NERL Research Abstract

EPA's National Exposure Research Laboratory

GPRA Goal 2.1.7
FY 2003 Annual Performance Measure (APM) #101

Significant Research Findings

Develop Methodology to Identify and Characterize
Caliciviruses and Sources of Human Pathogens in Water

Scientific Problem and The Safe Drinking Water Act, as amended in 1996, requires EPA to
Policy Issues	develop a list of unregulated microbiological and chemical

contaminants to aid in setting priorities for the Agency's drinking water
program. The initial list of contaminants, called the Contaminant
Candidate List (CCL), was published in 1998 in the Federal Register
(63 FR 10274). Caliciviruses were included in this list because they
have been responsible for drinking water-related outbreaks of acute
gastroenteritis in the U.S. Caliciviruses are divided into four
genera—Norovirus, Sapovirus, Vesivirus and Lagovirus. Vesiviruses
and lagoviruses infect only animals. Sapoviruses infect humans, but are
genetically and physically closely related to the two animal genera.
Noroviruses are the major cause of waterborne disease as they are
highly infectious to people of all ages. Recently, norovirus strains were
discovered in cattle with diarrhea. This discovery led to concern about
the possible zoonotic spread of these viruses between cattle and
humans.

The study's objectives were to determine if any sapovirus or norovirus
strains are present in local Ohio cattle herds and to determine the
relationship of any strains found to known human noroviruses. The
approach was to examine pooled fecal samples from local herds using a
molecular assay. For samples that contain viruses, this molecular assay
produces many DNA copies of a part of the virus genome. These DNA
copies were then sequenced. Because the sequences of the virus
genomes are unique for different strains, those found in the fecal
samples of local herds could be used 1) to prove that a positive result
was really caused by a sapovirus or a norovirus, and 2) to show the
relationship of the strains found to other animal and human strains.

Research
Approach

Results and
Implications

The molecular assay was used to screen RNA extracted from pooled
fecal samples from four veal calf herds. Noroviruses were found in

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three of the herds and sapoviruses in four. The sequences that were
found in these samples were used to improve the molecular assay, and
the modified assay was used to test 75 veal calf fecal samples. 72% of
these were positive for noroviruses. A subset of 21 of the positive
samples was sequenced and confirmed to be human-like noroviruses.
This study shows that noroviruses are frequently found in Ohio cattle
herds. Although the strains that were found were very similar to human
strains, they were not identical and are likely to be bovine-specific.
However, zoonotic transmission from cattle to humans cannot be ruled
out.

This research project directly supports ORD's efforts to improve the
scientific foundation for safe drinking water. The results of this
research support the Government Performance and Results Act Goal 2
("Clean and Safe Water"), Objective 2.1 ("Ensure Safe Drinking Water
and Recreational Waters"), and Sub-Objective 2.1.7 Long Term Goal 2
("By FY 2010, 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, and
implementation of rules by states, local authorities and water utilities").
It was done in support of an FY03 GPRA annual performance goal
("The Office of Water will have data, methods, assessments and
technology evaluations necessary to support scientifically sound risk
assessment and risk management decisions on unregulated
contaminants of potential public health concern") and annual
performance measure #101 ("Develop methodology to identify and
characterize H. pylori, caliciviruses and sources of human pathogens in
water").

Research Collaboration This research was performed under a collaborative agreement with the

and Publications	Ohio State University.

The findings of this research have been published (Publication No.
NERL-CI-MCEARD-02-059):

Smiley, J.R., A.E. Hoet, M. Traven, H. Tsunemitsu and L.J. Saif. 2003.
Reverse transcription-PCR assays for detection of bovine enteric
caliciviruses (BEC) and analysis of the genetic relationships among
BEC and human caliciviruses. J. Clin. Microbiol. 41, 3089-3099.

Future Research	An important outcome of this study is the finding that some molecular

assays designed to detect human noroviruses in environmental waters
can also detect the bovine strains. Laboratories analyzing these waters
need to take this into account and modify the assays to minimize this
source of potentially false-positive results. Additional research may be
needed to verify that the modifications are sufficient.

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Contacts for
Additional Information

Questions and inquiries can be directed to:
G. Shay Fout, Ph.D.

US EPA

National Exposure Research Laboratory
Cincinnati, OH 45268
Phone: 513/569-7387
E-mail: fout.shay@epa.gov

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