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Factors influencing virus adsorption to solids

Material Information

Title:
Factors influencing virus adsorption to solids
Creator:
Shields, Patricia Ann, 1958-
Publication Date:
Language:
English
Physical Description:
vi, 191 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Adsorption ( jstor )
Bacteriophages ( jstor )
Beef ( jstor )
Elution ( jstor )
Flocculation ( jstor )
pH ( jstor )
Quaternary ammonium compounds ( jstor )
Soils ( jstor )
Sulfates ( jstor )
Viruses ( jstor )
Adsorption (Biology) ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Microbiology and Cell Science thesis Ph. D
Viruses ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Bibliography: leaves 176-190.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Patricia Ann Shields.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
022306730 ( ALEPH )
14584647 ( OCLC )
ACZ3222 ( NOTIS )
AA00004871_00001 ( sobekcm )

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Full Text



















FACTORS INFLUENCING VIRUS
ADSORPTION TO SOLIDS













by




PATRICIA ANN SHIELDS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1986




FACTORS INFLUENCING VIRUS
ADSORPTION TO SOLIDS
by
PATRICIA ANN SHIELDS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986


Mom and Dad, and Tom, Sharon, Carrie and Jennifer


ACKNOWLEDGMENTS
I gratefully acknowledge the help and guidance given me by the
members of my committee, Dr. Samuel R. Farrah, Dr. Lonnie 0. Ingram, Dr.
Stephen G. Zam, Dr. Gabriel Bitton and Dr. Dinesh 0. Shah. I am forever
indebted to my chairman, Dr. Farrah, who has been my friend as well as
my boss. The example of scientific excellence he has set will never be
forgotten.
In addition, I extend thanks to those individuals who have helped,
in many ways, to make this journey enjoyable, especially Dr. Phillip
Scheuerman, Orlando Lanni, Sharon Berenfeld, Kathi Moody, Vivienne
Thompson, Dave Preston, Lena Dingier, Jane Strandberg and Gail Waldman.
In particular, I am beholden to Lena Dingier, and Jane Strandberg, both
of whom have shown me the true meaning of friendship.
Finally, I extend thanks to my family for the love and support they
have given me in this and all other endeavors. This dissertation is
dedicated to them.
iii


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS i ii
ABSTRACT v
CHAPTERS
I INTRODUCTION 1
II VIRUS ASSOCIATION WITH MEMBRANE FILTERS 7
Review of the Literature
Materials and Methods 25
Results 33
Discussion 65
III VIRUS ASSOCIATION WITH OTHER SOLIDS 76
Review of the Literature 76
Materials and Methods 95
Results 99
Discussion 123
IV CHARACTERIZATION OF VIRUSES 133
Review of the Literature 133
Materials and Methods 141
Results 143
Discussion 156
V SUMMARY 166
APPENDICES
A ROUTINE METHODS USED IN ANIMAL AND BACTERIAL VIRUS
PROPAGATION AND ASSAY 168
B COMPOSITION OF MEDIA AND SOLUTIONS USED IN CELL
CULTURE WORK 172
BIBLIOGRAPHY 175
BIOGRAPHICAL SKETCH 190
iv


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FACTORS INFLUENCING VIRUS
ADSORPTION TO SOLIDS
By
Patricia Ann Shields
May 1986
Chairman: Samuel R. Farrah
Major Department: Microbiology and Cell Science
Several factors influence the association of viruses with solids.
The natures of the adsorbent and virus studied are among the most
important factors that influence this association. Differences in virus
adsorptive behavior have been related to the relative strength of
hydrophobic and electrostatic interactions between the virus and solid.
The main objective of this study was to determine the relative
hydrophobic and electrostatic nature of viruses and various virus-
adsorbing solids.
Membrane filters used in virus concentration procedures were
characterized by contact angle and capillary rise measurements. Among
electropositive filters tested, Seitz S filters were found to be the
most hydrophobic, while Virosorb 1MDS filters were found to be the most
hydrophilic. Among electronegative filters tested, Millipore HA filters
were found to be the most hydrophobic, while Filterite filters were
found to be the most hydrophilic. These physical measurements were
found to correlate well with the ability of certain solutions to elute
v


viruses adsorbed to the filters. Based on these differences, several
two-step concentration procedures for viruses in water were developed
that used dissimilar filters for each stage.
Beef extract solutions are often used for the elution of viruses
adsorbed to membrane filters. The use of ammonium sulfate flocculation
to concentrate viruses in these solutions was compared to organic
flocculation. The recovery of seeded enteroviruses from sludge was 62%
using ammonium sulfate flocculation as compared to 34% when organic
flocculation was used as a concentration step. Ammonium sulfate
flocculation was also found to improve recoveries of viruses from
sewage effluents and soils.
The relative hydrophobic and electrostatic nature of viruses was
determined. Hydrophobic interaction chromatography and hydrocarbon
adherence tests were used to assess the relative hydrophobicity of the
viruses. Adsorption to DEAE-sepharose was used to assess the relative
electrostatic nature of the viruses. Based on these tests, echovirus-5
and bacteriophage MS2 were found to have the highest relative
hydrophobicity. Bacteriophages T2, T4 and MS2 were found to adsorb very
strongly to DEAE-sepharose, indicating these viruses had the strongest
relative electrostatic interactions of the viruses tested.
vi


