ASSESSMENT OF THE OCCURRENCE, FATE, AND TRANSPORT OF PATHOGENIC AND INDICATOR MICROORGANISMS IN THE ENVIRONMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: REVISED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0215717
• Project No.: CA-R-ENS-7772-H
• Project Start Date: Apr 1, 2013
• Project End Date: Mar 31, 2018

Project Director
Yates, M. V.

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
Environmental Sciences

Non Technical Summary
As the population of California grows, the competition for resources, especially water, among urban residents, agricultural enterprises, and environmental sectors is becoming increasingly fierce. The situation has been exacerbated by a number of factors, including drought and climate change. As it is apparent that new sources of freshwater are unlikely to meet the needs of the state, we are increasingly reusing material such as treated domestic wastewater, which was previously considered waste. However, even highly treated (e.g., tertiary) reclaimed water cannot be guaranteed to be free from disease-causing microorganisms originating in human waste (i.e., enteric pathogens). Despite significant advances in medicine during the last several decades, infectious disease still accounts for about 26% of deaths worldwide (Kindhause, 2003). Because the enteric viruses are shed in the feces, the possibility that sewage-contaminated, inadequately treated drinking water is the source of the viruses exists. Enteric viruses have been detected in environmental samples including wastewater, surface water, and ground water. Several enteric viruses, including the adenoviruses, enteroviruses, hepatitis A virus, and caliciviruses (including noroviruses) have been placed on the U.S. EPA?s drinking water Contaminant Candidate List (CCL) 3 for possible regulation. The only reliable method to detect infectious viruses is based on mammalian cell culture, which detects the production of visible cytopathic effects (CPE) upon virus infection of a living host cell. This method is difficult to perform and may take weeks before the viruses reach measurable amounts to allow detection. However, some epidemiologically important viruses such as noroviruses still cannot be cultivated or grow poorly in cell culture (Straub et al., 2007). This has prompted the need for new detection approaches that are rapid, sensitive and specific. These approaches must be quantitative, and be specific for the detection of only infective viruses (Yeh et al., 2009). In addition, the lack of rapid methods that can be used to detect very low numbers of infective organisms hampers our ability to make accurate assessments of risk to public health. In the last several years, molecular beacons (MBs) have been used for the construction of probes that are useful for real-time detection of nucleic acids (Tyagi and Kramer, 1996). MBs have been successfully used to detect several enteric viruses, including hepatitis A virus (Abd El Galil et al., 2004) and echovirus (Yeh et al., 2008). We have also developed a method to simultaneously detect two unrelated enteric viruses using a single cell line and probes with different fluorophores (Dunams et al., 2012). These methods, however, are somewhat laborious, as the fluorescent cells must be manually counted using a fluorescent microscope. Automation of the method is necessary for the method to receive widespread adoption. In addition, any methods that are developed must be tested using real environmental samples to ensure that the level of sensitivity and specificity observed in the laboratory is maintained.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0210	1103	100%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0210 - Water resources;

Field Of Science
1103 - Other microbiology;

Keywords

human pathogens

drinking water

wastewater

detection methods

environmental fate and transport

Goals / Objectives
Objectives The overall goal of this project is to learn more about the presence of disease-causing microorganisms so that management practices may be established to minimize the potential to contaminate water, where they may pose a public health risk through contamination of drinking water or food. The specific objectives are: Develop and refine methods to enable the rapid detection of infective microorganisms in environmental samples; Assess the potential for methods to detect low numbers of pathogens in environmental samples; and Examine microorganisms in urban runoff and assess associations with other water quality parameters

