DETECTION OF VIRUSES IN WATER
A REVIEW OF
METHODS AND APPLICATION
William F. Hill, Jr., Elmer W. Akin, and William H. Benton
ENVIRONMENTAL PROTECTION AGENCY
Water Quality Office
Division of Water Hygiene
Gulf Coast Water Hygiene Laboratory
Dauphin Island, Alabama 36528
1971
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Copyright(Cy1971. Reprinted by permission of the Board of Trustees
of the University of Illinois from
PROCEEDINGS
THIRTEENTH WATER QUALITY CONFERENCE
February 1971
VIRUS AND WATER QUALITY: OCCURRENCE AND CONTROL
Edited by
Vernon L. Snoeijink
Department of Civil Engineering
University of Illinois
Environmental Protection Agency
of the State of Illinois
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DETECTION OF VIRUSES IN WATER: A REVIEW
OF METHODS AND APPLICATION
WILLIAM F. HILL, JR., ELMER W. AKIN, AND WILLIAM H. BENTON
Environmental Protection Agency, Water Quality Office, Division of
Water Hygiene, Gulf Coast Water Hygiene Laboratory, Dauphin Island,
Alabama 36528.
ABSTRACT
One of the major problems facing environmental health officials
in regard to water quality is related principally to the unavail-
ability of reliable and standard methods to concentrate, detect, and
isolate low-multiplicities of virus from very large volumes of water.
The critical examination of all water supplies for the presence of
viruses (including waters used for drinking, recreation, and food
production) requires a quantitative approach. In order to be quanti-
tative, measurable quantities of water must be examined. This is the
only way in which a definitive assessment can be made as to the dis-
tribution and extent of virus contamination of our water resources.
The challenge to the virologist is related to the need for developing
new and/or improved techniques in the laboratory that have a high
likelihood for adaptation to the real world situation. In this regard,
a number of techniques have been shown experimentally to be good can-
didates for assessing the occurrence of viruses in various types of
water. The most promising methods are: (i) membrane-adsorption
technique; (ii) adsorption to precipitable salts, iron oxide, and
polyelectrolytes; (iii) aqueous polymer two-phase separation technique;
and (iv) soluble alginate filter technique. Most of these methods
have shown good-to-excellent virus recovery efficiencies as well as a
reasonable efficacy for concentrating viruses from water in controlled
laboratory experiments. Other methods such as (i) continuous-flow
ultracentrifugation; (ii) forced-flow electrophoresis and electro-
osmosis, and (iii) hydroextraction have also shown favorable virus
recovery efficiencies under laboratory-controlled conditions but fall
short as candidate techniques for real world virus-in-water problems.
From the data, it would appear that the most promising methods for
detecting and isolating low-multiplicities of virus in clean and
finished waters are those that rely on virus adsorption and/or retention
coupled with a flow-through sampling system. For waters that are mod-
erately or grossly turbid, it would appear that aqueous polymer two-
phase separation may be the better approach. In this review paper, the
above methods are briefly described in terms of mechanisms, procedure,
and efficiency. The methods are evaluated in terms of speed, simplicity,
and economy of application.
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INTRODUCTION
Environmental health security as related Co our water re-
sources is seriously jeopardized by the lack of standardized
methodologies for detecting viruses in water, The unequivocal
need for adequate and standardized virus methods for determining
the occurrence of viruses in water and waterways has been prof-
fered for a number of years (Public Health Service, Drinking Water
Standards, 1962; Kramer, 1965; Berg, 1967), According to the Com-
mittee on Environmental Quality Management of the Sanitary Engineer-
\
itig Division of the American Society of Civil Engineers, "the in-
adequacy of methods for detecting, identifying, and enumerating
viruses in samples of water constitutes an important Iserious]
gap in water quality control" (Committee report, 1970), The lack
of a method for the detection, of viruses in water then would seem to
be an enigma. Oversimplified, the total problem can be reduced to
the unavailability of reliable and standard methodology to sample,
recover, concentrate, and isolate viruses occurring at low-multi-
plicity levels in waters. There are a number of aspects of the
problem none of which are mutually exclusive. One aspect is con-
cerned simply with methods for sampling and detecting viruses in
water, Tor example, it is well documented that detecting viruses
in grossly polluted waters (sewage effluents) by the gauze-pad
technique has been phenomenally successful not because the technique
is particularly efficient but rather because of the high-multiplicities
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of viruses that occur in fecally polluted waters. Another aspect
of the problem is two-fold and is concerned with methods for
quantitative assessment of viruses in water. This involves (i)
collecting samples of water of sufficient volume which would have
a high likelihood of containing viruses, and (ii) concentrating
the viruses occurring at low-multiplicities therein for isolation.
It is the selection of a suitable method for concentrating viruses
from very large volumes of water that represents the greatest
challenge to a virological methods research activity. Consequently,
the greatest need in virus methods research is techniques that
efficiently concentrate viruses from very large volumes of water
regardless of whether the water is used for drinking, recreation,
or food production. Unfortunately, a reliable method for concen-
trating low-multiplicities of virus from large quantities of water
of the real world is not readily available for routine use, A
third aspect of the problem for detecting viruses in water is con-
cerned with laboratory isolation, identification, and enumeration
of recovered viruses. This requires the selection of susceptible
virus-host systems; e.g., a number of different cell cultures and/
or laboratory animals that will support the propagation of the
various virus isolants. Since 100 or more virus serotypes are
shed by the fecal route and therefore can be expected to occur in
domestic wastes and waterways, this is not a simplistic task. The
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availability of facilities and trained personnel to handle primary-
cell cultures, continuous-cell cultures, and suckling mice would
be mandatory. The final determinations of the laboratory require-
ments, however, would have to be made by a professional virologist.
In summary, one of the major problems facing environmental
health officials in regard to water quality control is related
principally to the unavailability of reliable and standard methods
to concentrate, detect and isolate low-multiplicities of virus from
very large volumes of water. The critical examination of all water
supplies for the presence of viruses (including waters used for
drinking, recreation, and food production) requires a quantitative
approach. And, in turn, the challenge to the virologist is related
principally to the need for developing new and/or improved tech-
niques in the laboratory that have a high likelihood for adaptation
to the real world situation. In order to be quantitative, measur-
able quantities of water must be examined. The smallest quantity
of water considered adequate for detecting low-multiplicities of
virus is probably 100 gallons (378.5 liters) for clean or finished
waters. For waters that are moderately or grossly polluted, a
minimum sample size of at least 1 liter or perhaps 1 gallon (3,78
liters)may be adequate. The point of departure of this paper con-
cerns methods that show promise for concentrating viruses from
very large volumes of water. The specific purpose of this paper is
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to describe and review a particular method and then expound briefly
on the advantages and disadvantages of that method in terms of
efficiency and application.
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BACKGROUND APPROACH TO THE PROBLEM
The approach to the methods research problem for concentra-
ting viruses from water stems from the physicochemical properties
of the virus particle itself and is related principally to those
procedures applicable to macromolecular proteins. "For example,
viruses are nucleoproteins and behave as colloidal hydrophilic
particles in suspension. Consequently, many of the physical and
chemical properties of viruses identify with the properties common
to proteins. In this regard, viruses manifest properties of solu-
bility which decrease with increasing concentrations of very soluble
salts such as ammonium sulphate. Viruses are amphoteric; i.e.,
capable of reacting either as electro-positive or as electro-negative.
They also have determinable isoelectric points (electro-neutral),
Since viruses exhibit polarity they are also immiscible in organic
solvents such as diethyl ether, n-butanol, chloroform, and fluoro-
carbons. Virus particles manifest unique surface properties as
exhibited in their ability to adsorb readily to a number of sub-
stances such as celite, alumina gel, tricalcium phosphate, starch,
and various resin and cellulose derivatives (Schwerdt, 1965),
Viruses have measurable molecular weights and sizes. For example,
a poliovirus virion has a molecular weight of 7 x 10® daltons and is
approximately 28 nm in diameter. Consequently, viruses also lend
themselves to sedimentation from solutions by ultracentrifugation
techniques.
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With regard to the nature of viruses, a number of reported
techniques have been shown experimentally to be good candidates
for assessing the occurrence of viruses in various types of water.
Likewise, from field studies, a number of these techniques have
also shown promise for adaptation to the real world situation.
The most promising techniques for concentrating viruses from water
involve, primarily, modifications of methods the principles of
which have been known for some time. For example, modification
of methods such as; (i) membrane-adsorption technique, (ii)
adsorption to precipitable salts, iron oxide and polyelectrolytes,
(iii) aqueous polymer two-phase separation technique, (iv) soluble
alginate filter technique, (v) continuous-flow ultracentrifugation,
(vi) forced-flow electrophoresis and electro-osmosis, and (vii)
hydroextraction procedures, are all considered potentially adaptable
to concentrating viruses from water. From a viruses-in-water
standpoint, many of the above methods are still developmental in
that they have been studied under rather rigidly controlled labo-
ratory conditions. Nevertheless, some of these methods are under-
going field evaluation by being applied, on a limited scale, to
suspect virus-contaminated raw water supplies.
Ideally, an acceptable method should satisfy the basic
criteria of a good method by being simple, rapid, sensitive, and
reliable. In addition, an acceptable method must be economical
from an application standpoint. Some methods that have shown good
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promise in controlled laboratory experiments, unfortunately, fall
short when they are applied to the field,, For example, most of
the methods mentioned above show good to excellent virus recovery
efficiencies as well as a reasonable efficacy for concentration
of virus from water in laboratory-controlled experiments. However,
the water sample size which has ranged from 10 ml up to 19 liters
has been most frequently used for the evaluation studies regardless
of whether the work was conducted in the field or in the laboratory.
Consequently, the adaptation of s'ome of these methods to very large
quantities of water of at least 100 gallons remains mere conjecture.
This is important, for if viruses occur at low-multiplicities in
clean and/or finished vraters then the expectation of success for
their detection and isolation is limited primarily by the sample
size, and for finished waters 100 gallons is considered minimal.
For moderately polluted waters, sample sizes of 1 liter to 1 gallon
may be sufficient as mentioned previously.
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QUANTIFICATION OF VIRUSES AND ENUMERATIVE RELIABILITY
There are two methods used to quantify or titer viruses;
the quantal assay technique and the plaque assay technique. The
one method which yields a quantal response (all-or-none) is
commonly called, in virology, the TCID^q assay method. The"ICID^q"
designation refers to tissue culture infective dose 50 percent.