2
Table 1. Human
enteric viruses
and associated diseases.3
Virus Group
Number
of Types
Disease
Poliovirus
3
Paralytic poliomyelitis, aseptic
meningitis
Coxsackievirus
Group A
24
Herpangina, aseptic meningitis,
paralysis, fever
Group B
6
Pleurodynia (Bornholm disease),
aseptic meningitis, acute
infantile myocarditis, rash,
fever, respiratory disease
Echovirus
34
Aseptic meningitis, fever,
rash, diarrheal disease,
respiratory disease
Hepatitis A
1
Infectious hepatitis
Reovirus
3
Fever, respiratory disease,
diarrhea
Rotavirus
4
Severe diarrhea, vomiting,
low grade fever, dehydration
Adenovirus
31
Respiratory and eye infections
Norwalk virus
3 (?)
Diarrhea, vomiting
a Adapted from 59, 60 and 68.


3
sludges and effluents are often disposed of on land (174). Once
disposed of in this manner, viruses associated with sludge floes or in
effluents interact with soils can lead to viral contamination of crops,
soils, and surface and groundwaters (65, 68, 172, 174).
While the number of viruses in raw sewage may be quite high, the
concentration of viruses after treatment, and in subsequently
contaminated solids and water supplies is low. Even at these low
numbers, enteric viruses are capable of causing disease and thus are
cause for concern. Because the numbers of viruses are low, it is not
always practical to screen samples for the presence of these pathogens.
Therefore, it is logical to screen for the presence of an indicator
organism whose presence may indicate fecal pollution of the sample. The
presence of coliform organisms, such as E. coli, has been used
extensively as a measurement of fecal contamination (2). However,
studies have shown that enteric viruses are more resistent to
environmental conditions and sewage treatment processes than are the
coliform organisms (7, 8, 88). Enteric viruses have been isolated from
water supplies which have met coliform limits (7, 31, 57, 76, 187).
Therefore, there appears to be a need to find another, more suitable
organism to serve as an indicator for enteric viruses.
The ideal microbial indicator should have the following
characteristics: i) It should be present when pathogens are present and
absent when pathogens are absent. ii) The growth characteristics and
persistence of the indicator should be similar to the pathogen, iii)
Both the pathogen and the indicator should occur in a constant ratio so
that counts of the indicator will estimate the amount of pathogen


4
present, iv) The indicator should be found in greater numbers than the
pathogen, v) The indicator should be at least as resistant to
environmental factors as is the pathogen, vi) The indicator should be
nonpathogenic, easily quantifiable, and be able to be tested for in a
variety of environments (63, 167). Several investigators have advanced
the idea of using bacteriophage as indicators for the presence of
enteric viruses in fecally contaminated water (35, 78, 98, 167).
Recently, Stetler monitored coliphages in conjunction with indicator
bacteria and enteroviruses in a drinking water plant (167). Statistical
analysis of the data indicated that coliphages were much better
indicators of enteroviruses than were coliforms or fecal streptococci.
On the other hand, other investigators have noted that bacteriophage
levels do not correlate well with the presence of enteric viruses in
field samples (91, 175). If bacteriophages are to serve as indicator
organisms, then simple and reliable methods must be developed to detect
and concentrate them from natural samples. Procedures used to
concentrate animal viruses often required extremes in pH, which are
unsuitable methods for phage concentration (149, 150). Investigators
have developed methods to concentrate phage using materials which do not
require these fluctuations in pH (67, 113, 150, 157). The development
of a method which could be used to detect both bacterial and animal
viruses in the same sample would simplify and speed up assay procedures
considerably.
Virus interactions with solid surfaces play a major role in their
removal by sewage treatment processes, as well as their response to,
transportation in and persistence in their environment. The phenomenon
of virus adsorption is also important in many methods used for the
detection and concentration of viruses in the environment (53) Thus,