Project Methods
Objective 1: Development and refinement of detection methods Development of new pathogen detection methods Carbon nanotubes (CNTs) have unique physical and chemical properties that make them very promising materials for use in nanotechnology. Most CNT fabricated devices have been used as field-effect transistors in which the change in electrical conductance on the binding of an analyte to a recognition molecule (32) is monitored. We developed a simple platform for the detection of bacteria and viruses based on a network of parallel aligned SWNTs bridging two gold electrodes acting as source and drain as the transducer element. The biosensor specificity relies on capture of the target microorganisms by functionalization of the biosensor with specific antibodies (Abs). We will extend this to detection of viruses of public health/environmental significance. SWNTs will be functionalized through covalent binding of the Ab to a pathogen-appropriate compound appropriate, which has adsorbed onto the SWNTs. The biosensor performance will be evaluated with selected pathogens. Use of flow cytometry to detect infective viruses Current methods are too labor intensive to allow widespread adoption, as the cells are identified by screening fields under a fluorescent microscope. Therefore, more automated are needed. The use of fluorescence activated cell sorter (FACS) for high throughput, sensitive, selective and rapid (e.g., less than 4 hours) analysis will be explored. We have demonstrated the use of this technique to detect and enumerate poliovirus-infected BGMK cells (3). However, our facility would not allow human pathogens to be used. We have acquired a FACS machine that allows use of pathogen-infected cells. The FACS' ability to sort the fluorescence intensity of each cell will count the number of cells that exhibit a minimum fluorescence level. It is expected that the number of fluorescent cells will increase with increasing infective doses and an accurate direct counting will be obtained. The validity of the assay will be tested by comparing with the plaque assay using samples with unknown amount of a test virus. Objective 2: Testing Protocols on Environmental Samples After methods are optimized, their efficiency will be tested and compared to standard plaque methods. Different concentrations of viruses will be seeded into samples to allow an assessment of the method's sensitivity and ability to accurately quantify infective virus particles. An assessment of the reproducibility of results and time required to achieve results will also be made. To assess the potential for false positive and false negative results, respectively, autoclaved samples and samples containing extremely low numbers of viruses will be used. Objective 3: Examine microorganisms in urban runoff A several-years-long study of the presence of pathogens, indicator microorganisms, and chemicals in residential urban runoff was recently concluded. The data from this study will be analyzed to determine whether there are associations between water quality parameters and the microbial data. This will be done using standard statistical analyses and neural network analysis.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The scientific community through publications, seminars, and presentations Undergraduate students who have been given lectures onthe topics Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?two graduate students were partially supported How have the results been disseminated to communities of interest?The results have been published in peer-reviewed, scientific journals. What do you plan to do during the next reporting period to accomplish the goals?same

Impacts
What was accomplished under these goals? Molecular beacons for the detection of dengue virus in situ have been developed. We developed a simple platform for the detection of bacteria and viruses based on a network of parallel aligned SWNTs bridging two gold electrodes acting as source and drain as the transducer element. The biosensor specificity relies on capture of the target microorganisms by functionalization of the biosensor with specific antibodies (Abs). We have extended this to detection of dengue viruses. SWNTs have been functionalized through covalent binding of the Ab to a pathogen-appropriate compound appropriate, which has adsorbed onto the SWNTs. This method was tested to detect dengue viruses in mosquitoes and in saliva. Manuscripts describing these research results were written for submission to scientific journals.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Intracellular Detection of Replication of Dengue Virus Serotype 1 using a Molecular Beacon Fluorescent Probe
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Biosens Bioelectron. 2017 May 15;91:811-816. doi: 10.1016/j.bios.2017.01.017. Epub 2017 Jan 10. A heparin-functionalized carbon nanotube-based affinity biosensor for dengue virus. Wasik D1, Mulchandani A2, Yates MV1.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Development of Carbon Nanotube-Based Biosensors to Detect Dengue Virus

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:scientists, community members Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students were partially supported. How have the results been disseminated to communities of interest?Through scientific publications and presentations to scientists and community members. What do you plan to do during the next reporting period to accomplish the goals?Continue to conduct research

