f/EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S1-81-061 Feb. 1982
Project Summary
Evaluation of the Tentative
Standard Method for Enteric
Virus Concentration and
Detection
Mark D. Sobsey
The tentative Standard Method
(TSM) for concentrating enteric viruses
from large volumes of drinking water
and other finished waters was evalu-
ated for nine enteric viruses using
100-gal (378-I) tap water volumes
experimentally contaminated with
about 100 plaque-forming units (PFU)
of virus. The mean recoveries of
poliovirus 1. coxsackieviruses A9 and
B3, echovirus 7, bovine enterovirus 1,
Minute Virus of Mice (parvovirus),
reovirus 3, adenovirus 5 and simian
adenovirus 11 were 22, 25, 2.5, 6.7,
22, 5.0, <1, <1 and <1 %, respectively
when the viruses were evaluated
individually in succession. Although
the recovery differences for the five
enteroviruses suggested variable TSM
effectiveness depending upon virus
type, subsequent TSM experiments in
which these viruses were simultan-
eously tested showed no significant
recovery differences, averaging 16%
(±2.7, 95% Cl) overall. TSM recoveries
for purified, dispersed mixtures of
enteroviruses obtained from raw
sewage and seeded into 100-gal
volumes of tap water averaged only
3.9% which was approximately the
same as poliovirus recoveries during
the same time period.
Enterovirus recoveries showed marked
seasonal variations that may be
related to water quality changes.
Reduced virus recovery was partly due
to interference with virus adsorption
to filters caused by soluble or colloidal
tap water constituents that were
removable by granular activated
carbon. The effects of these inter-
ferences could be overcome by adding
aluminum or magnesium chloride to
the tap water to enhance virus ad-
sorption.
Low virus recoveries by the TSM
were also due to virus inactivation by
the pH 11.5 glycine-NaOH used for
eluting adsorbed viruses from filters.
In this eluent the T-90 values (time for
90% virus inactivation) for Minute
Virus of Mice, poliovirus, reovirus and
the simian adenovirus SV-11 were 20,
1.8, <1 and <1 minutes, respectively.
Virus recoveries with TSM and other
negatively charged filters could be
markedly improved by various pro-
cedural modifications: (1) the use of
aluminum or magnesium chloride to
enhance virus adsorption, (2) the use
of less alkaline glycine or beef extract
both with and without EOTA for
elution of viruses from primary ad-
sorbents, and (3) recirculation of the
eluent through the adsorbent filters.
With the exception of MVM, viruses in
beef extract eluents could be efficiently
reconcentrated by organic flocculation.
Improved virus recoveries were also
obtained with adsorbent filters that
are more electropositive than TSM
filters. Zeta Plus filters that are
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positively charged up to about pH 5 to
6 adsorbed >99% of various viruses
from tap water at ambient pH levels of
7.0-7.5 without added polyvalent
cation salts. Most viruses could be
effectively eluted with glycine-NaOH,
pH >9.5 or beef extract. Low levels of
poliovirus in 100-gal (378-I) volumes
of tap water were more effectively
recovered with Zeta Plus filters than
with the TSM using Cox filters.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Cincinnati, OH, to an-
nounce 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 and Background
The potential transmission of human
enteric viruses via drinking water and
other waters is of considerable public
health concern. The viruses of major
concern in water are those that infect
the alimentary tract of man and in some
cases animals, and are excreted in large
quantities with the feces by infected
persons. These are the so-called enteric
viruses, and they include members of
the following taxonomic groups: (1) en-
teroviruses, (polio-, coxsackie- A and B
and echoviruses), (2) reo- and rotaviruses,
and (3) adenoviruses as well as the
Norwalk-type agents and Hepatitis type
A, which are not yet taxonomized due to
limited information on morphology and
chemical composition.
The continued occurrence of water-
borne outbreaks of Hepatitis A and
perhaps other virus diseases such as
gastroenteritis as well as the apparent
isolation of enteric viruses from drinking
water supplies in the U.S. and elsewhere,
indicate the need for a more thorough
and systematic evaluation of the po-
tential for virus disease transmission
via drinking water. Evaluation of this
problem requires the development and
utilization of simple and reliable methods
to quantitatively detect relatively small
amounts of enteric viruses in large
volumes of raw and finished drinking
water supplies.
Research efforts on methods to detect
enteric viruses in water have produced
two-step microporous filter adsorption-
elution methods that are sometimes
capable of concentrating small numbers
of enteroviruses from 100 or more
gallons of drinking water, and a tentative
method of this type was introduced into
the 14th edition of Standard Methods.
