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IMPROVING DETECTION METHODS FOR ENTERIC WATERBORNE VIRUSES
Impact/Purpose:
Overarching Objectives and Links to Multi-Year Planning
This task directly supports the 2003 Drinking Water Research Program Multi-Year Plan's long term goal 2 to "develop new data, innovative tools and improved technologies to support decision making by the Office of Water on the Contaminant Candidate List and other regulatory issues" under GRPA Goal 2 (Clean and Safe Water). The overarching objective is to provide the Office of Water, Agency risk assessors and managers, academics, the scientific community, state regulators, water industry and industry spokes groups the methods they need to measure occurrence of waterborne viral pathogens. The method improvements will facilitate the development of risk-based assessments and tools used by the Agency to set regulations, policies and priorities for protecting human health and allow the Agency to assure the public that the appropriate methods are being used to demonstrate that drinking water is safe from pathogenic agents.
Specific Objectives
Subtask A: Improving sample collecting, virus concentration and sample preparation
o Develop a less expensive alternative to the Virosorb 1 MDS filter.
Subtask B: Molecular and Cultural Assays
o Investigate methods to improve the reverse transcription step in reverse transcription-polymerase chain reaction (RT-PCR) assays by when, for what purpose, for what client.
o Development of complete real-time assays that can be used for screening environmental samples.
o Develop a reporter gene cell culture system to indicate if virus infection has occurred without the use of molecular assays.
o Use improved cell culture lines to develop assays for nonculturable or poorly growing viruses.
Description:
Waterborne viruses are a significant cause of illness, both within the US and worldwide. These illnesses can occur as the result of outbreaks, potentially affecting hundreds or thousands of people, or as a part of a background level of endemic infection. While many of these outbreaks cause acute, short-term illness, some viral pathogens such as hepatitis A virus can be life threatening, especially to sensitive subpopulations. Methods are needed to rapidly detect viral pathogens in the case of an outbreak and to determine the level infectious virus in environmental and treated water in order to more accurately assess the level of risk for different populations.
To address these issues, a significant effort has been made by the US EPA to develop sensitive, specific methods able to detect a wide variety of viruses in environmental waters, including ones that are not currently culturable. This effort has been very successful, with cell culture or molecular assays having been designed for most of the viruses associated with waterborne illness. Methods are now available for viruses currently on the CCL, such as adenovirus, calicivirus, coxsackievirus and echovirus, as well as viruses that are likely to appear on future CCLs, such as astrovirus, hepatitis E virus (HEV), rotavirus, poliovirus, reovirus, hepatitis A virus and enterovirus. However, in order to develop these methods, expensive reagents and equipment were often required and the methods themselves are often cumbersome as well as technically challenging.
The goal of this task is to begin to modify these methods so that ultimately more water treatment systems and water sheds can be studied using inexpensive and easily usable tools. Currently, environmental sampling assays can be broken down into two main areas: the upstream part of the process, which involves the sample collecting, virus concentration and sample purification steps, and the downstream part of the method, which includes the molecular or cellular testing assays. Projects to improve sample preparation and detection are described under subtasks A and B, respectively.