The expression for this method can vary depending upon the host
and the indicated response; i.e., lethal dose 50 percent (LD^q);
egg infective dose 50 percent (EID^q); and effective dose 50 percent
(ED50), to name a few. Briefly, the tissue culture procedure con-
sists of making serial logarithmic dilutions of the material to be
assayed and then inoculating a number of tubes (usually 4 or 5
tubes per dilution) containing cell-monolayers. Following an in-
cubation period, the occurrence or absence of cytopathlc effect
(CPE) in the cells of each tube is observed microscopically. The
50 percent endpoint may then be calculated by using the method of
Reed and Muench (1938) or some other acceptable method. The ex-
pected reproducibility of the quantal assay method is within +
0.3 log. The lack of precision of this assay method should be
recognized and caution exercised not to credit 50 percent endpoint
determinations with a precision they do not have; a 1.5- or 2-log
difference in titer is usually considered significant while a
1-log (or less) difference may be equivocal. The quantal assay
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method also permits the application of the statistical most-
probable-number (MPN) method for estimating virus multiplicities
(Chang, et at, 1958). The precision and accuracy of the adapted
MPN method is predicated primarily upon the dilution interval and
the number of tubes inoculated per dilution.
The other method is called the plaque assay method. This
method is considered by virologists to be the most precise method
to quantify viruses since a plaque or plaque-forming unit (PFU)
can be initiated by a single virion. Plaques appear as microscopic
circumscribed areas of cytopathology which result from the contig-
uous spread of virus in susceptible cells under a semi-solid overlay
(Figure 1). Some viruses do not produce plaques, however, and
some viruses exhibit a low-plaquing efficiency. However, when a
virus does possess a high-plaquing efficiency, it is inexcusable
to use any other method for quantification. Additionally, from
the standpoint of enumerative reliability, it is also known that
single virions as well as virion aggregates both possess the capac-
ity to produce single plaques. Consequently, it should be recognized
that factors, such as: (i) the number of virion aggregates; (ii)
the size of aggregates; and (ill) the aggregate-single virion ratio
in a given suspending medium contribute to the total enumerative
error term. Any virus handling techniques that enhance aggregation
or promote disaggregation should be recognized in order to maintain
enumerative reliability. In our laboratory, we found that selection
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FIGURE 1. Viral plaques produced in cell-monolayers of HEp-2 cells;
from left to right: echovirus type 6; coxsackievirus B—1; and
poliovirus type 1 (LSc2ab).
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of a suitable plaque assay diluent can be important under certain
circumstances (Hamblet, et al. 1967). A typical experiment is
shown in Table 1 where it may be observed that the plaquing effi-
ciency of poliovirus type 1 was increased almost 5-fold simply by
diluting the virus in nutrient broth when compared to diluting the
virus in Hanks' balanced salt solution. Furthermore, there is a
statistical notation that must be considered when quantitative
virus determinations are made by the plaque technique; i.e., the
number of plaques produced on a cell-monolayer follows a Poisson
distribution; assuming no error other than random sampling error
(Larson and Reinicke, 1965), Since enumerative reliability is
closely allied with the Poissonian statistical inference, experi-
mental designs must include a replicate sampling technique.
The analytical examination of these multiple observations (replicate
PFU counts) by statistical methods can then serve as a basis for
making valid interpretations of quantitative experiments. Data
handled in this manner will control and should ultimately minimize
enutneratlve error by measuring variability. It should be obvious,
however, that replicate sampling can only be expected to narrow
the margin of error not eliminate error. Under conditions of good
analytical control, the 95 percent fiducial confidence limits for
a single virus infectivity assay, as determined by replicate sam-
pling, are frequently greater than + 10% and may approach + 50% of
the mean plaque count. Moreover, the? expected error, based on the
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TABLE 1
EFFECT OF PLAQUE ASSAY DILUENT ON POLIOVIRUS MULTIPLICITY
ASSESSMENT FROM EQUAL VIRUS POOLS
Replicate
samples
HBSS
- Poliovirus poola
NB -
Poliovirus
noolk
PFV/ml
PFU/ml
1
470
580
270
2200
1100
2200
2
590
460
640
2500
2400
1600
3
440
510
460
2100
1600
3500
4
420
510
450
2400
3100
3100
5
540
430
480
2700
1400
2700
Mean + SE
483 + 22
2307 + 174
95% CI
435 to 532
1932 to 2681
Coefficient
of variation
18%
29%
£
HBSS, Hanks' balanced salt solution.
^NB, Nutrient broth (Difco).
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coefficient of variation, of replicate plaque counts usually
ranges from 10 to 30% and should not normally exceed 50 percent.
The precision and accuracy of the plaque assay method is
generally accepted to be of a high order. Nevertheless, if the
concept of enumerative reliability is to be maintained, then ran-
dom error must be included as one of the parameters inherent in
counting virus plaques. Furthermore, an awareness of the expected
variability incident to estimating virus multiplicities by replicate
plaque assays is mandatory for the professional virologist. The
interpretation of quantitative virological data must be meticulously
sophisticated and utmost caution exercised by the novice not to
consider single (or duplicate) plaque counts as absolute numbers.
This is particularly important when methods research concerns
quantitative virus assessments. In this regard, placing too much
emphasis on the percent of virus recovered, although mathematically
accurate, can often lead to not only distorted but meaningless
value judgments. For example, the interpretation of virus recovery
data when the initial input-multiplicity of virus is high (2 x 10^
to 2 x 10® PFU) or low (2 to 4 PFU), on the basis of percent recovery,
may be completely misleading because of the random error term and
the expected variability of enumerating viruses by the plaque tech-
nique, Consequently, to evaluate the efficacy of a virus concen-
tration method in a typical virus-input virus-output experiment, the
concentration of virus infectivity (in PFU) observed coupled with
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percent recovery are both important. The percent of virus recovered
as a single parameter may be nothing more than ancillary information
and in repeat experiments may often range, conservatively, from 20
to 90 percent and occasionally even exceed 100 percent; a calculated
artifact.
In summary, the plaque assay technique is the most precise
method for enumerating virus multiplicities, and as such is the
preferred quantitative method for laboratory-controlled experiments.
The quantal assay technique although lacking enumerative precision
*
is considered by many virologists to be more sensitive for detecting
very low (<1 PFU/ml) multiplicities of virus and, obviously, the
only technique applicable to virus detection for viruses that do
not form plaques. The quantal assay procedure would also be the
preferred detection method for mixed virus populations present in
a single sample. For best results in field trials, both the
plaque assay and the quantal assay techniques should be conducted
in parallel on all samples.
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SAMPLING WATER FOR VIRUS ASSESSMENT
Fundamentally, there are only two methods available for sam-
pling water for the presence of viruses. One method is an in eitu
entrapment technique and is called the gauze-pad or swab method.
This method is strictly qualitative and consists of suspending a
gauze pad (filled with cotton or other material) in the water to
be examined for a period of time, usually 24 hours to several days.
The pad is then treated with 1 N sodium hydroxide (pH8+) to enhance
elution of any 4entrapped viruses and the fluid expressed from the
pad. The other method is simply a water collection technique and
is called the grab- or dip-sample method. This method is quanti-
tative and consists of dipping a bottle or jug in the water to be
examined. Both methods have been used in the field, with varying
degrees of success, for detecting the presence of viruses in raw
waters. Some of the obvious advantages and disadvantages of both
methods have been reviewed recently (Grabow, 1968). It should be
pointed out that the efficacy of both methods is closely linked
with the nature of the water source. For example, when either or
both methods were applied by a number of workers to waste-waters,
sewage effluents, and other bodies of water that were grossly pol-
luted with treated or untreated domestic wastes, a nun^er of suc-
cessful virus isolations were made (Chin, et al, 1967; England,
et al. 1967). However, when the two methods have been tested or
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compared in parallel, the gauze-pad technique has been shown to be
the superior virus detection method. It should be noted that
dip-samples usually consist of 100 to 200 ml of sample and there-
fore bias does enter into the evaluation* Some typical observa-
tions are shown in Table 2.
In summary, if the water to be examined for viruses is grossly
polluted with fecal wastes then the application of the gauze-pad
technique will provide a high likelihood for virus detection. It
must be emphasized that the findings, however, will be strictly
qualitative not quantitative. If quantitative virus assessment of
waters is desired then the dip-sample method, or an equivalent
modification of some technique, where the volume of water is
measured, must be used. It should be noted here that it is only
the quantitative approach that will definitively assess the dis-
tribution and extent of virus contamination in our waterways
(including surface waters as veil as reclamation water and finished
drinking water) and thereby permit meaningful conclusions to be
made. Value judgements based on qualitative data will not resolve
the public health management of virus-in-water problems nor elucidate
the potential health threat of water-borne viruses and the epidemi-
ology of viral diseases transmitted by the water route.
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TABLE 2
COMPARISON OF GAUZE-PAD AND DIP-SAMPLE METHODS FOR ISOLATING VIRUSES
FROM SEWAGE
Virus Recovered
Reference
Gauze-
Number positive
"Number tested
-Pad
% Positive
Dip-Sample
Number positive
Number tested % Positive
Melnick et at, 1954
196/324
60.4
66/231 28.6
Mack et at, 1958 ,
66/602
10.9
26/651 4.0
Bloom et at, 1959
61/462
13.2
17/368 4.6
Mack et at. 1962
44/268
16.4
1/293 0.3
Lund and Hedstrom, 1969
74/84
88.1
32/84 38.1
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METHODS FOR CONCENTRATING VIRUSES IN WATER
Within the past several years, a number of reports have
appeared in the literature on methods for concentrating viruses
from water. Many of these methods show sufficient promise to permit
the quantitative assessment of viruses in waters of the real world.
Some of these methods have been mentioned previously in this paper
and have also been reviewed recently (Berg, 1967; Grabow, 1968).
These same methods were considered by the Committee on Viruses in
Water (Committee Report, 1969) to merit continued investigation.
In this section, a particular method will be briefly described
and then evaluated as to the advantages and disadvantages of that
method in terms of efficiency and application.
Merribrarie-adsprption technique.
The mechanism of the membrane-adsorption technique is related
principally to the unique surface properties of the virion; i.e.,
under specified conditions, viruses efficiently adsorb to a variety
of substances including microporous membranes. These membranes
which are composed of cellulose derivatives are commonly used to
partially purify or clarify crude virus-cell harvest material by
filtration. Membranes have also been used for sizing viruses (Ver,
et al, 1968). In virus filtration experiments, any observed losses
in virus titer in the filtrates after filtration have been attributed
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to (i) the presence of virion aggregates, and (ii) adsorption of
virions to the matrix of the menbrane.