Impacts
What was accomplished under these goals? Methods to detect polyomaviruses in water have been developed, and their relative resistance to inactivation by drinking water treatment process is being explored. The results of this research were published in Environmental Science & Technology, and other manuscripts describing these results are in preparation. The graduate student who conducted this research has completed his Ph.D. and was hired to work for the U.S.G.S. on water quality issues. Molecular beacons for the detection of dengue virus in situ are under development. We developed a simple platform for the detection of bacteria and viruses based on a network of parallel aligned SWNTs bridging two gold electrodes acting as source and drain as the transducer element. The biosensor specificity relies on capture of the target microorganisms by functionalization of the biosensor with specific antibodies (Abs). We have extended this to detection of dengue viruses. SWNTs have been functionalized through covalent binding of the Ab to a pathogen-appropriate compound appropriate, which has adsorbed onto the SWNTs. This method is currently being tested to detect dengue viruses in mosquitoes and in saliva. Manuscripts describing these research results are being written for submission to scientific journals.

Publications

• Type: Books Status: Published Year Published: 2016 Citation: Editors: Yates, MV (editor in chief). 2016. Manual of Environmental Microbiology, 4th ed. American Society for Microbiology. 1088p. (Refereed, Electronic, Invited)
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Yates, M.V. 2016. Drinking Water Microbiology. Manual of Environmental Microbiology, 4th ed. Editors: Yates, MV (editor in chief). American Society for Microbiology. 14p. (Refereed, Electronic)
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Yates, M.V. 2016. Risk Assessment Framework. Manual of Environmental Microbiology. Editors: Yates, MV (editor in chief). American Society for Microbiology. 10p. (Refereed, Electronic)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Reano, D., Yates, M.V. 2016. Determining the Solar Inactivation Rate of BK Polyomavirus by Molecular Beacon. ES&T. Vol. 50: p.7090-7094. (Refereed)
• Type: Books Status: Published Year Published: 2016 Citation: Water Science and Technology Board, Committee on the Beneficial Use of Graywater and Stormwater: An Assessment of Risks, Costs, and Benefits 2016. Using Graywater and Stormwater to Enhance Local Water Supplies: An Assessment of Risks, Costs, and Benefits (2016). National Academies Press. Washington, DC. 223p. ISBN: 978-0-309-38835-1 . Refereed, Electronic

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:As the Keynote Speaker at the Water Microbiology Conference, May 2015, at the University of North Carolina, Chapel Hill, I spoke to numerous graduate and undergraduate students about the newest methods being used to detect viruses in drinking water. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students are being trained. How have the results been disseminated to communities of interest?Not yet, although publications are being prepared. What do you plan to do during the next reporting period to accomplish the goals?Continue to develop and refine methods to enable the rapid detection of infective microorganisms in environmental samples.

Impacts
What was accomplished under these goals? A study describing the several-years-long study of the presence of pathogens, indicator microorganisms, and chemicals in residential urban runoff was recently concluded. The data from this study were analyzed to determine whether there are associations between water quality parameters and the microbial data. This was done using standard statistical analyses and neural network analysis. A manuscript descibing the studywas published in Water Research. We developed a simple platform for the detection of bacteria and viruses based on a network of parallel aligned SWNTs bridging two gold electrodes acting as source and drain as the transducer element. The biosensor specificity relies on capture of the target microorganisms by functionalization of the biosensor with specific antibodies (Abs). Wehave extended this to detection of dengue viruses. SWNTshave been functionalized through covalent binding of the Ab to a pathogen-appropriate compound appropriate, which has adsorbed onto the SWNTs. Methods to detect polyomaviruses in water have been developed, and their relative resistance to inactivtion by drinking water treatment process is being explored.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Long-term characterization of residential runoff and assessing potential surrogates of fecal indicator organisms Dane C. Reano, Darren L. Haver, Lorence R. Oki, Marylynn V. Yates. Water Research 74 (2015): 67-76.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three graduate students are bineg trained. How have the results been disseminated to communities of interest? a publication was submitted. What do you plan to do during the next reporting period to accomplish the goals? Continue to develop and refine methods to enable the rapid detection of infective microorganisms in environmental samples.