5
it is important to study the mechanism of and factors influencing virus
adsorption to solids.
Several factors may influence the association of viruses with
solids. These factors include pH (21, 121, 171, 183), the concentration
and nature of salt in solution (102, 123, 177, 178, 180), the presence
of organic compounds (17, 55, 183), the flow rate of virus through the
adsorbent (61, 103, 147, 174), the nature of the virus (53, 55, 56, 117,
189) and the nature of the adsorbent surface (11, 123, 124, 164, 174).
Accordingly, these factors have been studies to better understand
virus-solid interactions and determine the mechanism of viral
adsorption.
The adsorption of viruses to solids was though to be mainly
electrostatic in nature (94, 173). Since viruses are essentially
biocolloids, theories which describe colloidal behavior have been used
to describe viral interaction with solids.
If the charges on both the virus particle and adsorbing surface are
opposite, electrostatic attraction results in viral adsorption (94, 117,
190). Conversely, if both are of the same charge, electrostatic
repulsion occurs. In most natural water systems, virus particles are
above their isoelectric point and assume a negative charge. By altering
such factors as salt concentration and pH, adsorption to electronegative
surfaces can occur. This phenomenon can be explained by the double
layer theory (52, 53, 173). Essentially, this theory says that ions
associated with both surfaces will attract counterions from the medium
into a compact layer (called the stern layer) around the surfaces. This
layer of counterions will partially neutralize the charge on the
surface. In order for the system to maintain electrical neutrality,


6
there also exists a diffuse layer containing a lower number of
counterions some distance away. If the bulk solution of counterions
increases by altering the pH or the addition of cationic salts, the
thickness of this diffuse layer decreases. This is because less volume
is needed to contain enough counterions to neutralize the surface
charge. As the size of this layer is decreased, the two surfaces can
come closer to each other and short range attractive forces may take
over. Thus interactions between two disperse particles are thought to
result from a balance between repulsive double layer interactions and
attractive short-range forces, such as van der Waals forces.
Accordingly, altering the pH or addition of cations allows for increased
adsorption to occur (52, 53).
Many studies have confirmed that electrostatic interactions are a
primary mechanism for adsorption of viruses to solids (53, 183, 190).
However, such complex theories such as the double layer theory or simple
theories such as salt bridging fail to explain some of the experimental
results. For example, at high pH, viral adsorption to negatively charge
filters is maintained by the presence of multivalent metal ions.
However, the addition of a chelating agent, which should effectively
remove such ions and prevent salt bridge formation, does not promote
viral elution (40, 42). Furthermore, elution of adsorbed viruses at
high pH has been accomplished using neutral solutions such as urea or
nonionic detergents (42, 48, 180, 183). These variable effects of
different solutions on the elution of viruses adsorbed to solids cannot
be explained in purely electrostatic terms, indicating that additional
forces are involved in viral adsorption to solids. These forces
include hydrogen bonding and more importantly, hydrophobic interactions.


7
The role of hydrophobic interactions in virus adsorption phenomena
has been examined in several studies and results have shown that these
interactions are influenced by salts, and the nature of the virus and
adsorbent studies (32, 40, 49, 50, 154, 156, 177).
The overall objective of this study was to examine the
contributions of electrostatic and hydrophobic interactions in the
association of viruses with solids, in the hope of better understanding
the factors involved in viral adsorption.
Chapter II of this dissertation examines the association of viruses
to membrane filters. These filters are characterized by several
methods, and the differences in the nature of these filters are used to
explain the differences in viral elution patterns seen. In addition,
several concentration procedures are developed for the detection of
animal and bacterial viruses in water samples. Finally, a new
concentration procedure is shown that allows for simultaneous
concentration of animal and bacterial viruses from the same sample.
Chapter III examines the association of viruses to solids such as
clay, soil and wastewater sludges. The extent of hydrophobic
interactions involved in virus association to clays is examined.
Additionally, methods are developed to recover viruses adsorbed to soils
and sludges.
Chapter IV describes the characterization of viruses themselves.
Viruses are examined by adsorption to or elution from ion exchange gels
and hydrophobic gels. Viruses are further characterized by adherence to
hydrocarbons. These results are then used to explain differences in the
adsorption/elution patterns of different viruses.


CHAPTER II
VIRUS ASSOCIATION WITH MEMBRANE FILTERS
Review of the Literature
Concentration of Viruses on Membrane Filters
A wide variety of methods are available for the detection and
concentration of viruses in water samples, however, methods which employ
adsorption to and subsequent elution from membrane filters are still
considered to be among the best (53, 64, 136). In an early study,
Ellford (37) noted a decrease in bacteriophage titer when salt solutions
containing viruses were passed through nitrate cellulose filters. He
found that dilution of the phage into protein solution prior to passage
through the filter avoided loss of viruses onto the filter by
adsorption. In 1967, Cliver (25) found that some enteroviruses were
adsorbed to cellulose nitrate and cellulose acetate filters. In these
experiments, Cliver was attempting to rid solutions of bacteria so as to
assay these solutions for the presence of virus, and therefore this
adsorption was not desirable. He found, much the same as Ellford, that
proteins (in this case serum or gelatin) blocked this viral adsorption.
Later, work in the same laboratory indicated that 99% of enteroviruses
seeded into deionized water were adsorbed when passed through Millipore
(cellulose nitrate) filters (26). Over 80% of these viruses were
subsequently recovered by soaking the membrane in phosphate-buffered
saline containing 30-50% agamma chicken serum.


