Regulation of the Transcriptome of Rotavirus-Infected HT29.f8 cells by Arachidin-3
Hannah N Lockwood
The leading cause of severe diarrhea in infants and young children worldwide is rotavirus (RV) infections that can lead to dehydration and death. Although vaccines are available, some are cost prohibitive in emerging countries, and the efficacies are dependent on timing of vaccination and RV strain specificity. Continuing surveillance for newly emerging RV strains, assessment of vaccine efficacies, and development of cost effective antiviral drugs remain important strategies for the prevention of RV pathology. Previously, our laboratory has determined the antiviral activity of a stilbenoid, trans-arachidin-3 (t-A3), on inhibiting RV replication. The objective of this study was to determine host genes that are regulated in a RV-infected human intestinal cell line (HT29.f8) treated with t-A3. Microarray analyses provided insights into the regulation of the transcriptome of HT29.f8 cells at eight hours post RV/RV+t-A3 infections. Quantitative real-time PCR (qRT-PCR) validated the microarray experiments. One of the findings of this study demonstrates that the intrinsic apoptotic pathway that leads to cell death is activated with a RV infection.
Quantification of Rotavirus Particles in HT29.f8 cells treated with t-A1 and t-A3
Caleb M Witcher
Rotaviruses (RV) cause life-threatening gastroenteritis in children worldwide; the enormous disease burden has focused efforts to develop vaccines and led to the discovery of novel mechanisms of gastrointestinal virus pathogenesis and host responses to infection. Although the molecular mechanism(s) for the protective effect of trans-arachidin-1 and 3 (t-A1 and t-A3) are not known, the inhibition of viral replication could be attributed to the anti-oxidative and anti-inflammatory properties of the constituent stilbenoids. The objective of this study is to characterize virus particles by quantifying virus particles and determining structural differentiation during a RV infection in HT29.F8 cells, treated with/without t-A1 and t-A3. Three methods will be used in order to characterize the RV particles treated with t-A1 and t-A3. The first method used is verification via plaque assays. The second method will be the use of transmission electron microscopy. The last method is the use of tunable resistive pulse sensing technology with the use of the qNano by IZON. All of the data recorded by these techniques will be focused on the effects of t-A1 and t-A3 in addition to a rotavirus infection and will be verified by each other for accuracy and precision. The information gained from this study will help determine how t-A1 and t-A3 effect the maturation of infectious virus particles. Thus, these compounds potentially could be used to design and develop more efficacious RV therapeutic agents.
A Two-Dimensional gel proteomics analyses of a rotavirus-infected human intestinal cell line treated with a stillbenoid compound
Previously, our laboratory has shown stilbenoid compounds purified from peanut (A. hypogaea) hairy root cultures significantly decreases viral progeny numbers when added to a RV-infected human intestinal cell line, HT29.f8. Additionally, the decrease in the nonstructural RV protein, NSP4, observed in western blots, indicates the virus replication is affected by the stillbenoids. This study used a proteomic approach to identify and compare the protein expresssion profiles of a reference sample (uninfected) to RV-infected and RV-infected and stilbenoid-treated HT29.f8 cells using two-dimensional gel analyses. These data show which groups of cellular proteins are affected by an RV infection, and how they are altered with the addition of a plant natural product that exhibits antiviral activity. Using the reference sample, adjustments were made for the first dimensional isoelectric focusing gels (IEF) to resolve the pH range. The second dimensional gels, SDS PAGE, were employed to further isolate the cellular proteins by their mass. Likewise, these parameters were used with the two experimental groups, and the data were compared to distinguish differential protein expression patterns. Further testing is in progress to identify these proteins. This will advance the understanding of the cellular response mechanisms that are important in inhibiting a RV infection, and support the development of the stilbenoids as a potential RV therapeutic agent
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