Impacts
What was accomplished under these goals? A study describing theseveral-years-long study of the presence of pathogens, indicator microorganisms, and chemicals in residential urban runoff was recently concluded. The data from this studywere analyzed to determine whether there are associations between water quality parameters and the microbial data. Thiswas done using standard statistical analyses and neural network analysis. A manuscript descibing the study was submitted to Water Research. We developed a simple platform for the detection of bacteria and viruses based on a network of parallel aligned SWNTs bridging two gold electrodes acting as source and drain as the transducer element. The biosensor specificity relies on capture of the target microorganisms by functionalization of the biosensor with specific antibodies (Abs). We will extend this to detection of viruses of public health/environmental significance. SWNTs will be functionalized through covalent binding of the Ab to a pathogen-appropriate compound appropriate, which has adsorbed onto the SWNTs.

Publications

Progress 04/01/13 to 09/30/13

Outputs
Target Audience: The major target audiences have been scientific colleaugues as well as faculty and students at the Nelson Mandela African Institute of Science and Technology in Arusha, Tanzania. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three graduate students and 1 undergraduate students are being trained Two of the graduate students attended the meeting in Florianopolis and presented their research How have the results been disseminated to communities of interest? See above What do you plan to do during the next reporting period to accomplish the goals? Continue methods development for human polyoma viruses in water Continue methods development for dengue viruses Prepare and submit publication on microorganisms in urban runoff

Impacts
What was accomplished under these goals? Accepted an invitatino to serve on the NRC Committee on On-site Reuse of Graywater and Stormwater: An Assessment of Risks, Costs, and Benefits Spent 1 week in Arusha, Tanzania at the Nelson Mandela African Institute of Science and Technology presenting lectures and seminars on waterborne disease to students and faculty Attended biannual meeting of the Health-Related Water Microbiology group in Florianoopolis, Santa Catarina, Brazil; presented two posters Finished analyzing data on microorganisms in urban runoff for incorporation into a manuscript to be submitted for publication

Publications

• Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Quantitative Assessment of in vivo HIV Protease Activity Using Genetically Engineered QD-Based FRET Probes

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: There is a lack of information regarding the factors that control the fate and transport of many of these enteric pathogens in the environment, making it difficult to determine how best to protect drinking water source water from contamination. During this year, several steps were made that will allow us to make some substantial contributions towards addressing this issue. The most significant is the development of a method that allows the simultaneous detection of two different viruses (adenoviruses and enteroviruses) in one cell type. The method is extremely rapid (a few hours compared to several days), sensitive (as few as one plaque-forming unit can be detected), and is specific for infective viruses, as they are detected in living cells. We have also been analyzing the data that resulted from a several-year-long study on the presence of pathogens and indicator microorganisms in urban runoff water. This information is important for the development of BMPs to enable local communities to comply with TMDLs. We expect to have a manuscript ready for submission next year. PARTICIPANTS: Dr. Payal Sarkar - postdoctoral research associate, UCR Divya Sivaraman, Lakshmi Cella, Dane Reano, Ph.D. students, UCR TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Outcomes/Impacts During this year, my laboratory and I were involved in several activities in which we presented the work that was accomplished as a part of this project. Presentations included: Detection of waterborne viruses. Water Technology Conference, University of LaVerne, LaVerne, CA 1/26/2012. New methods for the detection of waterborne pathogens, Montana Rural Water Systems, 33rd Annual Conference, Great Falls, Montana, February 22, 2012. Public Health Issues, Montana Rural Water Systems, 33rd Annual Conference, Great Falls, Montana, February 23, 2012. Rapid Methods for the Simultaneous Detection of Infectious Human Enteroviruses and Adenoviruses in A549 Cells, XXIII Brazilian Congress of Virology, Foz du Iguacu, Brazil, September 29, 2012.