9
In 1967, Wallis and Melnick reported the first applied use of
membrane filters to concentrate viruses from water samples (180). This
report detailed several important discoveries. First, they showed in
this correspondence that the addition of salts (0.05 M MgCl2) greatly
enhanced virus adsorption to Millipore filters. The adsorbed virus was
recovered by grinding the filter with a mortar and pestle in the
presence of Melnick B media supplemented with 10% fetal bovine serum.
Over the course of several months, using this procedure, 2795 virus
isolates from sewage water were detected in the concentrate, while only
4 isolates were found in unconcentrated samples from the same period
(180). In addition, during the course of this study, Wallis and Melnick
noted interference with adsorption after the passage of about 100 ml of
sewage through the membrane filter. They suggested that this was due to
the high amount of organic material in the sewage. When the sewage was
passed through anion resins, the interfering material was removed and
virus adsorption was enhanced.
In an subsequent paper (181), Wallis and Melnick reported that
protein and other materials present in crude virus harvests interfered
with virus adsorption to Millipore filters. They called these materials
"membrane coating components" or MCC. These MCC could be removed by
adsorption to anion resins. Once the crude cell harvest was treated to
remove MCC, viruses in these harvests were efficiently adsorbed to
Millipore filters in the presence of salts at pH 5.0. Adsorbed virus
was eluted with a small volume of fetal bovine serum, and in virtually
all cases, 80 to 100-fold concentrations were achieved.
A study by Rao and Labzoffsky (138) confirmed and expanded on the
work of Wallis and Melnick. They found that low levels of calcium (200
ppm) facilitated virus adsorption to Millipore filters. In addition,


10
this was the first report of using a prefilter (Millipore AP25) in
series with the adsorbing filter. They suggested that in some water
samples, viruses attached to small solids were lost if the prefiltering
stage was separate from the adsorption stage. By using a filter series,
virus retained on the prefilter was eluted along with virus adsorbed to
the cellulose nitrate filter by treatment with 3% beef extract.
Since different eluting solutions had been used to recover viruses
adsorbed to membrane filters, a comparative study of these eluates was
conducted by Konawalchuk and Speirs (97). Coxsackievirus B5 and
poliovirus-1 adsorbed to Millipore filters were eluted using different
concentrations of beef extract, yeast extract and fetal bovine serum.
It was found that elution improved with increases in concentration of
eluting agent and the volume of the eluent. These researchers found
that the best eluate was undiluted fetal bovine serum, which recovered
over 80% of the adsorbed enteroviruses tested, and did not result in any
toxicity problems during viral assay procedures.
The ability of salts other than MgCl2 and CaCl2 to promote viral
adsorption was examined by Wallis et al. (178). They found that 0.5 mM
AlCl^ was as effective in promoting poliovirus adsorption to Millipore
HA filters as 50 mM MgCl^, and was therefore more cost-effective when
large volumes of water were processed.
The processing of large volumes of water generally requires larger
filters, and subsequently larger volumes of the eluting solution are
necessary for efficient viral elution. In order to reduce the amount of
sample to be assayed, there is a need for a second or reconcentration
step. Since the protein solutions commonly used for viral elution


11
interfered with viral readsorption (25, 37, 180, 181), Wallis et al.
(178) sought another eluate. They found that a high pH (pH= 11.5)
glycine buffer solution eluted viruses adsorbed to Millipore filters
effectively and these eluted viruses were easily readsorbed to a smaller
diameter filter of the same type by lowering the pH and adding AlCl^.
This study laid the groundwork for the development of an apparatus for
concentrating viruses from large volumes of water.
Wallis and coworkers (179) developed an apparatus for separating
virus from water contaminants so that the virus contained in the
clarified water could be readily concentrated on adsorbents at high flow
rates. In its final form, the apparatus consisted of five non-viral
adsorbing texile fibers in series, pretreated with Tween 80 to aid in
removal of contaminants without removal of virus. After passage of the
water through this filter series, the water was treated by passage
through an anion-exchange resin to remove organics which may interfere
with virus adsorption. Prior to virus adsorption onto cellulose nitrate
filters, the water was conditioned with MgCl^ to a final concentration
of 0.05 M to facilitate adsorption. Elution was accomplished using a
glycine buffer at pH 11.5, and virus was reconcentrated by adsorption to
and elution from a smaller diameter filter (178). Poliovirus-1 was
seeded at high levels into 150 gallons of tapwater, and using this
apparatus, 84% of the virus was recovered. WHen very low levels of
virus were added to the tapwater, virus recovery was still over 60%.
This original model was modified by Homma and coworkers (83) and
this prototype was developed for commercial use by the Carborundum
Company (Niagara Falls,NY) as the "Aquella Virus Concentrator" (64,
136). In this system, the water was passed through a series of


