Project Summary/Abstract Rotavirus and norovirus are well-known to lead to serious gastroenteritis in humans and great public health concerns. Both viruses are environmentally persistent, and they can be transmitted via direct contact with an infected person or contaminated surfaces, as well as polluted water and food. Disinfection can inactivate these persistent viruses and prevent infectious disease outbreaks. Almost all previous studies consider rotavirus and norovirus as individual, free viral particles in biomedical research, until a recent finding demonstrated that rotavirus and norovirus could transmit in vitro as viral clusters inside extracellular vesicles that have a lipid-rich membrane structure, i.e., vesicle-cloaked virus clusters (viral vesicles for simplicity). Vesicle-cloaked rotavirus and norovirus clusters are important emerging pathogens, because they are environmentally stable, widely present, and more infectious than their free viral particle counterpart; however, their inactivation by current disinfection strategies is unknown. Moreover, disinfection mechanisms are largely unclear, which prevents us from rational design of effective, robust, broad-spectrum, and low-cost disinfection processes. This project seeks to evaluate the performance of conventional disinfection strategies for inactivating vesicle-cloaked rotavirus and norovirus clusters, elucidate the mechanisms of viral vesicle inactivation, and develop new disinfection processes for effective viral vesicle inactivation. Our central hypothesis is that the vesicle-cloaked virus clusters are more persistent in disinfection compared to their counterpart of free viruses, and disinfection that damages multiple biological components in viral vesicles is more effective. Specific Aim 1 will evaluate the inactivation of vesicle-cloaked virus clusters by conventional disinfectants, i.e., bleach, ultraviolet light (monochromatic UV 254 nm), a surfactant (didecyldimethylammonium chloride), ethanol, and their combination. The dosage of disinfectants and their performance in disinfection will be determined to generate a dose-response relationship, which will be compared with that for free viruses. Specific Aim 2 will elucidate the mechanisms of inactivating vesicle-cloaked virus clusters by conventional disinfectants to understand the key factor that determines disinfection performance. The damage of biological components of viral vesicles and the impact on viral vesicles’ lifecycle in the host cells after disinfection will be evaluated. Specific Aim 3 will evaluate emerging disinfectants, i.e., polychromatic UV irradiation and activated persulfate, for inactivating vesicle-cloaked virus clusters. Both disinfection performance and mechanisms will be explored. Our study will shed light on the need for optimizing current disinfection paradigm and developing new disinfection strategies for rotavirus and norovirus, and it has a broader health impact because research outcomes can be tr...