Publications

• Dunams, D. P. Sarker, W. Chen, and M.V. Yates. 2012. Simultaneous detection of infectious human echoviruses and adenoviruses by an in situ nuclease-resistant molecular beacon-based assay. Appl. Environ. Microbiol., 78(5):1584-1588.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: There is a lack of information regarding the factors that control the fate and transport of many of these enteric pathogens in the environment, making it difficult to determine how best to protect drinking water source water from contamination. During this year, several steps were made that will allow us to make some substantial contributions towards addressing this issue. The most significant is the development of a method that allows the simultaneous detection of two different viruses (adenoviruses and enteroviruses) in one cell type. The method is extremely rapid (a few hours compared to several days), sensitive (as few as one plaque-forming unit can be detected), and is specific for infective viruses, as they are detected in living cells. We have also explored other methods to examine virus replication in living cells, using probes that are constructed with components that are more resistant to degradation by cellular enzymes or provide a more specific signal of the presence of the target. Our project with the Metropolitan Water District of Southern California was completed. We successfully adapted a plaque assay technique to allow the detection and quantification of infective enteric adenoviruses in the laboratory, making mine one of fewer than 5 in the country with that capability. This is significant because the adenoviruses are found more frequently in water and wastewater than are the enteroviruses (which have been the traditional focus of water virology studies). Studies on the survival of these viruses in surface and ground waters were completed. Our results have shown that these viruses remain infective, with very little loss of infectivity, in waters at environmentally-relevant temperatures for several months, which is much longer than most enteric viruses. A trip to Arusha, Tanzania to establish collaboration with scientists at the Nelson Mandela African Institute of Science and Technology was made in September. A joint proposal to study the effectiveness of constructed wetlands to remove waterborne pathogens was developed and submitted to PEER. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
During this year, my laboratory and I were involved in several activities in which we presented the work that was accomplished as a part of this project. Presentations included: Simultaneous Detection of Infectious Adenoviruses and Enteroviruses in Water. University of Puerto Rico, Rio Piedras, Puerto Rico. 08/2011 An in situ molecular beacon-based assay for the simultaneous detection of infectious human enteroviruses and adenoviruses in A549 cells. Dunams DD, Ganguli PS, Chen W, and Yates MV, Rotorua, NZ 9/2011

