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

轮状病毒和诺沃克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都存在于被称为水泡的充满液体的小囊中。这些水泡病毒簇广泛存在于粪便中，并已在废水中检测到。与非水泡病毒颗粒相比，轮状病毒和诺沃克病毒小泡更持久、更具传染性和更耐消毒。尽管有这些相关的发现，但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关于废水和天然水生系统中轮状病毒和诺沃克病毒囊泡的知识差距。为了实现这一目标，该小组将(I)识别和量化废水中的轮状病毒和诺沃克病毒囊泡，(Ii)在模拟的天然水和废水系统中调查它们的稳定性和传染性，以及(Iii)评估它们在阳光下的失活。该项目的成功完成将使人们对废水和自然水生系统中肠道病毒囊泡的分布、持久性、传染性和灭活有新的认识。通过将项目成果纳入课程模块的学生教育、培训和推广，将为社会带来进一步的好处。全国STEM劳动力的多样性将通过招募和指导来自代表性不足群体的高中、本科生和研究生来增加。最近在粪便和废水中发现了包裹着水泡的轮状病毒和诺沃克病毒颗粒，这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。尽管轮状病毒和诺如病毒囊泡比其组成的游离病毒颗粒更具传染性和更强的抗消毒能力，但关于它们在废水和自然水生系统中的去向、传输、传播、衰减和灭活的数据和机制了解非常有限。这项研究的目的是通过调查轮状病毒和诺沃克病毒囊泡的环境命运和传染性来解决这些知识差距。为了实现这一目标，研究团队将围绕三项具体任务开展综合实验计划。在任务1中，实验将侧重于使用反转录液滴数字聚合酶链式反应(RT-ddPCR)回收、表征和量化医院和市政污水中的轮状病毒/诺沃克病毒囊泡。在任务II中，PIS将使用一套分析工具和分析方法来表征轮状病毒/诺沃克病毒囊泡在有代表性的水基质中的稳定性和传染性，这些分析工具和分析包括动态光散射(DLS)、透射电子显微镜(TEM)和集成细胞培养-逆转录定量聚合酶链式反应(ICC-RT-qPCR)。在任务III中，PIS将使用与任务II类似的分析方法研究轮状病毒/诺沃克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对废水和自然水生系统中肠道病毒囊泡的环境命运和传染性的新的基础知识的发展。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.