In this method the water is pressure
filtered through microporous (0.45/u to
8.0 /urn pore size) filters having large
surface areas to which viruses adsorb
by a reversible reaction. The adsorption
reaction is thought to be primarily
electrostatic in nature, although weak
attractive forces, such as hydrogen
bonding, dipole-dipole interactions and
hydrophobic effects may also be in-
volved. In order to enhance virus
adsorption to the filter sufaces, appro-
priate ionic conditions are created in the
flowing water by the controlled addition
of acid and sometimes polyvalent cation
salts prior to passage of the water
through the adsorbent filter. After
processing the desired volume of water,
the adsorbed viruses that have accumu-
lated on the filter surfaces are eluted
with approximately one liter of an
alkaline buffer solution consisting of pH
11.5 glycine-NaOH. The eluate obtained
is readjusted by the addition of acid and
Aids to ionic conditions that enhance
virus adsorption: pH 3.5 and .0005 M
Aids. The viruses are then adsorbed to a
second, smaller (47-mm diameter)
microporous filter series composed of
fiberglass, asbestos and epoxy. The
adsorbed viruses are then eluted from
this filter with several milliliters of
alkaline buffer solution. The virus-
containing eluate is adjusted to a
physiological pH and isotonicity and
then assayed for viruses in cell cultures
using conventional virological tech-
niques.
The development of flow-through
filter adsorption-elution systems for
concentrating small quantities of viruses
from large volumes of water represents
a much needed technical achievement.
However, many of the key variables
involved in this type of methodology
have not been adequately evaluated,
and some of the procedures and
equipment in current use have been
somewhat arbitrarily selected. A rational
and systematic evaluation of the
methodology and materials is needed.
In particular, it has not been demon-
strated that the tentative Standard
Method (TSM) is capable of efficiently
detecting viruses of all major enteric
virus groups including Hepatitis A virus.
The method was developed and eval-
uated primarily for enteroviruses,
especially poliovirus, and there is
information in the literature indicating
that the method may be incapable of
recovering some other enteric viruses.
For example, in the application of filter
adsorption-elution methods to adeno-
viruses, one study found that adeno-
virus type 5 was inactivated by the
alkaline glycine-NaOH used for elution
of adsorbed viruses.
Another deficiency concerning eval-
uation of the TSM is that the method has
not been evaluated over a sufficient
time period with the same viruses and
water source to determine if changes in
water quality influence its effectiveness.
Dissolved and colloidal substances in
water may interfere with virus recovery,
and the types and concentrations ol
such interfering substances may
over time.
Objectives
General Objective
To rigorously and systematicall
evaluate the tentative Standard Methc
for concentrating and detecting enter
viruses in large volumes of drinkir
water and other finished waters.
Specific Objectives
To evaluate the tentative Standar
Method for detecting and concentratin
enteric viruses from tap water using th
four different types of microporous f ilte
systems that are currently recommendei (
as virus adsorbents and representative
viruses from the major enteric viru:
groups. The four primary virus adsorben
systems being evaluated are: (a)Balstor
fiberglass-epoxy filter tubes, (b) Co>
fiberglass-asbestos-epoxy filter discs
(c) Mi III pore cellulose nitrate mem-
branes, and (d) Commercial Filters
fiberglass cartridge filters and Cox
fiberglass-asbestos-epoxy filter discs in
series. A total of nine different viruses
are being tested: poliovirus, coxsackie-
viruses A9 and B3, echovirus 7, bovine
enterovirus 1, reovirus 3, simian
adenovirus SV-11, (human) adenovirus
5 and Minute Virus of Mice (an enteric
parvovirus).
After evaluating the tentative Standard
Method with the nine different laboratory
strains of enteric viruses, the method
will then be evaluated using mixtures of
naturally occurring enteric viruses
obtained from sewage.
If the present tentative Standard
Method is found to be incapable of
efficiently recovering certain enteric
viruses, then modifications of the
presently recommended methodology
will be evaluated for their ability to
efficiently detect representatives of all
enteric virus groups.
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Conclusions
1. The current tentative Standard
Method (TSM) for enteric virus
concentration from drinking water
and other finished waters has
serious deficiencies causing it to
give poor virus recoveries.
2. One major deficiency of the TSM is
that the pH 11.5 glycine-NaOH used
to elute adsorbed viruses from filters
rapidly inactivates adenoviruses and
reoviruses. It also inactivates en-
teroviruses and parvoviruses, but
not as rapidly. Alternative eluents
which employ less alkaline (pH 9.5-
10.5) conditions in glycine, pro-
teinaceous media such as 0.3-3%
beef extract, chelating agents such
as EDTA, or combinations of these
conditions show considerable
promise for all major enteric virus
groups. Virus elution efficiency is
further improved when these alter-
native eluents are recirculated
through the adsorbents, thereby
increasing the contact time.