Publications

• Biswas, P., L. N. Cella, S. H. Kang, A. Mulchandani, M. V. Yates and W. Chen. 2011. A quantum-dot based protein module for in vivo monitoring of protease activity through fluorescence resonance energy transfer. Chem. Commun., 47 (18): 5259-5261.
• Rigotto, C., Hanley, K., Rochelle, P.A., De Leon, R., Barardi, C.R.M., Yates, M.V. 2011. Survival of adenovirus types 2 and 41 in surface and ground waters measured by a plaque assay. ES&T, 45:4145-4150.
• Cantera, J.L., W. Chen, and M.V. Yates. 2011. A fluorescence resonance energy transfer-based fluorometer assay for screening anti-coxsackievirus B3 compounds. J. Virol. Meth. 171(1): 176-182.
• Sivaraman, D., P. Biswas, L. N. Cella, M. V. Yates, and W. Chen. 2011. Detecting RNA viruses in living mammalian cells by fluorescence microscopy. Trends in Biotechnology, 29 (7): 307-313.
• Ganguli, P., W. Chen, and M.V. Yates. 2011. Detection of murine norovirus-1 by using TAT peptide-delivered molecular beacons. Appl. Environ. Microbiol. 77(15):5517-5520.
• Haznedaroglu, B., M.V. Yates, M.F. Maduro, and S.L. Walker. 2011. Effects of residual antibiotics in groundwater on Salmonella typhimurium: changes in antibiotic resistance, in vivo and in vitro pathogenicity. J. Environ. Monit. 14:41-47.
• C. Garcia-Aljaro, L.N. Cella, D.J. Shirale, M. Park, F.J. Munoz, M.V. Yates, and A. Mulchandani. 2010. Carbon nanotubes-based chemiresistive biosensors for detection of microorganisms. Biosensors and Bioelectronics 26 (4): 1437-1441.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: There is a lack of information regarding the factors that control the fate and transport of many of these enteric pathogens in the environment, making it difficult to determine how best to protect drinking water source water from contamination. During this year, several steps were made that will allow us to make some substantial contributions towards addressing this issue. The most significant is the development of a method that allows the simultaneous detection of two different viruses (adenoviruses and enteroviruses) in one cell type. The method is extremely rapid (a few hours compared to several days), sensitive (as few as one plaque-forming unit can be detected), and is specific for infective viruses, as they are detected in living cells. We have also explored other methods to examine virus replication in living cells, using probes that are constructed with components that are more resistant to degradation by cellular enzymes or provide a more specific signal of the presence of the target. Our partnership with the Metropolitan Water District of Southern California continued. We successfully adapted a plaque assay technique to allow the detection and quantification of infective enteric adenoviruses in the laboratory, making mine one of fewer than 5 in the country with that capability. This is significant because the adenoviruses are found more frequently in water and wastewater than are the enteroviruses (which have been the traditional focus of water virology studies). Once the capability was developed, studies on the survival of these viruses in surface and ground waters were conducted. Our results have shown that these viruses remain infective, with very little loss of infectivity, in waters at environmentally-relevant temperatures for several months, which is much longer than most enteric viruses. A manuscript describing this work has been submitted and is under revision to address reviewer comments. We also completed studies conducted with Dr. Marc Deshusses, now at Duke University, on the inactivation of microorganisms during thermophilic anaerobic digestion of domestic wastewater. PARTICIPANTS: Dr. Paul Rochelle, Metropolitan Water District of Southern California, co-sponsored a student from Brazil, Dr. Caroline Rigotto-Borges, who worked in his laboratory and mine to develop the adenovirus plaque assay. Caroline did this work as part of her Ph.D. from the UNIVERSIDADE FEDERAL DE SANTA CATARINA - CENTRO DE CIENCIAS BIOLOGICAS in Florianopolis. Dr. Marc Deshusses, Duke University Dr. Jason Cantera, Dr. Payal Sarkar - postdoctoral research associates, UCR s. Daniela Dunams, M.S. student, UCR TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During this year, my laboratory and I were involved in several activities in which we presented the work that was accomplished as a part of this project. At the 2010 AWWA International Symposium on Waterborne Pathogens in May, our research was presented in four presentations. These are: Detection of Murine Norovirus in Groundwater Using TAT Peptide-Delivered Molecular Beacons (presented by Dr. Payal Sarkar, UCR) A Rapid, Sensitive Flow Cytometry Method to Detect Infective Viruses Based on Fluorescence Resonance Energy Transfer Technology (presented by Dr. Jason Cantera, UCR) Simultaneous Detection of Infectious Human Enteroviruses and Adenoviruses by Integrated Cell Culture RT PCR (presented by Ms. Daniela Dunams, UCR) Adenoviruses Are Highly Stable in Water and Resistant to UV, But Are They Significant Waterborne Pathogens (presented by Dr. Paul Rochelle, Metropolitan Water District)