12
prefilters consisting of 5 ym and 1 ym polyester depth cartridge filters
followed by a Tween 80 treated, 1 ym cotton cartridge. After passage
through these filters, the pH of the water was adjusted to 3.5 and AlCl^
was added instead of MgCl^ since previous studies had shown the use of
AlCl^ to be more cost effective than MgCl^ (178). Viruses were adsorbed
to a 1 ym fiberglass or cellulose acetate cartridge filter and eluted
with one liter of 0.5 M glycine buffer, pH 11.5, and reconcentrated onto
smaller diameter cellulose nitrate membrane filters.
Sobsey and colleagues (165) found that further modifications of the
Wallis-Melnick concentrator were in order. After adjustment of water to
pH 3.5, it was passed through a fiberglass depth prefilter (K-27)
followed by a 142 mm epoxy-fiberglass-asbestos Cox filter in series.
Elution of adsorbed virus was accomplished using one liter of high pH
glycine buffer solution and virus was reconcentrated to a 10 ml volume
by adsorption onto a smaller diameter (47 mm) Cox filter series. Using
this modified system, they were able to recover over 75% of poliovirus-1
seeded into 100 gallons of tapwater. Problems developed with this
system when it was scaled up to process larger volumes of tapwater (44,
46). Farrah et al. (44) found that there was clogging of the initial
virus adsorbent when large volumes of water were processed. This lead
to a reduced flow rate and organics, such as humic acids (46) were
concentrated along with the viruses and interfered with the
reconcentration of the viruses by adsorption to a smaller diameter
filter. The initial clogging problem appeared to be due to the use of a
flat filter at the primary adsorption step. This problem was eliminated
by using a pleated membrane filter cartridge (epoxy-fiberglass Filterite
filter). This cartridge filter provided a larger surface area for virus


13
adsorption and was capable of adsorbing virus in tapwater at
considerably higher flow rates. The adsorbed virus was eluted with 2 1
of 0.05M glycine, pH 10.5. To overcome the problem of organics
concentrated along with viruses interfering with readsorption to smaller
filters, this step was eliminated and replaced by an aluminum
flocculation procedure (44). THe initial eluate was neutralized and
AlCl^ was added to a 0.003 M concentration. This lowered the pH of the
water to 4. The solution was neutralized and a floe formed, which was
collected by centrifugation. Virus in the floe was eluted by mixing
the floe with an equal volume of 1.0 M glycine, pH 11.5. The complete
process could be done in 3 hours and virus recovery from 1900 1 of tap
water averaged between 40 and 50%.
Up to this time, most work done on the development of concentration
procedures to detect viruses had used tapwater seeded with virus. With
the development and subsequent modification of the Wallis-Melnick virus
concentrator, researchers began to examine its use with more polluted
water supplies. Using this concentration system, enteroviruses were
efficiently recovered from estuarine water (45, 120, 133) raw sewage
(54, 83, 151), and seawater (54).
Prior to 1978, work done on the concentration of viruses from water
supplies using membrane chromatography centered around the use of
negatively-charged filters such as Filterite, Millipore and Cox filters.
Efficient viral adsorption was accomplished by conditioning of the water
sample by addition of salts and acidification. In 1979, Sobsey and
Jones (164) reported the first use of electropositive filters in the
concentration of viruses from water samples. They tested two positively
charged filters: Seitz S, composed of cellulose-asbestos, and Zeta plus


14
grade S, composed of cellulose-diatomaceous earth-"charged-modified"
resin. These filters were compared with two negatively charged filters
used in virus concentration, Filterite and Cox filters. At pH values
near neutrality (pH 7.5), less than 20% of poliovirus-1 in unmodified
tapwater was adsorbed by either the Filterite or Cox filters, while
greater than 99% was adsorbed by the Zeta plus and Seitz S filters.
Zeta plus filters were used to concentrate poliovirus-1 from 100 gallons
of tapwater by adsorption to a 267 mm diameter 60S filter, and followed
by elution with 1 1 glycine buffer, pH 10. Virus in the eluate was
further concentrated by dropping the pH to approximately 7 and adsorbing
to a similar, smaller diameter filter followed by elution in a smaller
volume. Using this procedure, recovery of the poliovirus-1 equaled 75%.
Because these electropositive filters did not require acidification
of water samples, researchers began exploring the use of these filters
for the concentration of viruses sensitive to low pH, such as
bacteriophages (67, 113, 136, 149). While bacteriophages are of no
health concern to humans, this work has been done for several reasons.
Several investigators have advanced the idea of using bacteriophages as
indicators of fecally contaminated water (35, 78, 98, 167). Others have
indicated that concentration of naturally occurring phages is necessary
in order to learn more about bacteriophage ecology (136, 150).
Additionally, bacteriophages have been used as models to study the fate
of animal viruses in sewage treatment plants (140).
Logan et al. (113) evaluated the ability of positively charged
filters to adsorb coliphages at different pH values. They found that
greater than 90% of phages MS2 and T2 were adsorbed at pH 7 by the Zeta
plus filters. Efficient elution of virus was accomplished using a


