Citation:

VILLEGAS, L. F. AN IMPROVED METHOD FOR DETECTING VIRUSES IN WATER. Presented at 2006 EPA Science Forum, Washington, DC, May 15 - 18, 2006.

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:

Enteroviruses are important etiological agents of waterborne disease and are responsible for outbreaks of gastroenteritis. However, the prevalence and occurrence of these pathogens in raw drinking water sources is poorly understood. This is primarily due to the limited methods available to detect enterovirus in water. Current detection methods often rely on two techniques; cell culture assays, which require long growth periods, or molecular assays, which do not provide information about viability. Combining these two techniques could result in the rapid detection of viable virus. Several integrated cell culture/RT-PCR techniques have been developed, however the method described here has several unique improvements. This approach involved filtering 200 liters of surface water through a 1MDS cartridge filter and eluting twice using 1.6 liters of 1.5% beef extract. The elutions were concentrated using celite, and furter concentrated by ultracentrifugation. The concentrated samples were then innoculated into two culture tubes of BGM cell monolayers, and allowed to grow for 1-7 days post-infection. Aliquots of the cell lysate were taken at different time points, and tested by qRT-PCR or RT-PCR while the remaining cell culture supernatant was used to innoculate a new monolayer, allowiong for further virus replication. Using this new method, 10 PFU of poliovirus can be detected by qRT-PCR within 24 hours while as little as 1PFU can be detected after 2 days of growth in the culture. Similarly, environmental samples spiked with 30 PFU can be detected in 24 hours by qRT-PCR. The ICC/RT-PCR method presented here provides a faster and more sensitive approach to detecting viable virus from environmental samples as compared to previous detection methods. Moreover, this method greatly minimized the loss of virus in samples by using a serial passage technique during the cell culture phase of the method, and the use of qRT-PCR greatly reduces the risk of molecular contamination.

Record Details: