Investigating Underlying Mechanisms behind the Extreme Resistance of Adenoviruses to UV Disinfection

研究腺病毒对紫外线消毒极度耐药的潜在机制

• 批准号: 0933560
• 负责人: Karl Linden
• 金额: $ 39.73万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0933560&HistoricalAwards=false
• 关键词: Investigating; Underlying; Mechanisms; behind; Extreme

0933560LindenControl of viruses in drinking water is critical for public health, and disinfection is the primary barrier against disease-causing microorganisms. UV disinfection is now the method of choice for wastewater and is becoming a very important tool for disinfection of large and small drinking water systems due to the concerns over chlorination byproducts and the need to inactivate Cryptosporidium. Cell culture infectivity data generated over the past 10 years on UV treatment of adenoviruses using 254 nm low-pressure (LP) UV disinfection was used to set 2006 US EPA standards for disinfection requirements of all viruses at a level almost 5 times the typical UV dose of 40 mJ/cm2 used in practice. However, recent research by the PI and various co-investigators has found that use of newer polychromatic UV sources (medium pressure [MP] and pulsed UV) significantly improves the UV disinfection of adenoviruses. These differences between UV sources indicate that a fundamental understanding of how UV irradiation affects adenoviruses is lacking. Numerous authors have called for an increased understanding of the fundamental molecular mechanisms involved in viral response to UV as well as molecular methods for accurate pathogen detection. This research will enhance the understanding of the mechanisms behind UV disinfection of viruses and methods used will provide powerful tools for further disinfection investigations with important positive results for protection of public health. The objectives of the proposed research are 1) to adapt and apply molecular techniques to investigate the effects of low-pressure (LP) and medium pressure (MP) UV on adenoviral DNA and proteins, and 2) to compare the results obtained using the newly applied molecular methods to those obtained using classical cell culture infectivity assays. The techniques proposed here include 1) two methods to examine DNA damage: both general assessment of DNA damage using PCR, and specific detection of cyclobutane pyrimidine dimers (CPDs) using antibodies, 2) assessment of UV damage to the major adenoviral proteins using SDS-PAGE, and 3) assessment of the adenovirus capsid using flow cytometry and transmission electron microscopy. The hypotheses are that 1) LP UV and MP UV will be similar in their induction of DNA damage, 2) MP UV, but not LP UV, will cause significant damage to adenoviral proteins and loss of capsid integrity, and 3) MP UV, but not LP UV, will cause a decrease in cell culture infectivity which correlates with increased damage to capsid proteins. The broad impacts for society and the water disinfection community are improved UV disinfection of viruses and the associated public health benefits. If polychromatic UV systems are proven better able to inactivate viruses, they will be used in many small systems looking for an alternative to chlorine, and can be economically implemented on a larger municipal scale with lower UV dose requirements for viruses. The research plan is also ideally suited to bridging the emerging science of molecular biology with classical environmental engineering. Working with the Colorado Diversity Initiative, the research integrates (molecular biology-curious) undergraduate and graduate engineering students with a molecular biology trained post-doctoral researcher into an important engineering and fundamental science question, placing the team at the leading edge of both engineering disinfection technology and new tools for discovery to deepen the fundamental understanding of UV disinfection. Students will have the opportunity to interact with water engineers and utility operators, to present their research at national conferences and will expect to publish their work in respected journals. Establishing research leadership in this area will position these students for successful careers in academia where engineering for public health meets molecular biology. The research findings and techniques will be integrated into courses and laboratories on "Environmental Microbiology" and "UV Processes in Environmental Engineering". Finally, a workshop will be held in conjunction with a water technology conference in Nov. 2010/11 to more widely disseminate results to consulting engineers, utility decision makers, and regulators

0933560饮用水中病毒的溶解控制对于公共卫生至关重要，消毒是抵抗致病微生物的主要障碍。紫外线消毒现在是废水的首选方法，由于对氯化副产物的担忧以及需要灭活隐孢子虫的关注，因此正在成为对大小饮用水系统消毒的非常重要的工具。在过去的10年中，使用254 nm低压（LP）紫外线消毒在过去10年中生成的细胞培养感染数据用于设定2006年美国EPA标准所有病毒的消毒需求，几乎是使用40 MJ/CM2使用的典型uv剂量的近5倍。然而，PI和各种共同投资者的最新研究发现，使用新的多色紫外线源（中压[MP]和脉冲紫外线）可显着改善腺病毒的紫外线消毒。紫外线来源之间的这些差异表明，对紫外线辐射如何影响腺病毒的基本了解。许多作者呼吁人们对参与病毒反应涉及的紫外线反应的基本分子机制以及用于准确病原体检测的分子方法有更多的了解。这项研究将增强对紫外线消毒病毒和所使用方法背后的机制的理解，将为进一步的消毒调查提供强大的工具，并为保护公共卫生而重要的积极成果。拟议的研究的目标是1）适应和应用分子技术来研究低压（LP）和培养基压力（MP）紫外线对腺病毒DNA和蛋白质的影响，以及2），以比较使用新应用的分子方法对使用类别细胞培养培养物培养型感染性测量获得的结果获得的结果。此处提出的技术包括1）两种检查DNA损伤的方法：使用PCR对DNA损伤进行了一般评估，以及使用抗体对环丁烷嘧啶二聚体（CPD）的特定检测，2）使用SDS-PAGE评估紫外病毒蛋白对紫外线损伤的评估，并使用SDS-PAGE进行了sds-page和3）的传输循环。假设是1）LP UV和MP UV在诱导DNA损伤时将相似，2）MP UV，但不是LP UV会对腺病毒蛋白造成重大损害，而Capsid完整性的丧失以及3）MP UV的损害，而LP UV则不会导致细胞培养物的损害降低，从而降低了与蛋白质相关的损害，从而降低了与CAPSIDS相关的损伤。对社会和水消毒界的广泛影响是改善了病毒的紫外线消毒和相关的公共卫生益处。如果证明多色紫外线系统能够更好地灭活病毒，则将它们用于许多寻求氯选择性替代品的小型系统中，并且可以在较大的市政尺度上经济实施，对病毒的紫外线剂量要求较低。该研究计划也非常适合将新兴的分子生物学科学与经典的环境工程联系起来。该研究与科罗拉多多样性计划合作，将（分子生物学的）本科生和研究生工程专业的学生与分子生物学培训的分子生物学培训后的博士后研究人员融入了重要的工程和基本科学问题，将团队置于两种工程技术的领先地位，并在工程学技术的领先地位，并将新的工具进行了新的研究工具，以深化uv distection uv of uv distinfection of uv disninfection of uv disninfection of uv distinfection fornfuct of uv distinff。学生将有机会与水工程师和公用事业运营商互动，在国家会议上介绍他们的研究，并希望将其作品发布到受人尊敬的期刊上。在这一领域建立研究领导将使这些学生在学术界成功职业的职业生涯，在那里公共卫生的工程符合分子生物学。研究发现和技术将纳入有关“环境微生物学”和“环境工程中的紫外线过程”的课程和实验室。最后，将于2010年11月11日与水技术会议一起举行研讨会，以更广泛地传播结果，以咨询工程师，公用事业决策者和监管机构