The concentration of viruses by the membrane-adsorption tech-
nique depends on (i) adsorbing viruses to the membrane, and (ii)
then removing the viruses from the membrane by elution. Elution
is usually carried out at a high pH; i.e., pH 8 to 9. Substances
used to elute viruses from the -embrane are usually proteinaceous
in nature; i.e., whole serum, albumin, beef extract, casein, veal
infusion broth, and nutrient broth. Some of these substances have
also been used to pretreat membranes which enhances virus filtra-
tion efficiency. Many of the factors that influence the filtration
of viruses through the matrices of microporous membranes have been
described by Cliver (1965). Interestingly, Cliver also observed
that viruses adsorb to the membrane unless the membranes were
pretreated with serum or a gelatin solution, Cliver (1965) indi-
cated that whether virus passed through or failed to pass through
the filter membrane was probably the net result of several processes
i.e., (i) adsorption of virus to the matrix surfaces of the membrane
(ii) competition of macromolecules for adsorption sites on the mem-
brane, and (iii) the presence or absence of viral aggregates, to
name a few. In studies concerned with virus interactions with mem-
brane filters, Cliver (1968) su—larized that loss of virus in
membrane filtration was primarily due to adsorption of the virus to
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the membrane matrix surfaces. In turn, virus adsorption phenomena
was shown to be influenced by; (i) chemical composition of the filter
membrane, (ii) ratio of pore diameter to the diameter of the virion,
and (iii) the absence of substances such as those occurring in serum
that interfere with virus adsorption. In this regard, cellulose
triacetate membrane filters were shown to adsorb virus poorly when
the porosity exceeded the virion diameter by as much as 3 times.
Conversely, cellulose nitrate membrane filters were shown to adsorb
virus very efficiently even when the porosity exceeded the virion
diameter by as much as 285 times (Cliver, 1968). Working with virus-
seeded 1-liter samples of Chicago tap-water, Cliver (personal Qonmv.rsl-
aticmi 1970) indicated that he achieved about 260-fold virus concen-
tration and virus recoveries of 26 to 90% with the membrane-adsorption
technique.
Concentration of enteroviruses on membrane filters from crude
virus-cell harvest material was investigated by Wallis and Melnick
(1967a), They also observed that enteroviruses can be made to adsorb
or to pass through membrane filters simply by manipulating the virus
suspending medium. For example, the addition of salts to the virus
suspending medium, particularly salts containing divalent cations
such as MgC^ were shown to significantly enhance viral adsorption.
Additionally, the adjustment of the virus suspending medium to pH 5
(close to the isoelectric point of poliovirus type 1) was shown to
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markedly enhance viral adsorption. Conversely, the presence of
organic and proteinaceous substances in the virus suspending
medium was observed to interfere significantly with virus adsorp-
tion to the membrane matrices, presumably by competing for membrane
adsorption sites. They referred to these viral adsorption inter-
fering substances as membrane-coating components or simply as MCC.
These same authors reported that MCC was effectively removed by
passing the liquid sample through an anion-resin column [Dowex 1-X8
(Cl~) 100-200 mesh]. Other workers (Borneff, 1970; Schafer, 1970),
however, have had less than success in removing MCC in natural waters
with anion-resins and as a result have apparently abandoned the use
of microporous membranes for virus concentration and detection in
raw waters in Germany. Nevertheless, Wallis and Melnick (1967a)
state that with the use of membrane filters, virus can be quanti-
tatively recovered from crude virus-cell harvests and 80- to 100-fold
concentration of virus achieved. These investigators used serum as
the eluent. The membrane-adsorption method has also been applied to
field samples for concentration and detection of viruses from sewage
(Wallis and Melnick, 1967b). The method was tested in Houston, Texas
over a period of seven months. Briefly, samples which consisted of
1-gallon (3.78 liters) quantities of raw sewage were initially clar-
ified by prefiltration and then pressure-filtered through a 47-mm
or 90-mm HA Millipore membrane (0.45 ym porosity). During the period
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of the study, a total of 2,795 isolants were recovered from 10
separate samples (Range: 10 PFU to 701 PFU/gallon). These
results were compared with those observed from small samples of
unconcentrated sewage collected at the same time when only 4
isolants were recovered.
Rao and Labzoffsky (1969) investigated the efficacy of mem-
brane filters to detect low-multiplicities of viruses in what they
considered to be large volumes of surface waters (500-ml quantities).
They combined an AP 25 MF prefilter and an HA Millipore membrane
filter (0.45 vm porosity) as a single unit. Any virus adsorbed on
the prefilter was also eluted with a 3% beef extract, pH 8 eluent
¦in situ in a single operation. These same investigators indicated
that virus trapped in the prefilter was lost if prefiltration was
used to clarify the water as a preliminary step. These investiga-
tors observed experimental virus recovery efficiencies of 53 to
greater than 100 percent. They also determined the necessity of
having electrolytes (salts) such as CaC^ in the water to enhance
viral adsorption to the membranes. In their experimental studies,
they added 200 ppm Ca^+ to the raw water before conducting the
experiments.
Moore, et at. (1970) compared'various applications of a modi-
fied membrane-adsorption technique for concentration of viruses in
waste-water. They also observed that the apparent occurrence of
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KCC In waste-water adversely affected the efficiency of the mem-
brane filters (0.45 ym porosity) to adsorb poliovirus. They suc-
cessfully removed MCC from waste-water with the anion-resin pre-
treatment as described by Wallis and Melrtick (1967a) but failed to
remove MCC with a preliminary protamine sulfate flocculation tech-
nique. Experimentally, Moore and associates (1970) found that the
combination of aluminum hydroxide flocculation followed by membrane
filtration (retention of the virus-aluminum hydroxide complex) gave
the best results showing 81 to 100^ recovery of input (seeded)
poliovirus. These same investigators showed that the combination
of protamine sulfate flocculation followed by filtration through an
AP 20 MF prefilter and then concentrating the virus on a Millipore
membrane filter yielded a virus recovery of only 26 to 31 percent.
Hill, et al% (unpublished data) are currently evaluating the
adaptability of the inillitube MF cartridge filter (0.45 ym porosity)
to concentrate virus from very large volumes of water of 100 gallons.
Their preliminary findings indicated thct poliovirus can be concen-
trated up to 140- to 218-fold from demineralized-water and raw
estuarine-water (salinity 25 °/oo)( respectively. Virus recovery
from the demineralized-water was 56% while virus recovery from the
raw estuarine-water was 79 percent. Some virus penetrated the filter
when raw estuarine-water was used. In these studies, the pH of the
100-gallon water samples was adjusted to pH 4,5 before filtration.
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Divalent cations were added to the demineralized-water (500 ppm
Ca^+). No salts were added to the estuarine-water. Elution of
virus from the cartridge membranes was achieved by using 5 X
nutrient broth (Difco), pH 8.5+ as the eluent.
Berg (1970) has also been checking out the membrane-adsorption
technique for concentrating viruses from large volumes of water.
By the use of 1-liter volumes of Mcllvane's buffer (0,05 M Na2HP0^,
pH 7,0) seeded with virus (31 to 169 PFU/liter), Berg observed the
following virus" recoveries: (i) xeovirus type 1, 52 to 78%, (ii)
coxsackievirus B-3, 97%, (iii) echovirus type 7, 121%, and (iv)
poliovirus type 1, 103%, In these studies, virus was eluted from
the membranes with 3% beef extract and subsequently sonicated
before virus assay. In larger volumes of water; i.e., 25 gallons
(94,5 liters), virus recoveries have ranged from 50 to 75 percent.
Most of the large volume experimental work was done with distilled-
water. Virus was not consistently recovered quantitatively when
tap water was used, presumedly because of the presence of organic
substances competing for virus adsorption sites on the membrane
matrix.
Joseph (personal aommuniaation3 1970) has used the membrane-
adsorption technique in an attempt to isolate viruses from treated
drinking waters in Maryland, No virus, however, was isolated from
the drinking water source during the period of the study.
-------
-21
To summarize, the membrane-adsorption technique for concen-
trating and detecting viruses in various types of water holds
considerable promise as being an acceptable method. This state-
ment is in agreement with the conclusions of the Committee on
Environmental Quality Management (1970). Experimentally, volumes
of water from 10 nil to 100 gallons (378.5 liters) have been eval-
uated by the membrane-adsorption technique for virus concentration
and recovery efficiency with favorable results. Virus recovery has
ranged from 26% to greater than 100 percent. Equally significant,
virus concentration has ranged from 80- to 260-fold. Some typical
results are shorn in Table 3,
Real world application of the membrane-adsorption technique
has been rather limited. Wallis and Melnick (1967b) examined raw
sewage as it entered a treatment plant in Houston, Texas. This
field test was conducted over a period of seven months. One-gallon
size dip-samples were collected once or twice a month and a total
of 2,795 isolants were recovered from a total of 10 separate samples.
Fundamentally, the membrane-adsorption technique may be con-
sidered a simple technique. The efficacy of the technique would
seem to be predicated on (i) the proper selection of membrane
materials, (ii) the addition or presence of divalent cations, (iii)
the adjustment of the suspect virus-contaminated water to pH 5 or
below, (iv) the removal of MCC from raw water, and (v) the selection
-------
TABLE 3
RECOVERY OF VIRUS FROM WATER BY THE MEMBRANE-ADSORPTION TECHNIQUE
Reference
Type of
water
Volume of
water
Percent of
virus
recovered
Wallis and Melnick, 1967
Sewage
1.5 gallons
- 100
Rao and Labzoffsky, 1969
Surface-water
500 ml
53 - 100
Cliver, 1970
Tap-water
1 liter
26 - 90
Moore, et alOJ 1970
Waste-water
1 gallon
81 - 100
Berg, 1970
Distilled-vater
25 gallons
50 - 75
Hill, et al.3 ( )
Estuarine-water
100 gallons
56 - 79
-------
-22
of an efficient eluent. The major advantages of the membrane-
adsorption technique are simplicity, speed, and sensitivity.
The major disadvantages of the technique are related principally
to the types of water to be examined for virus. For example, the
occurrence of membrane-coating components in various types of raw
waters would adversely affect the efficiency of virus adsorption.
In addition, waters that are highly turbid would be expected to
clog the filter prematurely. Finally, there are cost consider-
ations; routine^ sampling of waters might be considered low to
moderate. Initial costs, however, for stainless-steel filter
holders capable of processing 100-gallon quantities of water could
exceed $1000.00 if more than one holder was required for the water
sampling system.
Adsorption to preoipitable salts, iron oxide, and poly electrolytes.
The mechanism of these techniques is similar to the membrane-
adsorption technique in that concentration of virus by the use of
precipitable salts, iron oxide, or polyelectrolytes relies on the
ability of viruses to adsorb efficiently onto the selected adsorbent.