15
solution of 1% beef extract in 50 mM arginine at pH 9.0. Next, they
seeded 65 1 of prefiltered pond water adjusted to pH 6.0 with various
coliphages. The pond water was passed through a 273 mm Zeta plus 60S
filter and adsorbed phage was eluted with 500 ml of the beef extract
solution. Recoveries of 87% for T2 and 93% of MS2 were obtained using
this method, while only 25% of 'fXlM was recovered. Use of this method
with river water resulted in 50-60% recovery of naturally occurring
bacteriophage.
Goyal and coworkers (67) also evaluated the use of Zeta plus
filters for the concentration of phage. They found that MS2, T2 and T4
were all efficiently adsorbed when suspended in tapwater, sewage and
lake water. Again, xl74 was found to adsorb less efficiently than the
other phage tested. Different eluting solutions for viruses adsorbed to
positively charged filters were tested and it was found that 4% beef
extract plus 0.5 M NaCl gave the best recoveries. To determine the
efficiency of this method, 500 ml of sewage water was passed through a
90 mm diameter Zeta plus 50S filter. Adsorbed indigenous bacteriophages
were eluted with 40-50 ml of the beef extract-NaCl solution with an
average efficiency of recovery of 56.5%.
The search group headed by Seeley and Primrose developed (148) and
modified (136) a portable concentrator using positively charged filters.
In this system, water is passed through a series of three prefilters,
precoated with 0.1% Tween 80 to prevent virus adsorption. The clarified
water was adjusted to pH 6.0 and pumped through a 500 mm diameter Zeta
plus filter. Adsorbed virus eluted with a solution of 1.0% beef extract
in 50 mM arginine. Recovery of naturally occurring bacteriophage in
pond water was greater than 60% (136).


16
The use of positively charged membrane filters for concentrating
viruses from very large volumes of water was hindered due to the lack of
availability of these filters in cartridge form. Previous work with
negatively charged filters had shown that these pleated cartridge
filters were capable of processing several thousand liters of tap water
without clogging (44, 46), while flat disc filters tended to clog
rapidly (44, 165). Sobsey and Glass (163) reported the use of a new
positively charged filter, Virosorb 1MDS, composed of fiberglass-
cellulose-"surface-modified resin" that was available as a double layer,
pleated-sheet cartridge. They compared these filters to the
electronegative Filterite pleated cartridge filters for the recovery of
poliovirus-1 seeded into 1000 1 of tapwater. Using beef extract as the
eluate and an organic flocculation procedure (92) for reconcentration,
they found virtually no difference in virus recovery between the
Filterite and Virosorb 1 MDS filter (33% and 30% respectively). However,
the Virosorb 1MDS filters were much easier for field use as no
conditioning of the water prior to adsorption was necessary.
Currently, both positively charged and negatively charged filters
are being used for the processing of large volumes of natural water
samples to detect low levels of viruses. Zeta plus 30S filters were
used to detect indigenous enteroviruses in activated sludge effluents
(23). Virus types isolated included poliovirus-1,-2,-3 and
coxsackievirus B3. During an outbreak of gastroenteritis and hepatitis
in Texas (76), concentration of water supplies by both Filterite and
Virosorb 1MDS filters resulted in the detection of several enteroviruses
such as coxsackieviruses B2 and B3, and Hepatitis A virus.


17
Factors Influencing Viral Adsorption to Filters
Several factors influence the adsorption of viruses to membrane
filters. These factors include the nature of the virus, the composition
of the filter, and the nature of the solution, including pH, the
concentration and type of salt in solution, the presence of organic
compounds such as proteins and humic acids, and the flow rate through
the filter.
Adsorption of viruses to membrane filters is greatly dependent on
the virus being studied (53). Viruses have protein coats composed of
many amino acids. These amino acids may be acidic, basic or
hydrophobic. The ionization of these amino acids is determined by the
pH values of the viral suspension. At the virus isoelectric point,
there is a net charge of zero. While determination of an isoelectric
point provides some insight to the charged nature of a virus, there are
many problems in drawing any conclusions based on this data alone.
First, studies have shown that the isoelectric point is not only type
dependent, but strain dependent (117, 189). In addition, isoelectric
point data tell us nothing about the charge density of the virus (53).
Little work has been done to elucidate data concerning the charge
density of viruses.
In addition to electrostatic forces effecting the adsorption of
viruses to filters, the hydrophobic nature of the virus may also play a
role. An examination of the amino acid sequence of the coat protein of
MS2 phage has indicated that most spans of amino acids along the
sequence are hydrophobic in nature (188). The relevance of this data