3. Another serious deficiency of the
TSM causing reduced virus recover-
ies is that soluble and/or colloidal
substances in finished tap watercan
interfere with virus adsorption to
conventional, electronegative TSM
primary virus adsorbents. There
appear to be temporal changes in
magnitude of these interfering
effects that may be related to
seasonal changes in the nature and
amount of interfering substances in
tap water. The effects of these
interfering substances can be over-
come by adding polyvalent cation
salts, such as MgCI2 and AICI3, to the
tap water to enhance virus adsorption
to TSM primary virus adsorbents and
the Filterite pleated cartridge fiber-
glass-epoxy filter.
4. The four alternative primary virus
adsorbents of the TSM do not differ
significantly in terms of virus recovery
efficiency, although recoveries with
the Balston filter were frequently
lower than those with the other three
adsorbent systems. The primary virus
adsorbent systems did differ in terms
of their susceptibilty to clogging,
with the Millipore and Carborundum-
Cox systems being more susceptible
than the Balston and Cox systems.
5. The results of this study suggest that
all enteroviruses are apparently
recoverable with equal effectiveness
by the TSM. This conclusion is based
on finding: (1) that the recovery
efficiencies for five different labor-
atory-grown enteroviruses did not
differ significantly in simultaneous
experiments and (2) that recovery
efficiencies for natural enterovirus
mixtures obtained from sewage
were not significantly different from
recovery efficiencies for exogenously
added, laboratory-grown poliovirus
during the same time period.
6. Microporous filters that are more
electropositive than the current TSM
filters and are composed of dia-
tomaceous earth-cellulose-"charge
modified" resin mixtures appear to
offer distinct advantages as virus
adsorbents because: (1) they effi-
ciently adsorb viruses from tap water
at neutral pH without added poly-
valent cation salts, (2) they are less
affected by soluble and/or colloidal
interferences in tap water that
reduce virus adsorption, and (3) with
• the possible exception of adeno-
viruses, adsorbed viruses can be
readily eluted with either pH 9.5 to
10.5 glycine-NaOH or 0.1 to 3% beef
extract, pH 9 to 10.
7. Highly electropositive microporous
filters composed of asbestos-cellu-
lose mixtures may also be effective
virus adsorbents because they effi-
ciently adsorb viruses over a wide pH
range without added salts and the
adsorbed viruses can be eluted with
pH 9 to 10 beef extract.
Recommendations
1. Because the current TSM for con-
centrating enteric viruses from
drinking water and other finished
waters is relatively ineffective,
improved filter adsorption-elution
methods that will be suitable and
effective in field use should be
developed and evaluated.
2. Further efforts should be made to
develop and evaluate alternative
eluents to pH 11.5 glycine-NaOH. In
particular, the use of glycine-NaOH
at lower pH levels and 0.1 to 3% beef
extract with and without chelating
agents such as EDTA or other
additives should be investigated.
3. Further studies should be done with
beef extract and other proteinaceous
eluents to improve the organic
flocculation procedure for parvo-
viruses.
4. Because polyvalent cation salts such
as MgCI2 and AICI3 are capable of
overcoming the effects of the soluble
and/or colloidal interferences in tap
water that reduce virus adsorption
efficiency, it may be prudent to
routinely use salt enhancement of
virus adsorption, unless it can be
experimentally demonstrated the
efficient virus recoveries from large
volumes of a particular water are
obtained without added salts.
5. Electronegative fiberglass-epoxy
pleated cartridge filters (Filterite)
should be considered as a fifth
alternative primary virus adsorbent
because: (1) they have a large
surface area and, hence, a large
filtration-volume capacity for water;
(2) they efficiently adsorb viruses
under the same conditions as TSM
filters, and (3) the adsorbed viruses
are elutable with pH 10.5 glycine-
NaOH and beef extract.
6. Additional studies should be done to
further develop and evaluate im-
proved and simplified filter adsorp-
tion-elution methods for virus con-
centration from water based on the
use of electropositive adsorbent
filters.
7. The physico-chemical mechanisms
of virus adsorption to and elution
from filters and other surfaces
should be investigated in order to
provide a rational basis for the
further development and optimization
of filter adsorption-elution methods
to concentrate enteric viruses from
water. In addition, the chemical
characteristics and behavior of the
dissolved and/or colloidal substances
in tap water that interfere with virus
adsorption to filters should also be
fully investigated in order to develop
optimum methods for their elimina-
tion or control.
The results of this investigation have
appeared in three publications: "Con-
centration of Poliovirus from Tap Water
Using Positively Charged Microporous
Filters" by M.D. Sobsey and B.L. Jones,
Appl. and Environ. Microbiol. 37(3):588-
95 (Mar. 1979); "Evaluation of the
Tentative Standard Method for Enteric
Virus Concentration from Large Volumes
of Tap Water" by M.D. Sobsey et al., J.
AWWA 72(5): 292-99 (May 1980); and
"Modifications of the Tentative Standard
Method for Improved Virus Recovery
Efficiency" by M.D. Sobsey et al., J.
AWWA 72(6):350-55 (June 1980).
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