United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-022 Jan. 1985
4MEPA Project Summary
Reoviruses in
Water Pollution Testing
Rex S. Spendlove, Bill B. Barnett, Dennis B. George, D. Jack Adams,
David N. Ridinger, John C. Roth, and Kamyar Zehedi
Removal of the outer reovirus coat
using proteolytic enzymes greatly
increases the infectivity of most
reoviruses prepared in cell culture. The
purpose of this study was to determine
the potential importance of
enzyme-enhanceable reoviruses in
water quality control testing. Proce-
dures were developed that would
recover and quantify infectious and
enzyme-enhanceable reoviruses in
sewage.
Twelve cell lines were tested to
determine their sensitivity to reoviruses
of three serotypes that had been
isolated from sewage. Madin-Darby
bovine kidney (MDBK) cells were the
most susceptible. Sewage-isolated,
protamine-precipitated reoviruses were
also used in conjunction with MDBK
cells in a comparative evaluation of
immunofluorescent cell count (ICC)
and plaque assay procedures.
Immunofluorescence assay is more
sensitive and more rapid than plaque
assay procedures for quantifying
reoviruses recovered from sewage.
A procedure for protamine sulfate
precipitation of reoviruses and
rotaviruses was developed. The use of
egg albumin with protamine sulfate
efficiently precipitates virus present in
sewage and allows assay of reovirus by
immunofluorescence. The virus-contain-
ing precipitate can be collected by low-
speed centrifugation rather than
filtration, which allows larger sample
volumes to be examined. The optimal
concentration of enzyme for enhancing
recovery of reoviruses and rotaviruses
precipitated from sewage was 200 fjg
of trypsin/ml.
During a one-year period, the
concentration of reoviruses was
compared with the concentration of
enteroviruses in sewage. The reoviruses
were consistently recovered in
approximately 5-fold higher
concentrations than the enteroviruses.
Reovirus infectivity is activated by
enzyme treatment in some sewage, but
not in other sewage. Some reoviruses
recovered from sewage were
inactivated by enzyme treatment.
Additional work is needed to fully
explore the conditions for enzyme
enhancement of reoviruses recovered
from sewage. However, even without
enhancement, reoviruses were isolated
from raw sewage in sufficiently high
numbers to suggest that reovirus
recovery procedures should be included
in the virological evaluation of
sewage-polluted water.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory, Research Triangle Park, NC. 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
Enteric viruses are known to occur rn
water that receives sewage effluents and
other wastewaters. There is, however, no
assurance that the procedures currently
being used for detecting viral pollution of
water are adequate or are the most
sensitive procedures available for quality
control testing. If the public is to be
protected from the hazards of viral
disease posed by waterborne exposure, it
is imperative that the best possible stand-
ard virus testing procedures be developed.
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This report describes a study to
evaluate reoviruses for their potential
usefulness in water quality testing. The
reasons for studying reoviruses were as
follows: (a) They occur abundantly and
consistently in sewage. (b)They are rarely
known to cause disease, so are relatively
safe to handle in the laboratory, and are
unlikely to become targets of
eradication by vaccination. Therefore,
standardized tests based on reovirus
detection will not have to be changed in
the future, (c) They have a broad host
range, so contamination by man and
lower animals is detected, (d) They are
amenable to low-cost isolation and iden-
tification procedures, (e) A single
antiserum will detect reoviruses of all
three serotypes. (f) Reoviruses are easily
cultivated to high infectious liters, (g)
Reovirus hemagglutination inhibition
tests can be used to assay antibodies in
epidemiologic studies, (h) The vast
majority of reoviruses are present in a
potentially infectious form which is not
normally detected by conventional
infectivity assays. These assays detect
only the infectious form unless the virus
is exposed to proteolytic enzymes which
remove the outer coat of the virus, and
convert potentially infectious virus (PIV)
to infectious virus (IV). The intact double
coat of potentially infectious reoviruses
makes them exceptionally resistant to
inactivation. Consequently, if they are
inactivated, other viruses present in
water are likely to be inactivated, (i) The
potential importance of enzyme-
enhanceable reoviruses as an indicator of
viral pollution in water had never been
assessed.
Conclusions
Madin-Darby bovine kidney (MDBK)
cells, rhesus monkey kidney cells (LLC-
MK2) and human embryonic intestinal
cells (lntestinal-407) were the most
susceptible cell lines, respectively, for
assaying reovirus serotypes 1, 2, and 3
previously isolated from sewage. The
MDBK cells were the most sensitive
when compared to LLC-MK2 cells and
Buffalo green monkey kidney (BGM) cells
for assaying reoviruses that had been
precipitated from sewage by protamine
and had never been exposed previously to
cells in culture.
The ICC method was found to be more
sensitive, more specific and more rapid
than the plaque assay procedure for
assaying reoviruses isolated from
sewage. The optimum inoculum size and
centrifugation conditions were an
inoculum of 0.25 mL centrifuged at 3000
rpm for 30 minutes in a Sorvall microtiter
plate rotor.
When some protamine sewage precip-
itates were assayed for reoviruses, the
potentially infectious viruses (PIV) were
present in higher concentrations thanthe
infectious viruses (IV). It appears that we
encountered some chymotrypsin
negative (CT~) mutants in sewage in our
study. The CT~ reovirus mutants are
inactivated by chymotrypsin treatment.
Some sewage appeared to convert PIV to
IV. This would explain why it was not
possible to enhance the infectivity of
some reoviruses that had been extracted
from sewage, i.e., the enhancement
occurred while the virus was ir> the
sewage.
During a one-year period, the concen-
tration of reoviruses was compared with
the concentration of enteroviruses in
sewage. The reoviruses were consist-
ently recovered in approximately 5-fold
higher concentrations than the entero-
viruses.
Recommendations
1. Reoviruses should be included
when a standard test procedure for
detecting viruses in water is
adopted since this study and
previous investigations have found
that reoviruses are consistently
present in sewage.
2. For recovery of reoviruses from
water, additional studies need to be
conducted to determine the best
procedure. The procedure should be
inexpensive and trouble free and
capable of processing large volumes
of water.
3. The immunofluorescent cell count
and MDBK cells should be used in
the assay of sewage for reoviruses.
4. The recommendation regarding
enzyme treatment of reoviruses in
water quality testing is that
additional studies be conducted.
The effect of adding EDTA directly to
the sewage at the time of collection
to protect any CT mutants from
inactivation by enzymes in the
sewage should be determined. This
procedure, unfortunately, would
inactivate rotaviruses.
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R. S. Spendlove. B. B. Barnett. D. B. George, D. J. Adams, D. N. Ridinger, J. C. Roth,
andK. Zehedi are with Utah State University, Logan, UT 84322.
Elmer W. Akin is the EPA Project Officer (see below}.
The complete report, entitled"Reoviruses in Water Pollution Testing," (Order No.
PB 85-125 847; Cost: $10.00, 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:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
•tf U S GOVERNMENT PRINTING OFFICE, 1983 — 559-016/7874
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
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