The concentration and purification of viruses by adsor/tion to a
variety of adsorbents has been commonly practiced In virology lab-
oratories for almost 40 years (Schwerdt, 1965). For example, Sabin
-------
-23
(1932) concentrated and partially purified poliovirus by adsorb-
ing the virus to alumina gel C and subsequently eluting the
virus at high pH. More recently, however, the application of
virus adsorption to concentrating viruses from water under experi-
mental conditions has been reported to be successful. Stevenson,
at. (1956) used an alum flocculation procedure for concentrating
coxsackievirus A-2. They added aluminum sulfate under specified
conditions to the virus-contaminated water and allowed a floe to
form -in situ. Virus was then eluted from the aluminum hydroxide
floe at an elevated pH (approximately pH 8+). These investigators
indicated that the alum-floc method permitted virus concentrations
of 100-fold or greater and was capable of detecting as little as
0.00625 LD^q/0.02 ml (volume inoculated per mouse).
Wallis and Melnick (1967c) concentrated a number of different
viruses by adsorption on aluminum phosphate, aluminum hydroxide,
and calcium hydrogen phosphate floes. In all cases, preformed
floes (precipitates) were added to the virus-contaminated aqueous
suspensions or sewage effluents. By using conditions found optimal
for virus adsorption, they observed that only acid-sensitive viruses
were concentrated on the aluminum phosphate floe; e.g., herpesvirus,
%
influenza, rubella, and vaccinia, to name a few. Conversely, all
viruses tested except reovirus and adenovirus were concentrated on
the aluminum hydroxide floe and the calcium hydrogen phosphate floe.
-------
-24
Among the viruses tested, enteroviruses were also included; e.g.,
polioviruses, echoviruses, and coxsackieviruses. Noteworthy,
adenovirus was adsorbed only to the aluminum hydroxide floe and
reovirus was not adsorbed to any of the salts tested. With four
of the viruses tested (herpesvirus, measles, poliovirus, and echo-
virus), quantitative recovery of the viruses was achieved from
1-liter volumes containing as few as 100 PFU/liter, Recovery of
virus exceeded 80% in most cases.. The use of aluminum hydroxide
floe as the adsorbent for concentration and detection of viruses
occurring naturally in sewage was also undertaken by Wallis and
Melnick (1967c). In sewage samples of 1-gallon quantities, 204
isolants were isolated over a period of four weeks. No viruses
were isolated from the same samples prior to concentration with the
aluminum hydroxide floe procedure. The method is apparently
limited, however, by sample size; i.e., being confined to about a
2-gallon quantity,
England (personal aomtunication, 1970b) has evaluated, over a
three year period, the aluminum hydroxide floe and the calcium
hydrogen phosphate floe procedures (modified after Wallis and
Melnick, 1967c) for concentrating and Isolating viruses from raw
sewage and sewage effluents. Concentration factors up to 100-fold
were achieved with the aluminum hydroxide floe procedure. Concen-
tration factors up to 300-fold were achieved with the calcium
-------
-25
hydrogen phosphate floe procedure. Virus recoveries with both
flocculation procedures have ranged from 80 to 100% with adeno-
virus and various enteroviruses. The procedures were observed
to be less efficient for recovering reovirus.
The concentration of virus (coxsackievirus A-9) by adsorption
to iron oxide was studied by Rao, et at• (1968). They evaluated a
number of different iron oxides and concluded that magnetic iron
oxide designated as M, 0. 2530 (available from Magnetic Tape
Division of Charles Pfizer Co,) was the best virus adsorbent. They
conducted their experiments by passing 500-ml quantities of virus-
contaminated water through iron oxide packed in columns containing
25 grams of iron oxide. They eluted the adsorbed virus from the
iron oxide in situ with 3% beef extract at pH 8 with an 87 to 90%
recovery. The researchers indicated that one of the limitations
of the method related to clogging of the iron-oxide bed with sus-
pended material. They concluded that the technique exhibited
excellent capacity for adsorbing virus and therefore merited addi-
tional investigation, particularly in regard to the method's
effectiveness for detecting low-multiplicities of virus in large
volumes of water, Rao (personal oorrmmioation3 1970) in additional
studies, has applied the method experimentally (virus-input studies)
to larger quantities of natural waters; i.e., 50 liters of river-
water and 150 liters of tap-water. The procedure has also
-------
-26
been modified. For example, the natural waters were first clari-
fied by filtration through a 47-mm, AP 25 MF fiberglass prefilter
pad and then filtered through an iron-oxide bed "sandwiched"
between two 47-mm AP 25 MF prefilter pads. Virus adsorbed to the
iron oxide was eluted by passing in situ 100 ml of 3% beef extract,
pH 8 through the system. Concentration of the virus in the beef
extract eluate was also accomplished. This was done by adding 1200
ppm Mg^ (as Mg in MgC^^I^O) to the 100 ml of eluate, adjusting
the acidity to pH 3 and then passing the fluid through a 47-mm,
0.45 um porosity Hillipore membrane filter. The adsorbed virus was
then again eluted in situ with 5 ml of 3% beef extract at pH 8. By
using the two-step adsorption-elution procedure, Rao claims that
virus recovery of 100% can be obtained from both types of water.
If these results can be confirmed, this two-step procedure may
represent a significant breakthrough since the second adsorption-
elution step has not been reported previously. Two aspects of these
findings are significant; (i) the physical concentration of the
system was 30,000-fold for the tap-water and 10,000-fold for the
river-water and (ii) the addition of Mg2+ and the adjustment to pH
3 may have solved the problem of interference of virus ^adsorption
to membranes as observed by other workers (Moore, at, 1970;
Wallis and Melnick, 1967a; Berg, 1970, and Hill, et aX%9 unpublished
data). The iron oxide virus-adsorption procedure was not applied
-------
-27
to the real world situation by Rao and associates.
Jakubowski and Hoff (personal oorrmuni cation, 1970) are currently
evaluating iron oxide as a virus adsorbent for adaptation to concen-
trating and detecting viruses from tap-water and estuarine-water.
They are using a thin-layer filtration procedure in which the iron
oxide is suspended in distilled-water and then filtered onto an AP
MF fiberglass prefilter pad to form the adsorbing layer. With
filter loads of 3 and 4 grams of iron oxide (using the 142-mm
Hillipore filtration unit), these researchers have observed virus
recoveries of 40 to 64% from 10-liter volumes of tap-water and 22
to 37% from 10-liter volumes of estuarine-water.
Metcalf (-personal comrumaabion1970) is also evaluating the
efficacy of iron oxide to concentrate and detect viruses from water.
By use of the 142-mm Millipore filtration system and the "sandv/ich"
technique, this investigator has observed virus recoveries ranging
from 80 to 97% in controlled laboratory studies. Additionally, he
has indicated that 5 to 10 PFU of enteroviruses can be recovered
from volumes ranging from 1 to 10 liters of water by the method.
The maximum flux of the iron oxide filtration system was about 5
gallons (18.9 liters) per hour. It was also observed that clogging
of the iron oxide filtering surface occurs in the presence of very
turbid water (e.g., clay, soil, or silt) and thereby impeded the
filtration process and significantly lowered the volume of water
-------
-28
capable of filtration.
SchSfer and Bomeff (personal communication, 1970) have used a
ferric chloride-flocculation method combined with the aqueous poly-
mer two-phase separation system for concentrating virus from water.
In their procedure, 1- to 5-liter samples of surface-water are
first clarified by filtration through an AP 20 MF fiberglass pre-
filter. To the virus-contaminated water is then added 200 mg FeCl^
per liter of water. The water is adjusted to pH 6.0 to 6.5 and
stirred for 60 tainutes. An in situ flocculation occurs with the
formation of Fe(OH)^. The floe is then entrapped on a DWAP Millipore
absorbent filter pad and subsequently the virus is eluted with 3%
beef extract (Difco), pH 8 by passing a 10-ml volume of eluent
through the ferric hydroxide layer on the filter pad. Two additional
elution steps are conducted using 5 ml of eluent each. In order to
examine several 5-liter samples at one time, all the eluates collect-
ed from the individual FeCl^ flocculation procedure are pooled. The
eluate pools are then further concentrated by the aqueous polymer
two-phase separation system (a discussion of this procedure appears
later in this paper). The results of the combined methods have
shown virus concentrations of 400-fold with virus recoveries of 25
to 30 percent. According to these researchers, the advantages of
the combined methods are vested in the fact that (i) eluates of
five, 5-liter samples can be pooled and then examined for virus
-------
-29
following the aqueous polymer two-phase separation system concen-
tration, and (ii) surface-waters do not require additional filtra-
tion to eliminate bacterial contamination. The major limitation
of the combined-methods technique is related to sample size; i»e.}
only 25 liters can be conveniently handled at one time.
England (1970) reported that the use of protamine sulfate
(salmine) flocculation followed by filtration through a Millipore
prefilter pad facilitated the recovery of reoviruses and adeno-
viruses from sewage effluents but was of little value for recover-
ing most enteroviruses (polioviruses, echoviruses, and coxsackie-
viruses) . This investigator indicated that concentration factors
up to 250-fold were readily achieved by the technique. Virus re-
coveries have ranged from 80 to 100% with reovirus and adenovirus
(England ,1970b)~
The concentration of viruses by adsorption to insoluble cross-
linked copolymers of maleic anhydride (polyelectrolytes) was de-
scribed by Johnson, et at, (1967). They reported that polymers
based on divinyl benzene-crosslinked styrene/maleic anhydride
copolymer can adsorb 100% of tobacco mosaic virus and more than
99.99% of poliovirus from aqueous suspensions. Virus was eluted
from the polymer-virus complex by the use of 1 M sodium chloride
with a 52% efficiency.
Wallis, et at, (1969) concentrated viruses from sewage by
adsorption onto an insoluble crosslinked copolymer of isobutylene
-------
-30
maleic anhydride designated as PE60 (produced by the Monsanto
Company, St. Louis, Mo.). In their initial studies, a batch-
technique was developed. Briefly, the batch-technique consisted
of adding washed PE60 (400 mg per gallon) to clarified sewage.
