus more to the body’s immune system and activate a response against the cells infected with EBV. This would ultimately lead to the decreased spread of the virus in the body and a decrease in the resulting symptoms caused by the viral flare-up.
Getting to this point where we could begin getting results was actually quite difficult and tedious. With the help of my professor and another graduate student, we had to run Polymerase Chain Reaction (PCR) experiments on multiple cell lines to find those that would express the genes like we needed them to. For a while, none of them seemed to be cooperating and expressing like we wanted. I had to also run many dilution experiments to find at which ratio our stock EBV virus would infect half the cells and leave half uninfected, so as to accurately determine the dilution to use in the real experiments for accurate infection. We found this value to be about 1:100. Next, I had to run Kalanchoe toxicity tests to the cells to make sure I didn’t put too much on our culture that would kill them. This way, I could suspend the cells in the appropriate medium with the extract, and then infect them with EBV and observe the trends. This Kalanchoe dilution ratio was found to be 1:10. Much of the time spent initially was in preparing for the actual experiments that would give us the necessary results.
What a great experience this research has been. I have learned a lot about the specific focus of our studies in the lab and especially about EBV and how to test for it in the body. I have become more familiar with tests run in the lab and how to perform them. Though we have found that Kalanchoe does not inhibit EBV gene expression, it may inhibit EBV itself in the body by causing a stronger immune response. To see if this is the actual mode of immunosuppression, I will continue to study this correlation. This most likely will involve the use of mice in the lab. More questions have opened up, and more avenues for research are clear in my search for a deeper understanding of this experiment. I am excited to see what more I can find and hope to publish a paper on my results as I continue to research.
Sources
- Medicine Net. “The Broad Spectrum of Epstein-Barr Virus”. 2008. October 26, 2009 http://www.medicinenet.com/script/main/art.asp?articlekey=89105>.
- Harley JB, James JA. “Epstein-Barr virus infection induces lupus autoimmunity.” Bull NYU Hosp Jt Dis. 2006; 64(1-2): 45-50. Review.
- Supratman U, Fujita T, Akiyama K, Ohigashi H, et al. “Anti-tumor Promoting Activity of Bufadienolides from Kalanchoe Pinnata and K. Daigremontiana*tubiflora.” Biosci. Biotechnol. Biochem. 65.4. (2001): 947-949.