Control 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