The sewage was clarified by filtration through an MF fiberglass
prefilter pad in order to remove particulate matter. To enhance
virus adsorption, the acidity of the sewage filtrate was adjusted
to pH 5.0 to 6.0. Virus was added and the mixture was stirred for
1 hour at 25° C. The polyelectrolyte-virus suspension was then
filtered through a 47-mm fiberglass prefilter pad. The virus-laden
polyelectrolyte was recovered from the filter pad with the aid of
a spatula. Virus was eluted from the polyelectrolyte with 10%
fetal calf serum, pH 8.0 or 9.0. In laboratory experiments, the
virus recovery efficiency was 93 percent. The insoluble poly-
electrolyte (PE60) virus-adsorption technique was also applied to
field samples of raw sewage from Houston, Texas during April and
May of 1968. In April, a total of 1,461 virus isolants were re-
covered. In May, a total of 205 isolants were recovered. In this
limited field study, the polyelectrolyte-virus-adsorption batch-
method was compared in parallel with the membrane-adsorption and the
aluminum hydroxide-adsorption methods for detecting vi/tuses in the
raw sewage. The authors concluded that the difference in virus
isolation frequencies among the three methods was not always
-------
-31
statistically significant. However, the polyelectrolyte-
adsorption method consistently gave the highest isolation rates
and was considered the preferable method because of economy and
the shortened time involved for processing the sewage samples.
In the same report, Wallis et al.j (1969) described a
modified thin-layer PE60 procedure in which the PE60 was
"sandwiched" between two fiberglass prefilter pads. This
"sandwich" modification prevented polyelectrolyte displacement
during the filtration process. The procedure included a double-
adsorption procedure in which eluted virus from the PE60 adsorbent
was readsorbed onto PE52 (crosslinked copolymer of ethylene maleic
anhydride). Virus was eluted from the PE52 adsorbent with physio-
logical saline. The double-adsorption-elution procedure resulted
in reducing a 1-gallon volume of sewage into a final volume of 3
ml with efficient recovery of virus.
Grinstein, et at. (1970) applied the polyelectrolyte-adsorption
method (batch-technique) to sewage and to a river stream receiving
sewage effluents in Houston, Texas during July and August of 1968.
Virus was isolated 5 miles downstream from the nearest sewage
effluent outlet. Overall, during two months of sampling 76, 1-gallon
samples were collected which yielded 12,855 virus isolants. The
average number of isolants per gallon sampled varied from 45 to
286 PFU. The authors concluded that with the polyelectrolyte-
adsorption method, it was now possible to monitor virus in natural
-------
-32
waters more effectively.
The application of the polyelectrolyte-adsorption technique
to very large volumes of water up to 100-gallon quantities or
greater has been studied by Wallis, et aZ. ( ). They used the
"sandwich" or thin-layer filtration system. Briefly, the thin-
layer of polyelectrolyte was prepared as follows: (i) an AP 20
MF fiberglass prefilter pad was placed on a filter-screen support
and 800 mg of PE60 suspended in 200 to 300 ml of distilled-water
were filtered onto the pad when the 90-mm Millipore filtration unit
were used; 10 grams of PE60 was used for the 293-mm filtration unit,
(ii) the polyelectrolyte thin-layer was examined for surface-covering
integrity and then a second AP 20 MF fiberglass prefilter pad was
placed on top of the polyelectrolyte layer to form the "sandwich".
By the use of the thin-layer filtration system (90-mm filtration
unit), 25, 50, 75, and 100-gallon quantities of virus-contaminated
tap-water were subjected to virus recovery experiments. The results
indicated virus recoveries ranging from 65 to 80 percent. The
method was also applied to a virus-seeded 17,000-gallon swimming
pool. By filtering 300 gallons (1135 liters) of the pool-water
through the thin-layer polyelectrolyte (293-mm filtration unit) an
efficiency of about 40% virus recovery was observed.
Jakubowski and Hoff (personal communication^ 1970) are currently
evaluating the polyelectrolyte-adsorption technique for concentrating
-------
-33
and detecting viruses in tap-water and estuarine-water (salinity,
27 °/oo). By use of the thin-layer filtration system (142-mm
filtration unit), 10-liter quantities of either tap-water or
estuarine-water were subjected to virus recovery experiments.
The filter load was either 1 or 2 grams of polyelectrolyte. In
the tap-water experiments, the results indicated virus recoveries
of 58 and 74% for the 1-gram and 2-gram filter loads, respectively.
In the estuarine-water experiments, the results indicated virus
recoveries of 12 and 17% for the 1-gram and 2-gram filter loads,
respectively. These researchers also tested the polyelectrolyte-
adsorption concentration system using naturally occurring virus
present in feces obtained from vaccinated infants. The adsorption
and elution results were similar to those obtained with cell
culture-grown virus.
Kalter, (personal communication3 1970) is also currently
investigating the polyelectrolyte-adsorption method for concentrating
viruses from water. By the use of PE60 as the copolymer, he has
observed virus recoveries of 60% from 1-liter quantities of water.
Berg (1970) has carried out experiments with the polyelectrolyte'
adsorption "sandwich" technique using PE60. By the use of 1-liter
volumes of distilled-water seeded with small amounts of virus (75
to 105 PFU/liter), relatively poor virus recoveries resulted. For
example, recoveries observed with poliovirus type 1 were 51 to 53
-------
-34
percent. With echovirus type 7, recoveries ranged from 25 to
30% and with reovirus type 1, 14 to 31 percent. Application of
the technique to 50-gallon (189-liters) samples of river-water
(field trial) was considered encouraging, however, since as much as
19 PFU of virus have been recovered from samples taken long
distances from outfalls along a fast-flowing river during the
winter months. Berg concluded by stating that despite its low
and erratic efficiency, the technique appears to be the most
sensitive presently available for large volume water studies.
England (personal aomimmvcation, 1970b) has evaluated the
polyelectrolyte-adsorption method (modified after Walliset a!.,
1969) for concentrating and isolating viruses from raw
sewage and sewage effluents. With sample volumes of 2-liter size,
concentration factors up to 500-fold have been achieved. In
laboratory-controlled experiments, recovery of enterovirus apd
reovirus was 90 to 100% when the input-virus multiplicities were
high; e.g., 1 to 10,000 PFU per ml. When the input-virus multi-
plicity was low; e.g., <0.2 PFU per ml, recovery was very poor
except for poliovirus.
To summarize, the use of precipitable salts, iron oxide, and
polyelectrolytes for concentrating and detecting viruses from water
holds good promise as being an acceptable method. Experimentally,
virus recoveries have ranged from 12 to 100% depending on the
-------
adsorbent used, the test virus, and the type of water being
examined. Experimentally, volumes of water from 400 ml to 300
gallons have been evaluated for virus concentration with favorable
results. Some typical results are shown in Tables A and 5. Appli-
cation of these virus-adsorption techniques to the real world
situation has not been extensive. By use of the aluminum hydroxide
floe technique and the polyelectrolyte-adsorption technique, however
good results were observed with l-.gallon (3.78 liters) field samples
of sewage and sewage effluents (Wallis, et at, 1969; Grinstein, et
at, 1970). The use of iron oxide in field studies has not been
studied. The advantages of using selected adsorbents for concentra-
ting and detecting viruses from water are related to their simplicity
speed, and economy. The major disadvantages of these methods are
related to the quantity and types of water to be examined for virus.
For example, the aluminum hydroxide floe technique is confined to
samples of about 2 gallons; the iron oxide and polyelectrolyte-
adsorption techniques are adversely affected by high turbidity which
clogs the systems. Cost considerations would be considered reason-
ably low except for the stainless-steel filtration assembly required
to support the fiberglass prefilter pads and adsorbent. These
filtration assemblies approach $900.00 for the 293-mm size unit.
-------
TABLE 4
RECOVERY OF virus from water by adsorption to precipitable salts and iron
OXIDE
Percent of virt
Reference
Wallis and Melnick, 1966
England, 1970
et altJ 1968
Rao, 1970
Jakubowski and Hoff, 1970
Wetcalf, 1970
SchJffer and Borneff, 1970
Volume
water
Adsorbent
used
1 liter
Sewage effluent
Al(OH) j
80 - X00
400 ml
Sewage effluent
Al(OH)
87 - 90
500 ml
£p —w9lt G t
Iron oxide
- 100
150 liters
Tap-water
Iron oxide
England, 1970
Iron oxide
Iron oxide
Iron oxide
FeClj
protamine
sulfate
Estuarine-water
¦jap—water
Tap-water
Surface-water
Sewage effl^ent
10 liters
10 liters
1-10 liters
5 liters
1 liter
22 - 37
40 - 64
80 -
45 - 100
80 - 100
-------
TABLE 5
RECOVERY OF VIRUS FROM WATER BY ADSORPTION TO POLYELECTROLYTE
Reference
Type of water
Volume
of water
Percent
of virus
recovered
Johnson, et at., 1967
Distilled-water
5 ml
- 52
Wallis, et alm> 1969
Sewage effluent
1 gallon
- 93
England, 1970
Sewage effluent
2 liters
90 - 100
Jakubowski and Hoff, 1970
Tap-water
Es tuarine-water
10 liters
10 liters
58 - 74
12 - 17
Kalter, 1970
Tap-water
1 liter
- 60
Berg, 1970
Distilled-water
1 liter
14 - 53
Wallis, et at( )
Tap-water
Swimming pool-water
100 gallons
300 gallons
65 - 80
- 40
-------
-36
Aqueous polymer two-phase separation method.
The mechanism of polymer two-phase separation is liquid-
liquid partitioning; a phenomenon closely related to adsorption.
Partitioning occurs as a result of differences in particle surface
properties and their distribution between two liquid phases while
adsorption occurs as a result of differences in particle surface
properties and their distribution between a solid phase and a
liquid phase (Schwerdt, 1965). Basically, the aqueous polymer
two-phase separation system consists of dissolving two different
polymers such as dextran and methylcellulose or dextran and
polyethylene glycol in water under specified conditions of salt,
pH, and polymer concentrations. Following a holding period,
usually 18 to 24 hours in the cold, two phases are produced. One
phase, the bottom dextran phase, is small in volume and should
contain the concentrated virus. The partitioning of particles in
aqueous polymer two-phase separation systems of dextran sulfate
and polyethylene glycol was reported by Albertsson (1958), The
application of Albertsson's two-phase separation system for
purification and concentration of viruses was described by
Philipson, et at, (1960).
