United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
*
Research and Development
EPA/600/S4-87/029 Mar. 1988
Project Summary
Improved Methods for
Hepatitis A Virus and Rotavirus
Concentration and Detection in
Recreational, Raw Potable, and
Finished Waters
Joseph L. Melnick
A nucleic acid hybridization test
for the detection of hepatitis A virus
and an immunoftuorescence test for
the detection of human rotavirus are
described. Also specifically detailed
are the sampling, concentration and
reconcentration steps for processing
water, wastewater and associated
solids for these viral pollutants.
Although the subject matter is
delineated in a concise step-by-
step form to expedite its usability,
the methods described are highly
sophisticated technology directed
toward the research structured
laboratory.
This Project Summary was
developed by EPA's Environmental
Monitoring and Support Laboratory,
Cincinnati, OH, to announce key
findings of the research project that
is fully documented in a separate
report of the same title (See Project
Report ordering information at back).
Introduction
This research project has resulted in
the development of detection
methodology for hepatitis A virus and
human rotavirus in the water
environment. The standardized
procedures developed by Baylor College
of Medicine are set forth in the report in
considerable detail, so that it can be
better utilized as a laboratory bench
guide. This product is part of the
Laboratory's program to upgrade
monitoring methodology for the detection
and enumeration of viral pollutants in
water, wastewater, and associated solids.
The development of these particular
procedures is important because human
rotavirus and viruses of the hepatitis A
type are difficult or impossible to detect
by current plaque assay procedures.
Recpgnizing the occurrence of
recreational water-associated type A
hepatitis has depended upon
development of clinical illness in
swimmers or bathers and probably does
not reflect the true incidence of this
epidemiologic route. In spite of
recognition and reporting deficiencies,
hepatitis A virus-associated illness is
still the most prevalent type of perceived
waterborne disease.
Human rotaviruses are excreted in
large numbers in the stools of infected
individuals. They survive sewage
treatment and are released in waste
treatment plant effluents. Availability of a
monitoring system for detection of
rotavirus is important because these
fastidious viruses appear to be a major
cause of childhood diarrhea.
Results
Procedures described in this report
include sample collection, sample
concentration and viral titration for the
detection of hepatitis A virus and human
rotavirus. They have been found to be
well suited to disclose the presence of
these virus types in varying qualities of
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water. The protocol is applicable in
surface and finished waters. Finished
water samples may be collected from
distribution system outlets (e.g. faucet) or
water mains at a municipal water
treatment plant. Recreational or raw
potable water samples that may be
collected include water, solids
suspended in water, or the uppermost
loose layers of bottom sediments, called
fluffy sediments. Water and suspended
solids may be collected and processed
as a single sample or collected and
processed separately. Fluffy sediments
are collected and processed separately.
The virus adsorption-elution (VIRADEL)
method as described in Chapter 6 of the
USEPA Manual of Methods for Virology
(EPA-600/4-84-013, February 1984)
was used successfully for the processing
of water samples. Water samples
processed for suspended solids or fluffy
sediments are passed through filters that
served as solids-collecting surfaces
Solids-associated viruses were
recovered by elution followed by
reconcentration of eluates.
Procedures for detecting rotaviruses
are dependent on cell culture
amplification of one or more viruses in
test sample. Assaying for their presence
is based on an immunofluorescence test
using a monoclonal antibody and
fluorescein-isothiocyanate-conjugated
antibody staining procedure to visualize
virus-infected cells.
The techniques described for
detecting hepatitis A virus are based on
the use of labeled nucleic acid (gene)
probes in hybridization tests to detect
virus nucleic acid. Guidelines are
provided for preparation of
complementary DNA and single-
stranded RNA probes used for their
detection.
Conclusions and
Recommendations
The described methods should enable
detection of hepatitis A virus and human
rotavirus in polluted surface waters to a
degree not currently possible. Where
found, solids-associated virus in
conjunction with the developed
methodology can be exploited for
separation and recovery of these viruses.
Improved methods for detection of
hepatitis A virus and human rotavirus in
water represent tools which can be used
to offer better protection against the
danger of waterborne virus infections.
Environmental assessment of
recreational, raw potable and shellfish
waters can be extended to include these
viruses. Decisions about the
effectiveness of water or wastewater
treatment procedures for elimination of
hepatitis A virus and human rotavirus can
be based on results of tests m which
these pathogens rather than simulant
viruses were used.
The use of labeled nucleic acid
probes for assaying for the type A
hepatitis virus places this work at the
cutting edge of virus detection
technology. It should, therefore, be
recognized that any undertaking of this
test requires laboratory personnel with a
background in molecular biology and a
knowledge of molecular cloning theory
and practice. Because of the
considerable degree of specialization
needed, this technology currently is
limited to highly specialized research
facilities and thus is not within the grasp
of today's water monitoring laboratory.
Nevertheless, it is an important step
forward in virus detection technology and
could form the foundation for the
production of commercially available,
off-the-shelf, gene probe test kits for
hepatitis A virus, as well as other enteric
viruses.
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Joseph L Melnick is with Baylor College of Medicine, Houston, TX 77030
Daniel R. Dahling is the EPA Project Officer (see below).
The complete report, entitled "Improved Methods for Hepatitis A Virus and
Rotavirus Concentration and Detection in Recreational, Raw Potable, and
Finished Waters," (Order No. PB 88-158 9441 AS; Cost: $14.95, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring and Support Laboratory'
U.S. Environmental Protection Agency
Cincinnati, OH 45268
I
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
JULI9-
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Official Business
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EPA/600/S4-87/029
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OFFICE: 1988— 548-013/8702(
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