18
has yet to be determined. It is possible that an understanding of the
relative degree of hydrophobicity of viruses may be useful in predicting
the adsorption behavior of different viruses to membrane filters.
The type and composition of a filter can influence the adsorption
of viruses to the surface. Early work by several investigators (25, 26,
180, 181) found that viruses adsorb efficiently to filters composed of
cellulose acetate, cellulose nitrate and cellulose triacetate. Oliver
(26) found that filters composed of nylon did not adsorb virus as
efficiently as others. This was explained as being due to the greater
hydrophilic nature of nylon compared to the other filters examined
(121). Mix examined various filters used in virus concentration (121)
and concluded that the relative contributions of ionic and hydrophobic
interactions by the filter determined the extent of virus adsorption.
Kessick and Wagner (94) evaluated the electrophoretic mobilities of
virus adsorbing filters as a function of pH, ionic strength, and salt
type. The filters studies were Millipore (cellulose nitrate), Filflo
W10A-7 (cellulose acetate) and Filterite (epoxy-fiberglass).
Electrophoretic mobility measurements through a pH range of 2.0 to 7.0
indicated that all three filters were negatively charged in this pH
range. They also found that increased concentration and valency of
salts worked to decrease the net negative charge of the filters tested.
This provided an explanation of why salt addition helped to promote
virus adsorption to membrane filters (178, 181).
Sobsey and Jones evaluated some positively charged filters using
electrophoretic mobility (164). They found that the electrophoretic
mobility of Zeta plus filters (composed of cellulose-diatomaceous earth-
"charge-modified" resin) and Seitz S (composed of cellulose-asbestos)


19
filters become more electropositive with a decrease in pH. The Zeta
plus filters had an isoelectric point between 5 and 6, while the Seitz S
filter was found to have an isoelectric point near 7.
Methods such as electrophoretic mobility measurements have limited
application. These measurements are done on very small particles of the
filter material obtained by shredding of the filter material (94). As
such, these measurements may not be indicative of conditions existing on
the intact filter surface. Indeed, Sobsey and Jones (164) data on the
isoelectric point of Seitz S filters was found to be considerably lower
than expected. They indicated that in the shearing process, fibers from
the inner layers of the filter become exposed, possibly lowering the
isoelectric point of the filter.
Despite the known effect of detergents (83, 179) and proteins (25,
37, 94) on viral adsorption to membrane filters, little or no work has
been done to measure the hydrophobic nature of these virus adsorbing
filters.
As discussed earlier, the pH of a solution can have a tremendous
effect on the adsorption of viruses to filters. At low pH values,
viruses are often below their isoelectric point and are thus positively
charged, and electronegative filters possess a net negative charge. The
resultant electrostatic attraction is likely a factor in promoting virus
adsorption (94, 117). The converse is true at high pH values, where
viruses are above their isoelectric point and are negatively charged.
Adsorption to electropositive filters is promoted by this electrostatic
attractive force (94).
Studies of virus adsorption to membrane filters have shown that
adsorption is enhanced in the presence of cations (181, 183). Moreover,


20
trivalent cations, such as aluminum, have been found to be as effective
in this promotion as divalent ions, such as magnesium, at a lower
concentration (178). Three possible mechanisms for the observed
enhancement of virus adsorption in the presence of cations have been
suggested. It has been proposed that these cations act as bridges
between the negative charges on the viruses and the filters (94). A
second suggestion is that the cations are adsorbed by the filters and
reverse the net charge of the filter (117). Electrophoretic mobility
measurements in the presence of cations (94, 163) tend to support this
theory. Another possibility is that the addition of cations (and
therefore an increase in ionic strength) reduces the diffuse layer of
ions surrounding the virus and the filter. This permits short-range
attractive forces to overcome the electrostatic barriers and permits
adsorption of the viruses (173). All of these proposals indicate that
salts influence electrostatic interactions between viruses and membrane
filters.
Recent work by Farrah and his research group (40, 49, 156) has
indicated that salts may also effect adsorption of viruses by
influencing hydrophobic interactions between filters and viruses.
Chaotropic salts are able to increase the solubility of certain proteins
(73). These chaotropic ions are relatively large, singly charged ions
such as trichloroacetate (TCA), thiocyanate, and iodide (74). It has
been suggested that hydrophobic interactions are a result of the
unfavorable interactions of apolar groups with water, and accordingly,
chaotropic ions have been viewed as decreasing the structure of water
and therefore making aqueous solutions more lipophilic. In contrast,
antichaotropic ions are generally small, singly charged ions such as