The concentration of enteric viruses from water by a single-
step and a two-step aqueous polymer two-phase separation system was
-------
-37
described by Shuval, et at. (1967). In their system, the single-
step, two-phase procedure was conducted as follows: (i) 640 ml of
test sample was added to a mixture containing 0.2% (w/w) sodium
dextran sulfate 2000 (A. B. Pharmacia, Sweden), 6.45% (w/w) poly-
ethylene glycol (carbowax 4000), and 0,3 M sodium chloride; (ii)
the mixture was shaken, transferred to a separatory funnel and
held overnight at 4° C; (iii) the small bottom phase was then drained
off together with the interphase; -(iv) potassium chloride was added
to a final concentration of 1 molar to the drained phases to precip-
itate the dextran sulfate; and (v) the mixture was then centrifuged
at 2000 rpm for 5 to 10 minutes and the supernatant fluid assayed
for virus. In the two-step, two phase procedure, sodium chloride
was added to a final concentration of 1 molar to the bottom phase as
obtained in the single-step procedure. The dextran sulfate was
not precipitated with KC1. Following an additional holding period
of 18 hours at 4° C, a new two-phase system developed; virus being
concentrated in the small top phase. In Shuval and associates
preliminary experimental studies, poliovirus was concentrated 52- to
200-fold by the single-step procedure with virus recovery effici-
encies ranging from 37 to 98 percent. The two-step, two-phase
procedure yielded a 274-fold virus concentration. The researchers
indicated that virus multiplicities as low as 0.066 PFU/ml were
detected by the two-phase system. They further speculated that
-------
-38
10 PFU/liter or less may be detectable by the method. Further
development and refinement of the polymer two-phase separation
system for concentrating enteroviruses in water was reported by
Shuval, et a1a in 1969. In the refined system, the procedure was
shown, experimentally, to achieve a median virus concentration of
520-fold with virus recoveries ranging from 35 to greater than 100%
from water. As few as 1 to 2 PFU per liter of sample were detected
about 85% of the time. The method was also evaluated by experimen-
tally seeding sewage effluent with poliovirus (2,7 to 1418 PFU/
liter). The average physical concentration factor was 224, The
virus recovery efficiency ranged from 62 to greater than 100 percent.
The aqueous polymer two-phase separation technique was also tested
in field trials by examining raw sewage, water from wells, springs,
and streams, effluent from oxidation ponds, and effluent from a
biofiltration plant. One-liter samples were collected. Viruses
were detected in virtually all of the field samples of sewage with
a maximum of 11,184 PFU/liter in raw sewage. Virus was also de-
tected in 2 samples of drinking water from a shallow municipal
well. The researchers concluded that the use of the aqueous
polymer two-phase separation method for field surveys of sewage
and water samples illustrated the value of the method for the
routine monitoring of potable water supplies for virus contamination.
The use of an aqueous polymer two-phase separation system for
-------
-39
concentrating enteroviruses from sewage has also been reported
by Lund and Hedstrom (1966). In their procedure, sodium dextran
sulfate 2000 was employed as a 20% solution by weight while poly-
ethylene glycol (carbowax 6000) was used as a 30% solution by
weight. Samples collected from the field survey were handled as
follows: (i) to 200 ml of sewaje were added 20 grams of 5 M NaCI,
58 grams of 30% polyethylene glycol, and 2.7 grams of 20% sodium
dextran sulfate. The acidity of the mixture was then adjusted to
pH 7.2. After'shaking, the sar.ple was held for 24 hours at 4° C
before being assayed for virus. During the field survey, weekly
samples were collected. From the end of June to December 1966,
a total of 40 virus isolations '.:ere made. The samples processed
by the aqueous polymer two-phase separation technique yielded 38
virus isolations while only 15 samples of untreated sewage samples
were positive for virus. The researchers concluded that the method
was efficient for concentrating virus from sex?age,
Lund (.-personal cormunioabi 1970) has also applied the
aqueous polymer two-phase separation system to water samples.
Samples were collected both by the swab-method (polyethylene sponge)
and by the dip-sample method. Swab samples were handled as follows:
(i) swabs were exposed to water to be examined for 24 hours; (ii)
200 ml of fluid were squeezed from the swab; and (iii) the sample
was then concentrated in the usual manner by adding sodium dextran
-------
-40
sulfate, polyethylene glycol, and sodium chloride. Dip-samples
were processed using 200-ml volumes. The physical concentration
was 100-fold, Following the two-phase separation, the bottom
phase and interphase were processed in parallel. Both samples
were decontaminated by ether treatment and then inoculated
directly into tissue culture. It was observed that with field
samples a greater number of virus-positive samples were obtained
by using the swab-method of sample collection. In laboratory-
controlled experiments, virus recovery from water samples was
observed to be 100 percent. Interestingly, Lund noted that with
sludge samples no concentration was obtained with the aqueous
polymer two-phase separation system.
Limitations of the aqueous polymer two-phase separation tech-
nique for detecting viruses in dilute aqueous suspensions'were re-
ported by Grindrod and Cliver (1969), Using primary rhesus monkey
kidney for virus quantification, 7 enteroviruses (poliovirus types
1, 2, 3; coxsackievirus A-9; coxsackievirus B-2 and B-3; and echovirus
type 6) were subjected to concentration by the aqueous polymer two-
phase separation technique. At a high-multiplicity of virus input
3 8
(1.4 x 10 to 6.5 x 10 PFU), all seven enteroviruses Rested were
efficiently concentrated into the bottom dextran phase. At a low-
multiplicity virus input (1 to 10 PFU), recovery of poliovirus type
1 was considered satisfactory while the recovery of coxsackievirus
-------
-41
A-9 was unsatisfactory (recovery <11%). During the course of
experimentation, it was observed the sodium dextran sulfate was
somewhat inhibitory to coxsackievirus A-9, and extremely inhibitory
to coxsackievirus B-2 and echovirus type 6. The researchers sug-
gested that inhibition of the model viruses used in quantitative
experiments might have escaped notice by other workers because the
bottom dextran sulfate phase was diluted before titrating the
viruses. The researchers summarized their results by stating that
the aqueous polymer two-phase separation technique was found to be
a significant aid to detection of all three polioviruses, coxsackie-
virus B-3, and in the presence of fluid maintenance medium only,
coxsackievirus A-9, The method, however, was considered worse than
no treatment at all for detecting coxsackievirus B-2, and echovirus
type 6. Cliver (personal oomrrwoxiaationj 1970) and in a recent pub-
lication (Grindrod and Cliver, 1970) indicated that there should
be a 50% probability of detecting virus in a sample containing 1
to 2 PFU per liter, depending on the virus type, of course, and if
dextran rather than sodium dextran sulfate is used in the two-phase
separation system. Recoveries of enteroviruses were observed to
range from 59 to 164% when tested with dextran as compared to
0,001 to 100% with dextran sulfate.
Liu, et at. ( ) have studied some of the parameters that
influence the efficacy of the aqueous polymer two-phase separation
-------
-42
system for concentrating and recovering viruses from water and
seawater. They worked with poliovirus type 1, coxsackievirus A-9,
echovirus type 11, adenovirus type 12, and influenza virus type A.
They observed that sodium dextran sulfate 2000 and 500 were equally
effective. Conversely, sodium dextran sulfate of smaller molecular
size and DEAE-dextran were totally ineffective. The optimal poly-
ethylene glycol as well as the sodium chloride concentration was
found to vary depending on the virus being studied. They con-
cluded that the4 use of the aqueous polymer two-phase separation
technique for separating a mixture of viruses could be a very
complex problem. Their results indicated that many facets of the
technique remained to be investigated; e.g., (i) more virus types
should be studied; and (ii) optimal concentrations of sodium
dextran sulfate, polyethylene glycol, and sodium chloride for each
virus type must be determined. Virus recovery from 2-liter volumes
in laboratory-controlled studies has ranged from 57 to 100 percent.
In a pilot field-survey study, Liu, at, ( ) observed that
sample sizes of 20 liters were impractical because a preliminary
volume-reduction step was required. This was done by hydroextrac-
tion with polyethylene glycol. They routinely processed samples
of 2-liter size by the aqueous polymer two-phase separation tech-
nique without difficulty,
Nupen (1970) has applied the aqueous polymer two-phase sepa-
ration technique in field trials for^virus detection in waste-water.
-------
-43
The field trials were undertaken in connection with the virus con-
trol program for testing the Windhoek advanced waste-water recla-
mation plant at Windhoek, South Africa. The technique as described
by Shuval, et at. (1969) was employed with the exception of the
following: (i) 2 grams sodium dextran sulfate 500 (A. B, Pharmacia,
Sweden), (ii) 6.45 grams polyethylene glycol (carbowax 20,000), and
(iii; 17.53 grams sodium chloride were added to each liter of water
sample. Virus recovery from 1-li.ter volumes in laboratory-controlled
studies was 40 percent. An evaluation of virus quantification from
the aqueous polymer two-phase separation technique by the TCID^q
assay method and by the plaque technique was also undertaken. In
this regard, the TCID^q method was considered to be superior to the
plaque method, experimentally, as determined by the coefficient of
variation among replicate titrations. The experiment was conducted
using poliovirus type 2 (P712) added to tap-water.
During the testing of the Windhoek reclamation plant, com-
posite water samples were prepared by collecting 500-ml samples from
each sampling point every 6 hours for a period of 48 hours. The
sampling points were: (i) settled sewage, (ii) humus tank effluent,
(iii) maturation pond effluent, immediately before advanced treat-
ment, (iv) Goreangab Dam effluent, and (v) the treated water. One-
liter quantities of water were concentrated by the aqueous polymer
two-phase separation technique. Virus was isolated from the settled
-------
-44
sewage, the humus tank effluent and after 14 days' retention in the
maturation pond effluent. No virus was detected after advanced
waste-water treatment. It was noted that, in the field samples,
a higher isolation rate was obtained by the TCID^q assay method
when a comparison was made with the plaque assay technique. The
researcher concluded by stating that the aqueous polymer two-phase
separation technique proved effective in testing of the virus
removal by the advanced waste-water treatment plant.
To summarize, the use of the aqueous polymer two-phase separa-
tion technique for concentrating and detecting viruses from water
holds promise as being an acceptable method. Experimentally,
virus recoveries have ranged from 35 to greater than 100 percent.
Some typical results are shown in Table 6. The method is limited
apparently to 1- or 2-liter volur.es of water. Application of the
method to field samples has been carried out by a number of workers
with favorable results (Shuval, 1969; Lund and Hedstrom, 1966; and
Nupen, 1970). Indications are that virus multiplicities as low as
1 to 2 PFU per liter of water can be detected with about 85%
reliability (Shuval, 1969). The major advantages of the technique
are simplicity and economy. One major disadvantage of the method
may be related to the inhibitory action of dextran sul/ate on
certain enteroviruses (Grindrod and Cliver, 1969) However, Liu,
et at, ( ) indicated that with KC1 precipitation, the dextran
-------
TABLE 6
RECOVERY OF VIRUS FROM WATER BY THE AQUEOUS POLYMER TWO-PHASE
SEPARATION METHOD
Reference
Type of water
Volume
of
water
Percent of
virus
recovered
Shuval, et al,3 1967
Distilled-water
640 ml
37 - 98
Shuval, et al. 3 1969
Sewage effluent
1 liter
35 - 100
Lund, 1970
Sewage effluent
200 ml
- 100
Cliver, 1970
Tap-water
1 liter
59 - 164
Nupen, 1970
Waste-water
1 liter
- 40
Liu, et al, j ( )
Tap-water
2 liters
57 - 100
-------
-45
sulfate is effectively eliminated from the bottom phase. The
small quantity of water that can be processed at a given time may
be another disadvantage; i.e., if the method is applied to rela-
tively nonpolluted or finished (drinking) waters. It would appear
that the method was more effective with moderately or grossly pol-
luted waters; with finished waters, the method may be equivocal.
