United States Environmental Protection Agency Office of Research and Development
National Exposure Research Laboratory
FY02 Research Abstract
Government Performance Results Act (GPRA) Goal 2.1.7
APM79
Significant Research Findings:
Method for the Detection of Caliciviruses in Water
The Safe Drinking Water Act, as amended in 1996, required the U.S.
Environmental Protection Agency (EPA) to develop a list of unregulated
microbiological and chemical contaminants to aid in priority setting for the
Agency's drinking water program. The initial list of contaminants, called the
Contaminant Candidate List (CCL), was published in the Federal Register
(63 FR 10274) in 1998. Caliciviruses were included in this list because they
have been responsible for drinking water-related outbreaks of acute
gastroenteritis in the U.S. They also are highly infectious to people of all
ages and are believed to be one of the major causes of waterborne disease.
The study of these hardy viruses has been hindered by the lack of a suitable
assay for their presence in drinking water. They have not been cultured or
grown in any animal model. The lack of an assay has prevented EPA from
accurately characterizing the risk and developing effective strategies to
safeguard individuals from waters contaminated with these viruses.
Research The study's primary objective was to develop a molecular method to detect
Approach caliciviruses in drinking water. The study approach was to combine a
procedure to concentrate caliciviruses from water with a new reverse
transcription-polymerase chain reaction (RT-PCR) technique and to
evaluate the performance and reliability of the method by participating in an
interagency waterborne calicivirus outbreak investigation.
Results and Human caliciviruses are divided into two genera, Sapovirus and Norovirus.
Implications The Sapovirus group causes gastroenteritis in young children, but has not
been associated with waterborne outbreaks. The Norovirus group consists
of two major genogroups, each with a number of distinct genetic clusters.
Members of this group have been shown to be responsible for waterborne
disease outbreaks. Norwalk virus is a key representative of genogroup I.
This virus caused a waterborne outbreak in children in Norwalk, Ohio during
1968 and was the first human calicivirus identified. Snow Mountain virus
caused a waterborne outbreak in Colorado in 1976 and is a key
representative of genogroup II.
The key result of this study has been the development of a molecular
Scientific
Problem and
Policy Issues
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procedure for detecting the Norovirus group of caliciviruses in water. The
method utilizes a modification of a previously developed virus concentration
technique. Viruses are concentrated from drinking water by passing the
water through a positively charged cartridge filter. Caliciviruses present on
the filter are eluted with a non-flocculating beef extract solution and
concentrated from the eluate using a standard celite procedure. Virus
particles are further concentrated and treated to remove inhibitors of RT-
PCR. The RT-PCR procedure that was developed uses primer sets capable
of detecting more than 90% of known noroviruses.
The optimized method was used to investigate an outbreak that occurred in a
small public groundwater setting. Viruses present in the groundwater were
concentrated from a 946 liter water sample at the time of the last reported
case of illness and analyzed for caliciviruses. RT-PCR results demonstrated
that the groundwater was positive for noroviruses. Isolates obtained were
confirmed by sequencing and found to have sequences that were identical to
those in clinical specimens from individuals who were affected by the
outbreak. The water and clinical isolates belonged to a common genetic
cluster within genogroup II.
The results demonstrate that the method is suited for waterborne outbreak
investigations and, therefore, should also be useful to the Office of Water and
stakeholders in both outbreak investigations and occurrence/exposure
studies. The results of these types of studies will give EPA a better
understanding of conditions which lead to calicivirus contamination of
drinking water.
This research project directly supports the Office of Research and
Development's research to improve the scientific foundation for safe
drinking water under the Government Performance and Results Act (GPRA)
Goal 2 ("Clean and Safe Water"), Objective 2.1 ("Ensure Safe Drinking
Water and Recreational Waters"), Sub-Objective 2.1.7 ("By 2003, provide a
stronger scientific basis for implementation of the Safe Drinking Water Act")
and an FY02 GPRA annual performance goal ("Produce scientific reports on
unregulated drinking water contaminants, in support of the development of
the next list of chemicals and pathogens for potential regulatory action [i.e.,
Contaminant Candidate List #2\. These reports will help ensure that future
drinking water regulations address the contaminants of greatest public health
concern"). The research also directly supports annual performance measure
(APM) 79 ("Method(s) for CCL related pathogens in drinking water for use
in the Unregulated Contaminant Monitoring Rule (UCMR)").
Research This research was a collaborative effort between National Exposure
Collaboration Research Laboratory scientists in Cincinnati, the State of Wyoming, EPA
and Publications region VIII and two Centers within the Centers for Disease Control (CDC).
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The State, EPA region VIII, and CDC conducted site and epidemiological
investigations. EPA region VIII collected the water sample, and NERL
scientists developed the calicivirus method and performed the analysis.
The results of this research have been presented at two conferences and in
one manuscript (Publication No. NERL-CI-MCEARD-02-039):
Anderson, A.D., Heryford, A.G., Sarisky, J.P., Higgins, C., Monroe, S.S., Beard, S.R.,
Newport, C.M., Cashdollar, J.L., Fout, G.S., Robbins, D.E., Seys, S.A., Musgrave,
K.J., Bartkus, J., Vinje, J., Bresee, J.S., Mainzer, H.M., and Glass, R.I. "A
waterborne outbreak of Norwalk-like virus among snowmobilers - Wyoming,
2001." Accepted by the Journal of Infectious Disease.
Seys, S.A., Mainzer, H.M., Heryford, A.G., Anderson, A.D., Fout, G.S., Sarisky, J.P.,
Musgrave, K.J. "Coordinating environmental public health practice with
epidemiology and laboratory analysis: a waterborne outbreak of snow mountain
virus in the Big Horn mountains of Wyoming," presented at the International
Conference on Emerging Infectious Diseases, Atlanta, GA, March 2002.
Willian-True, S., Parshionikar, S., Newport, C., Robbins, D.E. and Fout, G.S. "Detection
of outbreak-associated human caliciviruses in groundwater by RT-PCR"
presented at the American Society for Virology annual meeting, Lexington, KY,
July 2002.
Future Research Unregulated Contaminant Monitoring Studies (UCMR) and other research for
caliciviruses have not been possible under CCL list #1 because of a lack of
adequate detection and measurement methods. The calicivirus method
described herein is an effective, but costly method for detecting human
noroviruses. Future research will focus on modifications aimed at reducing
assay costs and at developing a cultural assay for these viruses. In addition,
a microarray Gene Chip® approach to typing calicivirus isolates is being
developed. This method will allow rapid identification of calicivirus
isolates from UCMR studies and provide for source typing of waterborne
isolates with clinical specimens. Also being developed is an immunological
method for surveying the degree of human infection caused by caliciviruses
through the waterborne route.
Questions and inquiries can be directed to:
G. Shay Fout, Ph.D.
US EPA Office of Research and Development
National Exposure Research Laboratory
Cincinnati, OH 45268
Phone: 513/569-7387
E-mail: fout.shay@epa.gov
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National Exposure Research Laboratory — November 2002
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