Publications

• Yeh, H.Y., M.V. Yates, A. Mulchandani, and W. Chen. 2010. Molecular beacon-quantum dot-Au nanoparticle hybrid nanoprobes for visualizing virus replication in living cells. Chem. Communic. 46 (22): 3914-3916.
• Popat, S.C., M.V. Yates, and M. Deshusses. 2010. Kinetics of inactivation of indicator pathogens during thermophilic anaerobic digestion. Water Res. 44 (20): 5695-5672.
• Shirale, D.J., M.A. Bangar, M. Park, M.V. Yates, W. Chen, N.V. Myung, and A. Mulchandani. 2010. Label-free chemiresistive immunosensors for viruses. Environ. Sci. Technol. 44 (23): 9030-9035.
• Cantera, J.L., W. Chen, and M.V. Yates. 2010. A simple, rapid and sensitive flow cytometry method to detect infective poliovirus based on fluorescence resonance energy transfer (FRET) technology. Appl. Environ. Microbiol. 76:584-588.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: There is a lack of information regarding the factors that control the fate and transport of many of these enteric pathogens in the environment, making it difficult to determine how best to protect drinking water source water from contamination. During this year, two very significant steps were made that will allow us to make some substantial contributions towards addressing this issue. One is that, through a partnership with the Metropolitan Water District of Southern California, we successfully adapted a plaque assay technique to allow the detection and quantification of infective enteric adenoviruses in the laboratory, making mine one of fewer than 5 in the country with that capability. This is significant because the adenoviruses are found more frequently in water and wastewater than are the enteroviruses (which have been the traditional focus of water virology studies). Once the capability was developed, studies on the survival of these viruses in surface and ground waters were conducted. Our results have shown that these viruses remain infective, with very little loss of infectivity, in waters at environmentally-relevant temperatures for several months, which is much longer than most enteric viruses. This information is currently being prepared for submission to a peer-reviewed journal for publication. The second major advancement was the adaptation of a plaque assay for the detection and quantification of noroviruses. Human noroviruses are estimated to causes 23 million cases of illness every year in the United States. However, studies on their environmental fate have been hampered by the lack of a suitable cell culture assay. Recent studies have demonstrated that murine noroviruses exhibit several of the characteristics of human noroviruses, and thus, are suitable surrogates for the human noroviruses. By developing this ability in the laboratory, we can now conduct studies on the behavior of these viruses. A manuscript describing this work is in preparation. In general, current methods for the detection of infective human viruses are still too labor intensive to allow for their widespread adoption, as the cells are identified by screening fields under a fluorescent microscope. Therefore, we need to develop methods that are more automated; work in this area has continued, with some excellent results. The use of fluorescence activated cell sorter (FACS) for high throughput, sensitive, selective and rapid (e.g., less than 24 hours) analysis was demonstrated using poliovirus as a model virus. This manuscript describes the results of our studies using flow cytometry to enable to early detection of infective polioviruses. As early as 12 hours post-infection, a significant number of infected cells were detected even when infected with 1 PFU. When applied to environmental water samples spiked with poliovirus, the FACS-based assay provided a similar level of sensitivity to the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assay, and can be used to detect other viruses that frequently do not form clear plaques on cell cultures. PARTICIPANTS: Dr. Paul Rochelle, Metrolpolitan Water District of Southern California co-sponsored a student from Brazil, Caroline Rigotto-Borges, who worked in his laboratory and mine to develop the adenovirus plaque assay. Caoline did this work as part of her Ph.D. from the UNIVERSIDADE FEDERAL DE SANTA CATARINA - CENTRO DE CIENCIAS BIOLOGICAS in Florianopolis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During this year, my laboratory and I were involved in several activities in which we presented the work that was accomplished as a part of this project. A presentation entitled, "Survival of adenoviruses in surface and ground waters measured by a plaque assay" was presented at the bi-annual meeting of the Health-Related Water Microbiologists in June 2009. This is a specialist group within the International Water Association, and represents the premier forum for the international dissemination of research in this field. Another presentation, entitled, "A rapid, sensitive method to detect infective viruses using flow cytometry" was also presented at this meeting. In addition, a presentation entitled, "Development of high-throughput and real-time methods for the detection of infectious enteric viruses" presented at the 2009 U.S. Environmental Protection Agency Workshop on Innovative Approaches for Detecting Microorganisms and Cyanotoxins in Water in Philadelphia in May 2009. A manuscript describing some of this research, entitled, "A simple, rapid and sensitive flow cytometry method to detect infective poliovirus based on fluorescence resonance energy transfer (FRET) technology" was submitted to Applied & Environmental Microbiology. In February, I was an invited participant in a workshop entitled, "Critical Research and Science Needs for the Development of Recreational Water Quality Criteria in Inland Waters Expert Workshop", sponsored by Water Environment Research Foundation, held in Dallas, Texas. I was also an invited participant in a workshop jointly sponsored by the U.S. EPA and the Centers for Disease Control and Prevention, The workshop, held in Atlanta in April, was entitled, "Dose-Response Relationships in Microbial Risk Assessment Expert Workshop". In April, I participated in a workshop organized by the Canadian Water Network. The purpose of the workshop was to provide a discussion frame for exploration of specific science-based discussion areas of potential value to end-users exploring "What the Science Supports in terms of Management Actions and Options for Source-Water Protection of Groundwater Sources from Pathogens" in preparation for a larger meeting held in May. In May, I was invited to be the Keynote speaker at the 2009 Indiana Environmental Health Summit, held in Indianapolis, Indiana. My presentation was a general discussion of pathogens in water: human health risk.

