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世界范围内水传播疾病的主要原因。在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，并已在废水中检测到。与非囊泡病毒颗粒相比，轮状病毒和诺如病毒囊泡更持久、更具感染性和耐消毒性。尽管有这些令人担忧的发现，迄今为止对肠道病毒囊泡的环境行为和命运的研究相对有限。该项目的目标是解决废水和自然水生系统中轮状病毒和诺如病毒囊泡的知识缺口。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）研究它们在模型天然水和废水系统中的稳定性和感染性，以及（iii）评估它们在阳光照射下的灭活情况。该项目的成功完成将导致对废水和自然水生系统中肠道病毒囊泡的分布，持久性，感染性和灭活的新知识。将通过学生教育、培训和外联活动，将项目成果纳入课程模块，进一步造福社会。通过从代表性不足的群体中招募和指导高中生、本科生和研究生，国家STEM劳动力的多样性将得到增加。最近在粪便和废水中发现的轮状病毒和诺如病毒颗粒的囊泡覆盖簇，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其组成的游离病毒颗粒更具感染性，并且对消毒更具抗性，但对其在废水和天然水生系统中的命运，运输，传播，衰减和灭活的数据和机理了解非常有限。本研究的目标是通过调查轮状病毒和诺如病毒囊泡的环境归宿和感染性来解决这些知识空白。为了实现这一目标，研究小组将围绕三个具体任务开展综合实验计划。在任务1中，实验将集中在医院和城市污水中的轮状病毒/诺如病毒囊泡的回收，表征和定量，使用逆转录液滴数字PCR（RT-ddPCR）。在任务II中，PI将使用一套分析工具和测定法（包括动态光散射（DLS）、透射电子显微镜（TEM）和整合细胞培养-逆转录定量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感染性。在任务III中，PI将使用与任务II相似的试验研究轮状病毒/诺如病毒囊泡在日光暴露下的灭活动力学和机制。该项目的成功完成将导致发展新的基础知识的环境命运和传染性的肠道病毒囊泡在废水和自然水生系统。该奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

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.