Description:
Enteric viruses cause waterborne disease outbreaks in the U.S. and worldwide. The primary focus of this task is to develop methods to measure the occurrence of enteric viruses in environmental and drinking waters. Cell culture- and molecular-based methods are being developed for the detection of the viruses currently on EPA's Contaminant Candidate List (CCL) and for emerging viruses likely to be on future CCL lists. These methods will give the Office of Water key tools needed to meet the legal requirements of the Safe Drinking Water Act and will provide the necessary procedures for conducting virus surveys under the Unregulated Contaminant Monitoring Rule (UCMR).
The viruses that are on the current CCL are the adenoviruses, caliciviruses, coxsackieviruses, and echoviruses. Viruses that are projected to be on future CCL lists include astroviruses, hepatitis A and E viruses and rotaviruses. Such viruses are on the CCL list because they can be found in sewage and environmental waters and because they have caused, or are likely to cause, waterborne disease outbreaks. Diseases associated with these viruses include: gastroenteritis, respiratory and ocular disease, encephalitis, meningitis, myocarditis, diabetes, and temporary paralysis.
This task is divided into three subtasks: subtask A - Virus Sampling, Processing and Culturable Methods; subtask B - Molecular and Integrated Cell Culture-PCR Method for Virus Detection; and subtask C - Development, Evaluation and Standardization of Bacterial Virus Detection Methods. Research under subtask A will lead to new or enhanced procedures designed to reduce the cost of concentrating viruses from water, to permit higher virus recovery rates from various water matrices and to enable the culturing of fastidious viruses. Research under subtask B will lead to standardized molecular reverse transcription-polymerase chain reaction (RT-PCR) assays. Research will also be directed towards procedures for determining whether a virus is infectious. Methods will be optimized for maximum sensitivity while minimizing false results and these will be field evaluated using water samples collected from different regions. Research under subtask C will be to evaluate bacterial virus detection methods in support of Agency regulatory efforts (Ground Water Rule).
Keywords:
ADENOVIRUS, CALICIVIRUS, CCL, COLIPHAGE, COXSACKIEVIRUS, ECHOVIRUS, EMERGING PATHOGENS, HEPATITIS A VIRUS, HEPATITIS E VIRUS, HUMAN ENTERIC VIRUSES, METHODS, NORWALK VIRUS, REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR), ROTAVIRUS, WATER,
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Project Information:
Progress
:Subtask A - Virus Sampling, Processing and Culturable Methods
Positively-charged 1MDS filters are used to concentrate human enteric viruses from environmental and drinking waters. A new method to elute virus from the filters and reconcentrate them with celite was developed and published. The method gave a consistent 95±3% recovery of seeded poliovirus.
Two new chapters describing virus detection technology have been appended to the USEPA Manual of Methods for Virology. Chapter 14 describes the use of a positively charged filter to concentrate viruses from water. Chapter 15 describes a quantal method for assaying culturable waterborne human enteric viruses that is more sensitive than the plaque assay. The methods described in Chapters 14 and 15 primarily detect infectious enteroviruses and reoviruses and includes the detection of coxsackieviruses and echoviruses, two groups listed on the CCL.
The development of a method to reuse the 1 MDS filter has been completed and will be submitted for publication in the near future. This approach has been demonstrated to be an effective way to reduce the overall cost per assay.
Several cell lines and culture methods have been evaluated for their ability to produce CPE in response to adenovirus infection. A paper describing this work will be submitted in FY05.
Subtask B - Molecular and Integrated Cell Culture-PCR Method for Virus Detection
A new method to remove inhibitors of the RT-PCR assay was developed and published. The effectiveness of the new RT-PCR method was demonstrated in a cooperative study with the U.S. Geological Survey using five of their National Water Quality Assessment sites. A manuscript describing the study has been accepted for publication. A rapid molecular method to identify hepatitis E viral strains in drinking and source waters was developed and published.
A rapid molecular method developed to detect caliciviruses water was used to identify Norwalk-like caliciviruses in well water following two waterborne outbreaks. In each case the calicivirus strain from well water was sequenced and found to be genetically identical to patient isolates from the outbreak. The results of both outbreak studies have been published.
Internal controls for RT-PCR reactions have been successfully developed for enteroviruses (e.g., coxsackieviruses and echoviruses), rotaviruses, hepatitis A and E viruses and Norwalk virus. The results of this study were presented at the American Society for Microbiology Annual meeting and a manuscript has been submitted for publication.
An integrated cell-culture/RT-PCR method to detect astrovirus has been developed and published. In it the relative sensitivity of RT-PCR, real-time RT-PCR and integrated cell culture RT-PCR was compared. In addition, the sensitivity of the molecular assay was determined.
Subtask C - Development, Evaluation and Standardization of Bacterial Virus Detection Methods
A method on the detection of coliphages has been reviewed and accepted for publication in Standard Methods for the Examination of Water and Wastewater. The method includes the following protocols: somatic coliphage assay, male-specific coliphage assay using Escherichia coli Famp, male-specific coliphage assay using Salmonella typhimurium WG49, single agar layer method, and membrane filter method for male-specific coliphages.
A new chapter describing bacteriophage detection technology have been appended to the USEPA Manual of Methods for Virology. Chapter 16 describes procedures for the detection of coliphage in water matrices. Two quantitative procedures and one qualitative presence-absence procedure are presented. The procedures described have an important consideration as a general indicator of water quality.
Relevance
: The methods being developed under this task will allow EPA to collect occurrence and exposure data on human enteric viruses, which is an identified need in EPA's Research Strategy for the Drinking Water Contaminant Candidate List. In addition, the research under Subtask C supports the Groundwater Rule by improving the sensitivity of the current coliphage methods.
Clients
:Office of Ground Water and Drinking Water (Dr. Paul Berger)
Project IDs:
ID Code
:12485
Project type
:OMIS