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DETECTION OF OUTBREAK-ASSOCIATED HUMAN CALICIVIRUSES IN GROUNDWATER BY RT-PCR
Citation:
WillianTrue, S., S. Parshionikar, C Newport, D. Robbins, AND G S. Fout. DETECTION OF OUTBREAK-ASSOCIATED HUMAN CALICIVIRUSES IN GROUNDWATER BY RT-PCR. Presented at American Society for Virology Annual Meeting, Lexington, KY, July 20-24, 2002.
Impact/Purpose:
Overarching Objectives and Links to Multi-Year Planning
This task directly supports the Drinking Water Research Program Multi-Year Plan's long term goal to "develop scientifically sound data and approaches to characterize and manage risks to human health posed by exposure to waterborne pathogens and chemicals" 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 with exploratory occurrence and exposure data on human enteric viruses. These data 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 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 Conduct an exploratory occurrence studies on emerging human waterborne pathogenic viruses and viruses on the Contaminant Candidate List (CCL) in water (Subtask A; to be completed by 9/05 in support of LTG 1 (due 2010)).
o Determine the relationship of bacterial virus indicators to human enteric virus occurrence in the above studies (Subtask A; to be completed by 9/05 in support of LTG 1 (due 2010)).
o Develop a non-invasive assay for measuring human exposure to viruses (Subtask B; to be completed by 9/05 in support of LTG 1 (due 2010)).
Description:
Human caliciviruses (HuCV) are a major worldwide cause of food and waterborne outbreaks of acute nonbacterial gastroenteritis, and have been placed on the U.S. Environmental Protection Agency's (U.S. EPA) Contaminant Candidate List (CCL) of agents to be considered for regulatory action. This activity has led to the development of a method to measure HuCV occurrence in drinking water. Viruses are considered the likely agent in over half of the waterborne outbreaks, but they have rarely been detected. HuCV are non-culturable and can only be detected in concentrated water samples using RT-PCR. In 2001, the U.S.EPA participated in the investigation of two HuCV outbreaks in the state of Wyoming. The first outbreak was recorded at a wintertime vacation lodge in northern Wyoming and the second during the summer at a restaurant in central Wyoming. For each outbrak a well water sample was concentrated for virus using a 1MDS positively charged cartridge filter. Filters were eluted with beef extract and the eluates concentrated with celite. The concentrated eluates were treated to remove PCR inihibitors and then assayed by RT-PCR using several HuCV-specific primer sets. HuCV were detected in the concentrated water samples from both outbreaks. Sequencing of the cloned RT-PCR products demonstrated the presence of a genogroup II, subtype 1 HuCV in the first outbreak and a genogroup II, subtype 3 HuCV in the second outbreak. These results demonstrate the utility of the RT-PCR method for investigations of waterborne outbreaks of suspected viral origin.