Citation:

FOHL, L. AND G. FOUT. DEVELOPMENT OF AN INTEGRATED CELL CULTURE/RT-PCR METHOD FOR THE DETECTION OF ENTEROVIRUS IN WATER. Presented at US EPA Post-doc Poster Session, Cincinnati, OH, March 30, 2005.

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 1 for "regulated contaminants" and long term goal 2 for "unregulated contaminants and innovative approaches" 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 methods developed will improve the quality of risk-based assessments and tools used by the Agency to set regulations, policies and priorities for protecting human health and will 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 Subtask Objectives:

o Evaluate techniques for enhancement of growth of human enteric viruses in support of CCL #2 and #3 and for use in the UCMR (Subtask A; to be completed by 9/05 in support of LTG 2)

o Develop a multiplex RT-PCR method that incorporates internal controls for use in the UCMR (Subtask B; completed 9/03 in support of LTG 2)

o Develop and evaluate new molecular technologies for use in the UCMR. Included will be real-time RT-PCR methods for Norwalk virus and astroviruses, and integrated cell culture/molecular procedures for detection of infectious viruses (Subtask B; to be completed by 9/05 in support of LTG 2)

Description:

Virus contamination in environmental samples is believed to be underestimated due to the limitations in the methods available for detection. A major detection method is based upon the formation of cytopathic effect (CPE) in cell culture. The main limitations to this method are the amount of time that is required to detect CPE formation and the fact that not every virus produces CPE or is culturable. Molecular assays have been developed for the detection of virus genomic material; however, the presence of virus RNA does not indicate that infectious virus is present. Also, water samples often contain substances that inhibit molecular assays.

One method which will overcome the major limitations of the CPE-based and molecular assays is integrated cell culture-RT-PCR (ICC/RT-PCR). This work describes a method for doing ICC-RT-PCR on water samples of 200L and greater. This involves using 1MDS filters to collect the virus from water, eluting the virus from 1MDS filters with beef extract, and concentrating the sample using EPAs celite method. Secondary concentration is performed by ultracentrifugation and the resulting pellet is resuspended and placed on a cell monolayer. Cell lysates are collected and used as template for RT-PCR and quantitative real time RT-PCR (qRT-PCR) assays for the detection of virus RNA.

This method has resulted in an 89.8 and 66.4% total recovery after the ultracentrifugation step on two independent trials. Once these pellets were inoculated onto cell monolayers, virus RNA was detected in 24 hours using both qRT-PCR and RT-PCR. CPE was not detected in these samples until day 3 and was not present in all the samples until day 4. This study was performed using spiked distilled water and will be validated using environmental samples.

Record Details: