The Addition of Arachidin 1 or Arachidin 3 to Human Rotavirus-infected Cells Inhibits Viral Replication and Alters the Apoptotic Cell Death Pathway

Rotavirus (RV) infections are a leading cause of severe gastroenteritis in infants and children under the age of five. There are two vaccines available in the United States and one in India that can be administered early in childhood, however they only protect against specific strains1. From our previous work, both arachidin-1 (A1) and arachidin-3 (A3) from peanut (Arachis hypogaea) hairy root cultures significantly inhibit simian RV replication2,3,4. The purpose of this study was to determine if a human intestinal cell line, HT29.f8, infected with a human RV, Wa, was affected by A1 and A3. Cell viability assays were utilized to determine if A1 and A3 affect the HT29.f8 cells with/without RV infections. At eighteen hours post infection (hpi), supernatants from the RV-infected HT29.f8 cells with/without the arachidins were used in plaque forming assays to quantify and compare the amount of infectious RV particles that are produced during an infection. Transmission electron microscopy (TEM) was used to visualize cell ultrastructure and individual RV particles. Additionally, tunable resistive pulse sensing technology (TRPS) using the qNano system by IZON was employed to quantify and measure virus particle sizes, and display the size distribution of RV particles. Likewise, quantitative real time polymerase chain reactions (qRT-PCR) were performed to determine if A1 and A3 regulated cell death pathways in the HT29.f8 cell line. This data will guide our future studies to determine the antiviral mechanism(s) of action of A1 and A3

Arachidin-1 and Arachidin-3 Modulation of Rotavirus-infected MA104 Cells

Rotavirus (RV) causes severe life-threatening diarrhea in young children and immunocompromised individuals. There are several licensed attenuated vaccines for young children, but there are no vaccines or antiviral therapeutics for immunocompromised patients of any age. Previously, our laboratory demonstrated that arachidin 1 (A1) and arachidin 3 (A3) decreases the number of infectious simian RV particles and RV non-structural protein 4 (NSP4) in a human intestinal cell line which suggests effects on RV replication. This study examined the effects of the arachidins on the human RV (Wa)-infected African green monkey kidney cell line, MA104. The addition of either A1 or A3 did not decrease the viability of MA104 cells, however plaque forming assays measured significant decreases in the number of infectious RV particles with the addition of the arachidins. Correspondingly, western blot analyses revealed a change in the presence of VP6 and NSP4 (structural and nonstructural RV proteins, respectively). This implies that like the simian RV, Wa replication is also affected by both A1 and A3. Additionally, tunable resistive pulse sensing technology (TRPS) measured changes in the population distribution of released nanoparticles between 60-140 nanometers. Additionally, TEM morphometric analyses showed ultrastructural changed in RV-infected cells treated with A1 or A3. This included nucleus to cytoplasm ratios that were determined by TEM and whole cell fluorescent assays that disclosed significant nuclear size alterations with the addition of RV which implied modifications of the apoptosis and autophagy pathways. Moreover, the increased presence of autophagic vesicles seen with RV+A1 reinforced the model of a switch from the apoptosis to the autophagy pathway. In addition, immunoblot assays reveal the presence of cannabinoid 1 and 2 receptors on MA104 cells. These receptors bind A1 and A3 and are important in signaling in the endocannabinoid system. This implies a role for Witcher et al.: Arachidin-1 and Arachidin-3 Modulation of Rotavirus-infected MA10 2 A1 and A3 in modulating cannabinoid receptor cell signaling in RV-infected cells which indicates a mechanism of action of A1 and A3 with potential RV therapeutic activity

A Time Course Study of Rotavirus-Infected Intestinal Cells Treated with Stilbenoids and the Regulation of Apoptosis

This is a time course study of virus –host interactions that are modified with the addition of two small natural products. They appear to effect virus replication and the host response to the infection

Remote Antarctic feeding ground important for east Australian humpback whales

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