Collaborative Research: Presence, Persistence, and Inactivation of Vesicle-Cloaked Rotavirus or Norovirus Clusters in Water

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Rotavirus and norovirus are major causes of waterborne diseases worldwide. In 2018 it was discovered that both of these enteric viruses are often found in small fluid-filled sacs called vesicles. These vesicle virus clusters are widely found in stool and have been detected in wastewater. Rotavirus and norovirus vesicles are more persistent, infectious, and resistant to disinfection compared to non-vesicle viral particles. In spite of these concerning findings, relatively limited research on the environmental behavior and fate of enteric virus vesicles has been performed to date. The goal of this project is to address this knowledge gap on rotavirus and norovirus vesicles in wastewater and natural aquatic systems. To achieve this goal, the team will (i) identify and quantify rotavirus and norovirus vesicles in wastewater, (ii) investigate their stability and infectivity in model natural water and wastewater systems, and (iii) evaluate their inactivation under exposure to sunlight. Successful completion of this project will result in new knowledge on the distribution, persistence, infectivity, and inactivation of enteric virus vesicles in wastewater and natural aquatic systems. Further benefits to society will be achieved through student education, training, and outreach integrating project findings into course modules. The diversity of the Nation’s STEM workforce will be increased through the recruitment and mentoring of high school, undergraduate, and graduate students from underrepresented groups.The recent discovery of vesicle-cloaked clusters of rotovirus and norovirus particles in stool and wastewater presents a challenge to our current understanding of the environmental fate and health risks of waterborne viruses. Although rotovirus and norovirus vesicles are more infectious and are more resistant to disinfection than their constituent free viral particles, there is very limited data and mechanistic understanding of their fate, transport, transmission, attenuation, and inactivation in wastewater and natural aquatic systems. The goal of this research is to address these knowledge gaps by investigating the environmental fate and infectivity of rotovirus and norovirus vesicles. To achieve this goal, the research team will carry out an integrated experimental program organized around three specific tasks. In Task 1, experiments will focus on the recovery, characterization, and quantification of rotavirus/norovirus vesicles in hospital and municipal wastewater using reverse-transcription droplet digital PCR (RT-ddPCR). In Task II, the PIs will characterize the stability and infectivity of rotavirus/norovirus vesicles in representative water matrices using a suite of analytical tools and assays including dynamic light scattering (DLS), transmission electron microscopy (TEM), and integrated cell culture-reverse transcription quantitative PCR (ICC-RT-qPCR). In Task III, the PIs will investigate the kinetics and mechanisms of inactivation of rotavirus/norovirus vesicles under sunlight exposure using similar assays as in Task II. Successful completion of this project will lead to the development of new fundamental knowledge on the environmental fate and infectivity of enteric virus vesicles in wastewater and natural aquatic systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.