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

0933560 linden控制饮用水中的病毒对公共卫生至关重要，消毒是抵御致病微生物的主要屏障。紫外线消毒现在是废水处理的首选方法，由于对氯化副产物的关注和对隐孢子虫灭活的需要，紫外线消毒正在成为大型和小型饮用水系统消毒的非常重要的工具。在过去10年中，使用254 nm低压（LP）紫外线消毒对腺病毒进行紫外线处理，产生的细胞培养感染性数据被用于制定2006年美国环境保护局（EPA）对所有病毒的消毒要求标准，其水平几乎是实际使用的典型紫外线剂量40 mJ/cm2的5倍。然而，PI和多个合作研究者最近的研究发现，使用较新的多色紫外线源（中压[MP]和脉冲紫外线）可显著改善对腺病毒的紫外线消毒。这些紫外线来源之间的差异表明，对紫外线照射如何影响腺病毒缺乏基本的了解。许多作者呼吁增加对病毒对紫外线反应的基本分子机制的理解，以及准确检测病原体的分子方法。这项研究将加深对紫外线消毒病毒的机制和方法的理解，为进一步的消毒研究提供有力的工具，对保护公众健康具有重要的积极作用。本研究的目的是：1)适应并应用分子技术来研究低压（LP）和中压（MP）紫外线对腺病毒DNA和蛋白质的影响；2)将新应用的分子方法得到的结果与传统细胞培养感染试验得到的结果进行比较。本文提出的技术包括：1)两种检测DNA损伤的方法：利用PCR对DNA损伤进行一般评估，并利用抗体对环丁烷嘧啶二聚体（CPDs）进行特异性检测；2)利用SDS-PAGE对紫外线对主要腺病毒蛋白的损伤进行评估；3)利用流式细胞术和透射电镜对腺病毒衣壳进行评估。假设是1)LP UV和MP UV在诱导DNA损伤方面是相似的，2)MP UV而不是LP UV会对腺病毒蛋白造成明显的损伤和衣壳完整性的丧失，3)MP UV而不是LP UV会导致细胞培养感染性的降低，这与衣壳蛋白损伤的增加有关。对社会和水消毒界的广泛影响是改善紫外线对病毒的消毒和相关的公共卫生效益。如果多色紫外线系统被证明能够更好地灭活病毒，它们将被用于许多寻找氯替代品的小型系统中，并且可以经济地在更大的城市规模上实施，对病毒的紫外线剂量要求较低。该研究计划也非常适合弥合分子生物学与经典环境工程的新兴科学。与科罗拉多多样性倡议合作，该研究将（分子生物学好奇）本科和研究生工程专业的学生与分子生物学训练有素的博士后研究员整合到一个重要的工程和基础科学问题中，将团队置于工程消毒技术和新工具的前沿，以加深对紫外线消毒的基本理解。学生将有机会与水工程师和公用事业运营商互动，在国家会议上展示他们的研究成果，并有望在受人尊敬的期刊上发表他们的研究成果。在这一领域建立研究领导地位将使这些学生在公共卫生工程与分子生物学相结合的学术界取得成功。研究成果和技术将整合到“环境微生物学”和“环境工程中的紫外线处理”的课程和实验中。最后，将在2010/11年11月举行一个与水技术会议相结合的研讨会，以更广泛地向咨询工程师、公用事业决策者和监管机构传播结果

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