Kylene Kehn-Hall, George Mason University. Viral infections can cause a significant change in the overall host gene expression landscape, leading to a more ...
Next Generation Sequencing for Biodefense Applications
Characterizing Host Transcriptomic Responses to Viral Infections through Next Generation RNA Sequencing Chelsea Pinkham, George Mason University Bibha Dahal, George Mason University Cynthia de la Fuente, George Mason University Brett Beitzel, United States Army Research Institute of Infectious Disease (USAMRIID) Aura Garrison, United States Army Research Institute of Infectious Disease (USAMRIID) Connie Schmaljohn, United States Army Research Institute of Infectious Disease (USAMRIID) Gustavo Palacios, United States Army Research Institute of Infectious Disease (USAMRIID) Aarthi Narayanan, George Mason University Catherine Campbell, DCE Consulting Kylene Kehn-Hall, George Mason University Viral infections can cause a significant change in the overall host gene expression landscape, leading to a more favorable environment for viral replication. Transcriptomics analysis through Next Generation RNA sequencing can be used to identify alterations in the host transcriptome in diseased states, revealing crucial pathways that are essential for the viral life cycle and/or host immunity. These datasets can be key to obtaining a bigger picture of the events occurring throughout the course of infection. Our goal is to use RNA sequencing to identify critical pathways that are important for viral pathogenesis, which can then be targeted using therapeutics or used as biomarkers of infection. Specifically, we analyzed differentially expressed genes following Rift Valley fever virus (RVFV) and Venezuelan equine encephalitis virus (VEEV) infections. Due to the presence of competent vectors and the potential for weaponization, these viruses remain a threat to the United States and the warfighter. Transcriptomics analysis was used to investigate the host response to various virulent and attenuated strains. Human small airway epithelial cells (HSAECs) were utilized in our studies of RVFV in order to mimic an infection in humans via the aerosol route, while U937 monocytes were used in VEEV infections. These cells were mock-infected or virally-infected and collected for RNA sequencing at early, mid, and late time points post infection. Fold changes of differential gene expression were calculated and Qiagen's Ingenuity Pathway Analysis was used to explore highly altered pathways. Several pathways that are critical to RVFV infection were identified that have previously been confirmed by our group, including MAPK signaling and p53 signaling. Several novel signaling pathways were upregulated at the RNA level following RVFV infection, such as calcium signaling and integrin-linked kinase signaling (ILK). Calcium signaling regulates some of the most fundamental processes, acting as a second messenger to induce signal transduction and gene expression. Calcium signaling was also highly altered in VEEV infection. Moving forward, our group has employed the use of pharmacological inhibitors to determine the significance of specific pathways in the replication of multiple viruses. Manidipine, an FDA-approved calcium antagonist used against hypertension, reduced both RVFV and VEEV titers. While our studies are focused on RVFV and VEEV, Manidipine reduced Zika virus titers as well, indicating that Manidipine could be effective across multiple virus families. In addition, ILK signaling is responsible for connecting the extracellular matrix to the actin cytoskeleton within the cell. Compound 22, an integrin-linked kinase inhibitor, reduced RVFV titers. Compound 22 also reduced viral titers in cells only pretreated with the inhibitor, indicating that ILK may be important for an early viral event, such as entry. Collectively, our data demonstrate that transcriptomic analysis can be used to identify pathways important for RVFV and VEEV replication. Furthermore, this is the first known large-scale RNA sequencing dataset acquired for RVFV, giving novel insights to viral pathogenesis.
Funding provided by Defense Threat Reduction Agency grant "Molecular Pathogenesis of Select Agent Viruses and their Attenuated Vaccine Derivatives", HDTRA1-13-1-0006, to KK and AN. We also thank Dr. Shinji Makino (University of Texas Medical Branch) and Dr. Friedemann Weber (Institute for Virology, Philipps-University Marburg) for MP12 and ZH548 reverse
genetics systems, respectively. Disclaimer: Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the U.S. Army or the Department of Defense.
