Human rotavirus egress and infection in association with extracellular vesicles

Rotaviruses (RVs) are important pediatric gastrointestinal pathogens that can be released and infect in association with extracellular vesicles (EVs). EVs are membrane-bound structures released from ‘donor’ cells that transport cargo to ‘recipient’ cells. EVs vary by size, composition, and biogenesis, and they may differ by donor cell. Virus association with EVs can enable non-lytic virus egress, shield viruses from the immune system, and promote efficient multiparticle infection. Murine RV enclosure in EVs increases the number of cells infected and the severity and duration of disease in mice compared with free RV particles. Simian RV SA11 is released from human colonic epithelial Caco-2 and HT29 cells in association with EVs. Our results suggest that Caco-2-derived EVs fail to protect SA11 from neutralization, while EVs from SA11-infected HT29 cells confer protection. SA11 visually associates with EVs from Caco-2 and HT29 cells, in some cases through external adhesion. Small and medium EVs from HT29 cells aggregate with one another and RV particles. Addition of neuraminidase, which cleaves SA11 sialic acid receptors, reduces aggregation and RV association. EV-associated SA11 in Caco-2-derived small EVs and HT29-derived large and medium EVs can enhance multiparticle RV infection of recipient cells. While it is known that enclosure within EVs can enhance RV infection, our findings suggest that RV egress, shielding, and infection properties differ by donor cell, and RV adhesion to EVs may contribute to protection and infection efficiency. Due to tropism restrictions, human RV association with EVs is poorly characterized, and it is unclear which culture models are optimal for such studies. Although more studies of RV release in EVs have been conducted using Caco-2 cells, HT29 cells more closely resemble the targets of RV infection in humans. Human H69 cholangiocytes release RV non- lytically in EVs. Human intestinal enteroids, biopsy-derived cultures that differentiate into multiple cell types, are the most biological model for human RV infection. We will use Caco-2, HT29, and H69 cells and human intestinal enteroids to test the hypothesis that association with EVs, in some cases through external adhesion, differentially influences human RV egress, protection, and infection properties in a donor cell-specific manner. In Specific Aim 1, we will elucidate contributions of EVs to human RV egress and protection. In Specific Aim 2, we will elucidate contributions of EVs to human RV infection of recipient cells. Elucidating the complex interplay between human RVs and EVs will enhance an understanding of fundamental human RV biology and inform selection of culture models to study human RV egress and infection in the laboratory. This work also may generate new hypotheses regarding human RV pathogenesis mechanisms and prevention strategies. Project Number: 1R21AI190445-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Kristen Ogden | Institution: VANDERBILT UNIVERSITY MEDICAL CENTER, NASHVILLE, TN | Award Amount: $481,250 | Activity Code: R21 | Study Section: Viral Dynamics and Transmission Study Section [VDT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19044501