Publications

• Hsiao-Yun Yeh, Marylynn V. Yates, Wilfred Chen, and Ashok Mulchandani, Real-time molecular methods to detect infectious viruses: a mini-review, Seminars in Cell & Developmental Biology, 20, 49 54, 2009. (Invited Contribution)

Progress 08/20/08 to 12/31/08

Outputs
OUTPUTS: Land application of biosolids is another potential source of water contamination due to the presence of enteric pathogens in this material. The federal rule that regulates the land application of these biosolids, 40 CFR Part 503, requires that the materials meet certain microbiological standards prior to application. Since this regulation was developed, a number of pathogenic microorganisms have emerged as being of potential public health concern. However, data on their ability to survive and be transported in the environment after land application is lacking. Thus, there are questions about potential health hazards associated with the land application of biosolids. There is a lack of information regarding the factors that control the fate and transport of many of these enteric pathogens in the environment, making it difficult to determine how best to protect drinking water source water from contamination. Laboratory and field-derived data were processed to enable a statistical analysis of the data. As numerous factors that may affect fate and transport were examined (soil type, temperature, moisture content, presence or absence of biosolids) for several microorganisms, it was necessary to enlist the assistance of statisticians to ensure that the appropriate statistical analyses are performed. The analyses are on-going. Current methods are still too labor intensive to allow for their widespread adoption, as the cells are identified by screening fields under a fluorescent microscope. Therefore, we need to develop methods that are more automated. The use of fluorescence activated cell sorter (FACS) for high throughput, sensitive, selective and rapid (e.g., less than 24 hours) analysis was explored using poliovirus as a model virus. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
A presentation entitled, "Fate and Transport of Pathogens from Biosolids-Amended Soils" describing the research on the fate and transport of pathogens in biosolids-amended soils was made to an international group of researchers at a meeting on Future Directions in Pathogens in Groundwater Research, sponsored by the Canadian Water Network in Calgary, Alberta, Canada in September 2008. A manuscript entitled, "A simple, flow cytometry-based assay for detecting poliovirus infection using mammalian cells expressing CFP-YFP protein pair undergoing FRET" by J. Jason L. Cantera, Wilfred Chen, Yu-Chen Hwang, and Marylynn V. Yates is being prepared for submission to a peer-reviewed scientific journal. This manuscript describes the results of our studies using flow cytometry to enable to early detection of infective polioviruses. As early as 12 hours post-infection, a significant number of infected cells were detected even when infected with 1 PFU. When applied to environmental water samples spiked with poliovirus, the FACS-based assay provided a similar level of sensitivity to the plaque assay for detecting and quantifying infectious virus particles. This approach, therefore, is more rapid than plaque assay, and can be used to detect other viruses that frequently do not form clear plaques on cell cultures.

Publications

• No publications reported this period