Inhibition of Epstein-Barr virus infection by Kalanchoe pinnata extract

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Lance A. Stutz and Dr. Brian Poole, Microbiology and Molecular Biology

Introduction

Epstein-Barr virus (EBV) is a common infectious agent which infects more than 95% of adults worldwide (1). EBV is related to a number of diseases, including cancers such as Burkitt’s lymphoma and nasopharyngeal carcinoma, and is the causative agent of infectious mononucleosis (2). Upon infection, EBV invades antibody-producing immune cells known as B cells. Most people do not show signs or symptoms of the infection because EBV tends to hide in B cells in a latent state. Latent EBV infection contributes to the development of EBV-associated cancers. However, EBV can also be in an active or lytic state. In this state, EBV infects and then destroys B cells. Such damage to the immune system is often made manifest as prolonged fatigue, which is the main symptom of mononucleosis. The immune system is usually able to overcome an active infection after a period of time, but no drug is known to effectively inhibit EBV in vivo.

In a recent experiment by Greer et al., Kalanchoe pinnata acetone extract was found to have antiviral activity against herpes simplex virus types 1 and 2 (3). K. pinnata grows in temperate climates such as India and South America, and is commonly used in traditional medicine as an anti-inflammatory and antiseptic treatment for coughs, ulcers, and sores (4). As EBV is also a member of the human herpesvirus family, K. pinnata extract is a prime candidate to provide antiviral treatment for mononucleosis and EBV-associated cancers.

Materials and Methods

Using conventional techniques in molecular biology, we sought to assess the ability of K. pinnata extract to inhibit EBV infection. First, we determined the toxicity of K. pinnata extract (obtained from Dr. Brent Johnson at Brigham Young University) by varying the concentration of extract in duplicate samples of Ramos cells—an EBV-negative B cell line. The number of living cells in each sample was counted using a flow cytometer.

After determining the optimal concentration of extract for use in future experiments, we sought to produce an effective method to measure EBV infection. We obtained a bacterial artificial chromosome (BAC) from Dr. Wolfgang Hammerschmidt at the National Research Center for Environment and Health in Munich, Germany, which contains the EBV genome with the addition of a gene for green fluorescent protein (GFP). Using this BAC, we transfected Human Embryonic Kidney (HEK) 293 cells via calcium phosphate precipitation and selected for transfected cells with the antibiotic hygromycin. After allowing the transfected cells to grow for several weeks, we extracted supernatant from the cells using a sterile filter and measured the quantity of our viral supernatant using qPCR. Thus, EBV infection could be detected by viewing cells under ultraviolet light or with the laser of a flow cytometer. Finally, we infected several B cell lines, including Raji, Ramos, and primary B cells, with our viral stock. Certain samples of the Ramos cells were also treated with K. pinnata extract.

Results

K. pinnata extract is toxic to cells at high concentrations. The optimal concentration of the extract for our infection assays was determined to be 2.5 μg/mL. We produced a stable transfected HEK 293 cell line which produced GFP-EBV. Green fluorescence in these cells was easily detected when viewed under ultraviolet light (see Figure 1). The concentration of our viral supernatant was calculated to be 5×108 virions/mL. Differences in the amount of green fluorescence between infected and uninfected B cells could not be detected qualitatively using an ultraviolet microscope or quantitatively using a flow cytometer. Hence, the effect of K. pinnata extract on EBV infection could not be determined from our experiments.

Discussion and Conclusion

The production of infectious GFP-EBV proved to be a difficult task. Although we were able to establish a stable cell line which produces these genetically modified virions, the viral supernatant we obtained was either not concentrated enough or contained very few particles that were actually infectious. Upon further review of Dr. Hammerschmidt’s publication on GFP-EBV (5), we noticed that he stimulated viral production in GFP-EBV HEK 293 cells by transfecting them with a plasmid containing an EBV gene called BZLF1. This gene activates the lytic stage of the virus and would produce higher concentrations of viral particles in the cell supernatant. Our future infection assays will include the use of a BZLF1 plasmid and the treatment of Raji, Ramos, and primary B cells with K. pinnata extract.

In retrospect, we should have more carefully reviewed published scientific literature before beginning the process of producing GFP-EBV and more rigorously tested our viral supernatant before performing experiments with K. pinnata extract. Due to obstacles in generating an effective measure for EBV infection, we were not able to accurately test the ability of K. pinnata extract to inhibit EBV infection. However, this project provided many opportunities to learn new techniques in molecular biology and to acquire new skills in research and planning methods which will be valuable in our future research pursuits.
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References

  1. Luzuriaga K., & Sullivan J. L. (2010). Infectious Mononucleosis. New England Journal of Medicine, 362, 1993-2000.
  2. Pattle S. B., & Farrell P. J. (2006). The role of Epstein–Barr virus in cancer. Expert Opinion on Biological Therapy, 6(11), 1193-1205.
  3. Greer M. R. J., Cates R. G., Johnson B. F., Lamnaouer D., & Ohai L. (2010). Activity of acetone and methanol extracts from thirty-one medicinal plant species against herpes simplex virus types 1 and 2. Pharmaceutical Biology, 48(9), 1031–1037.
  4. El Abdellaoui S., Destandau E., Toribio A., Elfakir C., Lafosse M., Renimel I., André P., Cancellieri P., & Landemarre L. (2010). Bioactive molecules in Kalanchoe pinnata leaves: Extraction, purification, and identification. Analytical and Bioanalytical Chemistry, 398(3), 1329-1338.
  5. Delecluse H.J., Hilsendegen T., Pich D., Zeidler R., & Hammerschmidt W. (1998). Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells. Proceedings of the National Academy of Sciences, 95(14), 8245-50.