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

轮状病毒和诺如病毒是全世界水传播疾病的主要原因。2018年，人们发现这两种肠道病毒通常存在于被称为囊泡的充满液体的小囊中。这些囊泡病毒簇广泛存在于粪便中，也在废水中检测到。与非囊泡病毒颗粒相比，轮状病毒和诺如病毒囊泡更具持久性、传染性和耐消毒性。尽管有这些令人担忧的发现，但迄今为止对肠道病毒囊泡的环境行为和命运进行的研究相对有限。该项目的目标是解决有关废水和天然水生系统中轮状病毒和诺如病毒囊泡的知识差距。为了实现这一目标，该团队将(i)识别和量化废水中的轮状病毒和诺如病毒囊泡，（ii）研究它们在模拟天然水和废水系统中的稳定性和传染性，以及（iii）评估它们在阳光照射下的失活能力。该项目的成功完成将对废水和天然水生系统中肠道病毒囊泡的分布、持久性、传染性和灭活产生新的认识。通过将项目成果整合到课程模块中的学生教育、培训和推广活动，将进一步造福社会。通过招募和指导来自代表性不足群体的高中生、本科生和研究生，将增加美国STEM劳动力的多样性。最近在粪便和废水中发现的囊泡状轮状病毒和诺如病毒颗粒簇，对我们目前对水传播病毒的环境命运和健康风险的理解提出了挑战。虽然轮状病毒和诺如病毒囊泡比它们的游离病毒颗粒更具传染性和更耐消毒，但关于它们在废水和天然水生系统中的命运、运输、传播、衰减和失活的数据和机制理解非常有限。本研究的目的是通过调查轮状病毒和诺如病毒囊泡的环境命运和传染性来解决这些知识空白。为了实现这一目标，研究团队将围绕三个具体任务开展一个综合实验计划。在任务1中，实验将集中于利用反转录液滴数字PCR （RT-ddPCR）回收、表征和定量医院和城市废水中的轮状病毒/诺如病毒囊泡。在任务II中，pi将使用一套分析工具和检测方法，包括动态光散射（DLS）、透射电子显微镜（TEM）和细胞培养-反转录定量PCR (ICC-RT-qPCR)，表征代表性水基质中轮状病毒/诺如病毒囊泡的稳定性和传染性。在任务III中，pi将使用与任务II中类似的实验研究轮状病毒/诺如病毒囊泡在阳光照射下失活的动力学和机制。该项目的成功完成将导致有关废水和天然水生系统中肠道病毒囊泡的环境命运和感染性的新基础知识的发展。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.