Soluble alginate filter technique.
The mechanism of soluble alginate filters that permits the
concentration of viruses has not been precisely proffered in the
literature but undoubtedly involves a sophisticated virus entrap-
ment and may even involve a cor.bination of retention and adsorption.
Nevertheless, the unique feature of the alginate filter is related
to its solubility in sodium citrate. And, for the most part, the
alginate-citrate solution is not virus-inactivating nor cytotoxic.
In the early work, filters were prepared by the individual investi-
gator. This was done, for exar.ple, by placing a solution of 1%
sodium alginate sol onto a piece of filter paper previously soaked
with an electrolyte consisting of 0.5 M lanthanum nitrate [La(NC^y
6H2o3 and 0.5 M aluminum chloride (AlCl^I^O). Following filtra-
tion, a 3.8% solution of sodium citrate is used to dissolve the
alginate filter. Tissue culture or mice are then inoculated di-
rectly with the alginate-citrate solution (G&rtner 1967). Alginate
-------
-46
filters are now available commercially in the United States
(Sartorius Membranes Division, Brinkman Instruments, Inc.,
Westburg, N.Y. 11590) and also in Germany (Sartorius).
The efficiency of the laboratory-prepared alginate filters
was reported by Gartner in 1967. Working with poliovirus as the
model virus in filtration studies, Gartner presented evidence that
the alginate filter completely retained the input-virus; no virus
being found in the filtrate. The experimental recovery of polio-
virus from dissolved filters ranged from 25 to 100% being somewhat
influenced, perhaps, by the initial input-multiplicity of virus.
Field trials were also undertaken by G&rtner in which untreated
sewage and sewage effluents from purification plants were examined
for virus content by use of the homemade alginate filters, A com-
parison of the direct inoculation method to the soluble alginate
filter technique yielded 55% and 87% virus-positive isolations,
respectively. This indicated the superiority of the soluble
alginate filter method over the direct inoculation method. Gartner
(personal communication, 1970) has further indicated that 1-liter
volumes of clean water can be filtered in a short time. With pol-
luted (turbid) waters, however, the filtration flux is noticeably
reduced, G'drtner (1970) recognized that the soluble alginate filter
technique may not be the ultimate method but he feels that it is
the best method presently available for concentrating and detecting
-------
-47
viruses from water.
Witt (1965) presented information that up to 10 liters of
water could be filtered through the soluble alginate filter and
then by dissolving the filter in 1 ml of isotonic citrate solution,
a concentration of 10,000 to 1 could be achieved. In laboratory
experiments using poliovirus tyje 3, he was able to detect as
little as 0,001 LD^/ml, Mitt observed some difficulties in the
beginning when the alginate method was applied to field samples
of drinking water. With increasing volumes of water, he found a
corresponding increased filtration flux that negated the filtra-
tion process. This difficulty vas overcome in later field studies
by combining the alginate filter with a membrane support filter.
In these field studies, no viruses were isolated from the drinking
water,
Nupen (1970) has applied tr.e soluble alginate filter technique
in field trials for virus detection from waste-water# The field
trials were undertaken in connection with the virus control program
for testing the Windhoek advanced waste-water reclamation plant at
Windhoek, South Africa, By the use of sterile Sartorius-membrane
filters (GmbH 50-trm) under 500 m Hg negative pressure, 1-liter
volumes of waste-water were filtered through the alginate-membrane
supported filter system. The virus-containing-alginate film was
floated off the supporting membrane with 0.9% sodium chloride and
then dissolved in 3 ml of 3,8% sodium citrate solution. During the
-------
-48
field trials, virus was isolated from the settled sewage, the humus
tank effluent and after 14 days1 retention in the maturation pond
effluent. No virus was detected after advanced waste-water treat-
ment. These results were the same as those observed with the
aqueous polymer two-phase separation technique. A laboratory eval-
uation of the soluble alginate filter technique by Nupen showed an
average virus recovery of 40.05% and a coefficient of variation
of 41.69 percent. According to the researcher, this further indi-
cated that the alginate method was comparable to, and of equal
efficiency as, the aqueous polymer two-phase separation technique.
However, Nupen (personal oo^runioation^ 1970) also indicated that
the soluble alginate filter technique was impractical time-wise
on turbid water samples because of clogging problems.
Poynter, (personal convr.cnioation^ 1970) has used the soluble
alginate filter technique over the past six years and is currently
using the method for isolation of viruses from river waters in
England, Alginate filters were at one time prepared by hand at
Poynter's Laboratory but now are obtained commercially from
Sartorius (Germany). The cornmercial filters have been reinforced
so that pressures of up to 700 mm Hg may be used. According to
Poynter, the alginate filter technique provides the simplest and
yet the most effective method for examining environmental waters
for viruses. In practice, turbid waters are prefiltered through
-------
-49
Oxoid cellulose acetate membrane filters (47-mm, 0.45 ym porosity)
pretreated with broth (10 ml of Hartleyfs digest broth or a
protein-containing fluid such as serum) to prevent virus adsorp-
tion. The crystal clear water is then filtered through the
alginate filter. Up to 2 liters per alginate filter may be pro-
cessed at one time. Filtration is done in a cold-room at 5° C.
Following filtration, the alginate filter is detached from the
reinforcing backing-filter piece and dissolved in isotonic sodium
citrate (3.8%). Viruses present in 1 to 2 liters of water are
thus concentrated into a 1-ml volume. After addition of antibiotics,
the alginate-citrate solution is inoculated directly onto tissue
culture. Raw water samples of 19-liter size have also been pro-
cessed with a special pressure vessel. Rupture of the alginate
filter with the larger volume has not been observed. During the
past several years of field experience, Poynter has observed that
enteroviruses are regularly recovered from the River Thames and
the River Lee at counts of 0.5 to 10 PFU/liter. In laboratory
studies, Poynter (1970) indicated that virus recoveries have
ranged from 60 to 70% to over 100 percent. Viruses have never
been found in the filtrates following filtration.
To summarize, the use of the soluble alginate filter technique
in laboratory experiments and in field trials indicates that the
soluble alginate filter method holds good promise for being an
-------
¦50
adequate and acceptable method for concentrating and detecting
viruses from water. Experimentally, virus recoveries ranging from
25 to 100% have been reported. Some typical results are shown in
Table 7. Volumes of water normally subjected to virus assessment
have beet» 1 or 2 liters. However, 19-liter volumes have also
been processed successfully. Application of the soluble alginate
filter technique to the real world situation has been carried out
for over six years by British investigators (Poynter, 1970) and by
investigators in Germany (GHrtner, 1970) and South Africa (Nupen,
1970) with favorable recovery results. The advantages of the
soluble alginate filter technique for concentrating and detecting
viruses in water are related to its simplicity, speed, and econ-
omy with particular emphasis being placed on the solubility feature
of the filter material. The major disadvantage of the method mani-
fests itself when turbid waters are subjected to virus examination
because the filters clog rather easily. Consequently, waters con-
taining suspended particulate matter must be prefiltered before
filtration by the alginate filter,
Continuous-floi) v.Itracentrifugaticn method.
The mechanism of this method is related to the sedimentation
characteristics of virus particles (virions) subjected to a cen-
trifugal force. Today, commercial ultracentrifuges have capacities
-------
TABLE 7
RECOVERY OF VIRUS FROM WATER BY THE SOLUBLE ALGINATE FILTER
TECHNIQUE
Percent of
Volume of
virus
Reference
Type of water
water
recovered
Gartner, 1967
Drinking-water
10 liters
25 - 100
G&rtner, 1970
Surface-water
1 liter
- 100
Nupen, 1970
Waste-water
1 liter
- 40
Poynter, 1970
River-water
2 liters
60 - 70
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-51
of 100- to 1000-ml and are capable of speeds yielding average
centrifugal fields of 40,000 to 120,000 times gravity. Small
viruses, the size and density of poliovirus virions can be complete-
ly sedimented within 1 to 1.5 hours from an aqueous suspension by
a centrifugal field of 120,000 times gravity (Schwerdt, 1S65).
Depending upon the size, shape, and density of virus particles, the
centrifugal force, viscosity, and density of suspending medium, it
is possible to fractionate classes of particles by either rate-
zonal or isopycnic-zonal centrifugation. In rate-zonal centrifu-
gation, particles are separated on the basis of differences in
sedimentation rate in a density gradient. The density gradient
provides gravitational stability to the system. In isopycnic-
zonal centrifugation, particles having the same sedimentation rate
are separated on the basis of different buoyant densities. The
adaptation of isopycnic-zonal centrifugation to concentration of
viruses from water relies on the fact that this procedure can be
carried out as continuous-flow ultracentrifugation whereby a con-
tinuous stream of water is flowed over a density gradient in a
special rotor (Anderson, et al. 1967).
The application of routine ultracentrifugation for concentra-
tion and detection of enteroviurses in dilute suspension was
described by Oliver and Yeatman (1965), By use of a Spinco Model
L Preparative Ultracentrifuge with both the number 30 and 50 rotors,
-------
-52
quantitative recovery of poliovirus type 1 and coxsackievirus B-2
was achieved. Samples of less than 10 ml can be concentrated in
120 minutes in the number 50 rotor. The researchers concluded
that there was a 50% probability of detecting enterovirus by
means of the number 50 rotor when the initial virus multiplicity
was as low as 0.12 PFU/ml. The same findings were proffered with
the number 30 rotor when the initial virus multiplicity was as
low as 0.025 PFU/ml. Cliver (personal eotrmuni.oationt 1970) indi-
cated that by means of the number 50,1 rotor, 840 ml of sample
t
can be processed per day. Concentration of virus has ranged from
35- to 70-fold. Experimentally, virus recoveries have ranged from
an average of 60 to 70% with a high of 118 percent. The method
has not been applied to field samples.
