1. Danielle V. Reenstra York College of Pennsylvania, Department of Biological Sciences The Interferon Response to Epstein-Barr Virus Virus containing supernatant was prepared from B95-8 cell line PBMCS were isolated from healthy individuals 1:2 dilution of virus incubated with samples for various times Prepared RNA and cDNA Performed qrtPCR for type I and type II IFN Samantha K. Dunmire 1, Oludare A. Odumade 1, Eliana Coccia 2, Kristin A. Hogquist 1 Carolyn Mathur 3, and David Singleton 3 1 Lab. Medicine and Pathology, University of Minnesota, Minneapolis, MN 2 Istituto Superiore di Sanità, Milan Italy 3 3 York College of Pennsylvania Image from http://www.bio-rad.com Funding provided by the Life Sciences Summer Undergraduate Research Programs at the University of Minnesota. Epstein-Barr Virus (EBV) of the Herpesviridae family, causes chronic infection in approximately 90% of the world population and is associated with cancer and autoimmunity. The purpose of this study was to determine the cell types producing a type I interferon (IFN-α and IFN-β) response to EBV. Type I IFN is highly important for inducing antiviral immune responses. Peripheral blood mononuclear cell (PBMC) samples from healthy individuals were depleted of plasmacytoid dendritic cells (pDC) and/or monocytes. Samples were then stimulated with EBV or control stimuli and left to incubate. The interferon regulated genes (IRG) of each sample were measured via quantitative real-time PCR (qrtPCR). Preliminary data showed pDC and monocyte depletions had minimal effect on IRG expression. These results suggest that while pDCs and monocytes may lead to an IFN response, there are likely other cells involved, such as B cells. Results I: EBV can stimulate a robust IFN response in human PBMC. Multiple cell types are likely involved in the IFN response such as pDCs, monocytes, and other cells The viral supernatants produced a transient response to type I and type II IFN. This may be due to sub-optimal virus preparation. Figure 3 Of 43 genes initially analyzed, 5 were selected for further study in titration and time course analysis. Three of these were induced by all stimuli (IFI27, MX1, and OTOF). Two (CXCL9 and IFNG) were stimulated primarily by EBV. These genes were then studied further in a time course. IRG Expression in Response to Stimuli Harvested PBMCs from healthy donor Depleted pDCs, monocytes or both Cells were incubated with EBV or TLR7 or TLR9 agonists for 18 hours Prepared RNA and cDNA Performed qrtPCR on 43 genes) Results II: Figure 2 The fold changes of 43 genes were compared between depleted and non depleted samples for all stimuli. Only combined depletion with EBV stimulation is shown. Interpretation: A strong type I IFN response was induced by EBV (similar to TLR7/9 stimulation controls—data not shown). Neither pDC, monocyte, or combined depletion of samples had a major effect on IFN response induced by EBV. Figure 4 Viral supernatants were prepared and incubated with PBMC for various periods of time. qrtPCR performed the genes IFNG and IFNB are shown. Interpretation: The viral supernatants we prepared stimulated the highest IFN response (type I and type II) at the 3 hour time point. This may be because the virus preparation was sub-optimal (we did not determine the titre of viable virus). Figure 1 There are multiple mechanisms by which EBV may stimulate an interferon response. If EBV is exposed to monocytes, it may signal through TLR2 to generate type I IFN production. Through pDCs, viral DNA may bind to TLR9, leading to type I IFN production. EBV gp350 infects B cell through CD21. In infected B cells, EBV-encoded RNAs (EBER) are synthesized, which bind to RIG-I, which leads to type I IFN production. EBV is a double stranded DNA virus. The primary mode of transmission is through saliva. EBV causes infectious mononucleosis and is linked to autoimmunity and cancer (Odumade, et al, 2011). EBV directly infects epithelial cells in the tonsil and B cells in the blood (Savard, 2000). IFNs are important in creating an antiviral response. IFNs can be produced by any cell (Sen, 2001). Possible Ways EBV Could Stimulate an Interferon Response Requires exposure to virusRequires productive infection TLR2 TLR9 IFN α/β CD21 gp350 MonocytepDC EBER RIG-I IFN α/β DNA IFN αβ B cell The University of Minnesota is getting an EBV viral strain that encodes green fluorescent protein. This will facilitate both viral production and testing. This virus will be used in future experiments with sorted PBMC populations to determine which cell types respond to EBV. AbstractAbstract BackgroundBackground Methods I Methods II Future Directions ConclusionsConclusions Literature Cited AcknowledgementsAcknowledgements Odumade, O., Hogquist, K., Balfour Jr, H. 2011. Progress and Problems in Understanding and Managing Primary Epstein-Barr Virus Infections. Clinical Microbiology Reviews 24(1):193-209. Savard, M., Celanger, C., Tardif, M., Gourde, P., Flamand, L., Gosselin, J. 2000. Infection of Primary Human Monocytes by Epstein-Barr Virus. Journal of Virology 74(6):2612-2619. Sen, G. 2001. Viruses and Interferons. Annual Review of Microbiology. 55:255-281.