The application of continuous-flow ultracentrifugation for
concentrating viruses in water was presented by Anderson in 1965
at a national symposium (Anderson, et at. 1967), A high perfor-
mance continuous-flow centrifuge was described which removed over
95% of suspended poliovirus at a flow rate of 2 to 3 liters per
hour. The sedimented virus was pelleted directly onto the wall
of the rotor and removed by resuspension. It was noted by Anderson
that certain types of virus, however, were inactivated after pel-
leting. This difficulty was overcome by trapping virus particles
isopycnically in stationary density gradients. The experimental
-------
-53
systems built were successfully tested with adenovirus type 2
and respiratory syncytial virus. According to Anderson (1970)
continuous-flow centrifuges for large-scale isolation of viruses
from dilute suspensions are being developed. For example, the
K-II zonal ultracentrifuge at flow rates above 25 liters per hour
has yielded influenza virus recoveries of 80 to 90 percent.
Higher-capacity centrifuges are also being designed. These cen-
trifuges not only concentrate the virus but band it isopycnically
at the same tiifle.
To summarize, the use of preparative ultracentrifugation for
concentrating and detecting viruses from waters of the real world
does not hold too much promise as a routine method. The major
disadvantages of the method are: (i) the ultracentrifuges are
too costly to buy and maintain; and (ii) processing large volumes
of water would be prohibitively time-consuming. The application
of ultracentrifugation techniques to virus-in-water problems of
the real world may involve their use as a secondary-procedure for
final virus concentration prior to viral assessment in tissue
culture. Preparative ultracentrifugation could be particularly
useful for concentrating viruses iij eluates from virus adsorption-
elution methods. Continuous-flow ultracentrifugation, at the
present time, must be considered as developmental, the application,
of which remains to be determined.
-------
-54
Forced-flew electrophoresis and electro-osmosis.
The mechanism of electrophoresis and electro-osmosis is
related principally to the amphoteric nature of viruses in that
viruses exhibit mobility in a predictable direction at a given
pH under a direct current electrical field. In forced-flow
electrophoresis, electrophoretic transport brings about selective
adsorption of viruses onto dialyzing membranes. The electrophoretic
cells used in this method are essentially filtering devices. The
apparatus which' consists of semipermeable dialyzing membranes and
a microporous filter separated by suitable plastic spacers has
been described in detail by Bier, et at• (1967). In electro-
osmosis, water diffuses from one side of a membrane to the other
under the influence of a difference of electrical potential. For
example, when water is confined in a capillary tube, adsorption of
hydroxy1 ions (0H~) imparts a negative charge to the walls of the
tube. Adjoining the hydroxyl ions is a layer of hydrogen ions
(H+) in equal number producing an electrical double-layer. If a
difference of electrical potential exists between the two ends of
the tube, water migrates toward the negative electrode by a pro-
cess referred to as electroendosmos'is. The presence otf anions
such as viruses (suspended at a pH above their isoelectric point)
are firmly adsorbed to the walls of the tube during the process.
-------
-55
The application of forced-flow electrophoresis to concentra-
tion and detection of viruses in water was reported by Bier, et
at, (1967). When using bacteriophage as the model virus, and
flow rates of 60 to 240 ml per hour, quantitative recovery of
virus was achieved. Concentration of bacteriophage was observed
to be approximately 100-fold. According to the researchers,
forced-flox^ electrophoresis has several advantages: (i) large
volumes of water can be processed in a short time; (ii) the proce-
dure and equipment are simple; and (iii) bacterial contamination
can be eliminated from the virus suspension. Interestingly, it
was noted in forced-flow electrophoresis that the application
of electric current prevents clogging of the microporous membrane
filter and decreases adsorption of virus onto the filter matrix.
The method has not been applied to field samples. According to
Bier (personal communiaation3 1970), the sensitivity of the tech-
nique, as determined experimentally, is of the order of a few
PFU per ml while recovery of virus is near 100 percent. Complete
retention of all the virus within the electrophoretic cell has
been obtained,
McHale, et at, (1970) described the concentration of virus
from water by forced-flow electrophoresis and electro-osmosis.
They used an electrophoretic filter/concentrator modified after
Bier. When using poliovirus as the model virus and flovr rates of
-------
-56
approximately 300 ml per hour, virus was concentrated nearly
3-fold by forced-flaw electrophoresis. By the use of electro-
osmosis, concentration of virus was 5-fold; water being removed
at a rate of 0,8 ml per hour per cm of membrane area. Under the
conditions of experimentation, the investigators concluded that
forced-flow electrophoresis and electro-osmosis were more rapid
and gentle than other methods for concentration of virus.
To summarize, the use of forced-flow electrophoresis and
electro-osmosis techniques for concentrating viruses from water
t
appears to be developmental but may have some application. Con-
centration of virus from water has ranged from 3- to 100-fold.
The small volumes of water that can be processed in a reasonable
time period, however, would tend to malce these methods of little
value as primary virus concentration techniques for very large
volumes of water. Their application as secondary virus concentra-
tion procedures may be worth considering. Neither technique has
been applied to the real world situation.
Other methods,
Cliver (1967) described the use of polyethylene glycol in a
hydroeKtraction method for concentrating virus from water. In
this method, a 100-ml sample is placed into a dialysis tube which
is surrounded by 100 grams of polyethylene glycol {carbowax
20,000) dissolved in 100 ml of wat-ir. Concentration Is allowed to
-------
-57
proceed at room temperature. After 2 to 3 hours, the volume of
the 100-ml suspension was reduced to 1 ml, achieving, of course,
a 100-fold physical concentration. Virus recoveries have ranged
from 10 to 30% (Oliver, personal oommunication, 1970), Indications
are that there is a 50% probability of detecting virus in liter-
quantities of water if the contamination level is at least 10 PFU
per liter.
Hoff, et alB (1967) designed and evaluated a flow-through
gauze sampler-device for concentration of viruses from water. The
sampler-device was tested during some field trials on a stream
remote from human habitation. In the field studies, 1 gallon per
minute was pumped through the sampler-device for 7 days (1440
gallons per day). No virus was detected during the course of the
field trials. Experimental laboratory studies indicated virus
recoveries ranging from 0.6 to 3.5% depending upon whether the
water sample was clear or turbid; higher virus recoveries being
observed when the water was turbid. Hoff (personal QormunioaHont
1970) indicated that the main advantage offered by the flow-through
gauze sampler-device was that large volumes of natural waters could
be sampled over extended periods of time (1440 gallons per 24 hours).
Nevertheless, the efficiency and quantitative recovery of virus
from water was discouragingly low in experimental studies.
Liu, et at. (1970) reported on a preliminary study that was
carried out on the flow-through gauze sampler-device designed by
-------
-58
Hoff and associates for recovering virus from tap-water and seawater.
The amount of virus (poliovirus) recovered by the sampler-device
was 2% when tap-water was used. In seawater, virus recovery ranged
from 15 to 19 percent. The addition of 3% sodium chloride to the
tap-water increased the virus recovered to 47 percent. Volumes of
water up to 90 gallons were tested by the sampler-device. The flow
rate of the sampler-device was 1-gallon per minute. The virus con-
centration factor achieved when seawater was used was 100-fold;
while in tap-water the virus concentration factor achieved was
about 10-fold, ' The researchers concluded that the flow-through
gauze sampler-device should be further developed and evaluated.
GENERAL DISCUSSION
A number of methods adapted to concentrating and isolating
viruses from water have been described. Several of the methods
hold good promise as being acceptable for routine use in the real
world situation. For example, the membrane-adsorption technique,
adsorption to iron oxide or polyelectrolytes, aqueous polymer
two-phase separation, and the soluble alginate filter technique all
have shown good-to-excellent virus recovery efficiencies under
laboratory-controlled conditions. Under field conditions, common
-------
-59
deficiencies have been observed with some of these methods when
the water was turbid (muddy); a common feature of many surface-
waters. For example, the membrane-adsorption technique, the iron
oxide or polyelectrolyte "sandwich" technique and also the soluble
alginate filter technique all fall short for examining moderately
turbid water because of clogging problems. This difficulty
can be overcome to some extent by clarification of turbid water
samples by prefiltration before applying the particular virus con-
centration method. The need to prefilter turbid waters places an
ill-defined limitation, however, on a particular method since pre-
filtration may result in significant losses of virus in the water
sample. If low-multiplicities of virus occur in the water sample
their presence may be undetected. It would appear that the aqueous
polymer two-phase separation technique is best suited for quantita-
tively detecting and isolating viruses from moderately turbid
waters. The major disadvantage of this method is related to the
small volume of water that can be processed at a given time; e.g.,
1- to 2-liter samples. Therefore, the waters must be moderately
or grossly polluted with fecal wastes in order to achieve a high
number of successes (virus isolations). The major advantages of
the aqueous polymer two-phase separation technique are simplicity
and economy.
In clean water or finished waters, the problem of clogging
would be nil but the expected occurrence of viruses at extremely
-------
-60
low-multiplicities would demand that very large volumes of water
of at least 100 gallons be processed. It would appear that the
virus-adsorption and/or virus-retention procedures; i.e., virus
adsorption to microporous membranes, iron oxide, and polyelectro-
lytes; and virus retention by soluble alginate and possibly the
gauze flow-through sampler-device are best suited for quantita-
tively detecting and isolating viruses from clean or finished waters.
In addition to the very large volumes of water that can be pro-
cessed by the virus-adsorption and/or virus retention procedures,
they have the common advantage- of speed, simplicity, and economy.
These features will be particularly appealing when a technique is
eventually selected as a candidate for routine use. Initial costs,
however, may seem relatively high when considering the cost of the
stainless-steel filtration assemblies or holders. These can often
approach $900.00 for the larger size holders.
The shortcomings of contir.uous-flow ultracentrifugation for
routine use for quantitatively detecting and isolating viruses
from waters are several-fold, the least of which is very high cost.
The application of continuous-flow ultracentrifugation to the real
world problems of viruses-in-vater would appear to be impractical.
Likewise, forced-flow electrophoresis and electro-osmosis would
appear to be primarily research tools rather than having any
practical application for quantitatively detecting and isolating
viruses from naturally contaminated waters.
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Acknowledgements
We thank Abner C. Jones III for preparing the photographic
material. We also thank Vida H. Hartfield for typing the manus
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-61
REFERENCES
Anderson, N. G,, Cline, G. B., Harris, N. W., and Green, J. G. Q967)
"Isolation of Viral Particles from Large Fluid Volumes" in
Transmission of Viruses by the Water Routes G. Berg, ed.
Interscience Publishers, New York.
Anderson, N. G. (1970) Public Health Aspects of Viruses in Watera
N. A, Clarke, ed, Cincinnati, Ohio, 1, p, 1.
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