21
floride, or multivalent ions such as citrate, calcium or magnesium ions.
These antichaotropic ions have been found to promote hydrophobic
interactions, presumably by increasing water structure (73, 74). Farrah
et al. (49) found that at high pH, solutions of chaotropic salts, such
as TCA, eluted over 95% of poliovirus-1 adsorbed to Millipore filters,
while an antichaotropic salt, sodium phosphate, eluted little adsorbed
virus. The authors hypothesized that the ability of chaotropic salts to
elute adsorbed virus indicated that hydrophobic interactions were a
major factor in maintaining virus associations to filters at high pH.
In a further study (40), antichaotropic salts, such as magnesium
sulfate, efficiently promoted adsorption of MS2 to Zeta plus filters at
pH 6.0, while TCA effectively blocked adsorption, again indicating that
hydrophobic interactions were involved. The use of agents involved in
promoting or weakening hydrophobic interactions has yet to be
incorporated in a concentration method for the recovery of viruses from
water samples.
The presence of organic materials, such as humic acids, serum, and
other proteins can effectively reduce virus adsorption to filters. In
early work, Ellford (37) and Cliver (25) both prevented virus adsorption
to filters by pretreating the filters with protein solutions. Wallis
and Melnick found that materials in crude virus harvest fluids (MCC)
interfered with viral adsorption to Millipore filters (181).
Later, the research group headed by Melnick (83, 178, 179) observed
that organics in water samples were adsorbed to membrane filters and
influenced viral adsorption. Elution of viruses from these filters also
resulted in elution of these organic impurities, which interfered with
readsorption of viruses to a smaller filter (44, 45). These impurities,


22
humic acids and other organic compounds, were characterized in a
subsequent publication (46). They found that these organics adsorbed at
low pH, were eluted at high pH, formed floes at low pH and could be
removed by anion-exchange resins.
Recently, humic and fulvic acids were tested for their ability to
interfere with adsorption to some positively charged filters (69). It
was discovered that fulvic acid had little or no effect on the
adsorption of poliovirus-1 to Zeta plus or Seitz S filters. Humic acid,
while interfering with viral adsorption to these filters to some extent,
still allowed up to 40% of the virus to be recovered.
Organic compounds are believed to interfere with viral adsorption
to membrane filters by competing for membrane adsorption sites (53, 64).
Since organics can compete with viruses for adsorption sites, they have
been used to elute viruses from filter surfaces. Some such solutions
used for viral elution include beef extract (64, 107, 136, 138, 183),
yeast extract (97), tryptose phosphate broth (70, 159), casein (16, 42),
Tween 80 (154, 156), and fetal bovine serum (138, 180, 181).
The final factor that can effect virus adsorption to membrane
filters is the flow rate of water through the adsorbing filters, Scutt
(147) examined the adsorption of poliovirus-1 and reovirus-1 to
Millipore filters at different flow rates. While reovirus-1 was
adsorbed at all flow rates tested, breakthrough was observed with
poliovirus-1 as the flow rate was increased. These results indicate
that a minimum contact time is required for an electrostatic bond to
form between the virus and the adsorption site (64, 147).
The flow rate at which virus adsorption becomes diminished depends
on factors influencing the strength of the electrostatic forces between


23
viruses and filters. During the development of the Wallis-Melnick virus
concentrator, it was discovered that the flow rate of sewage through
glass filters effected virus adsorption (83). At low flow rates (1.9
1/min), 96% of virus as adsorbed compared to only 68% adsorption when
the flow rate was increased to 9.5 1/min.
Reconcentration Procedures
In concentration procedures, as the volume of water processed is
increased, larger filters, and subsequently larger primary eluates, are
used. As such, a second, or reconcentration step is often necessary.
This second step may lower virus recovery, but it reduces the amount of
eluate to a manageable volume for subsequent assays (64). Some
procedures used to reconcentrate viruses after initial adsorption to and
elution from membrane filters include: hydroextraction,
ultrafiltration, ultracentrifugation, readsorption to and subsequent
elution from smaller filters of the same type, and inorganic and organic
flocculation.
One means of reducing the volume of the initial eluate and thus
reconcentrate viruses is hydroextraction. The principle of
hydroextraction is simple. An aqueous solution containing virus is
placed in a semipermeable membrane, often dialysis tubing, and the
tubing is placed into a hygroscopic material overnight. Water passes
through the membranes while large macromolecules, such as viruses are
retained and concentrated (43). Farrah et al. (45) used hydroextraction
with polyethylene glycol as a reconcentration step in the recovery of
enteroviruses from estuarine water. Using this procedure, 80% of seeded


24
poliovirus-1 was recovered. Others (149) found that hydroextraction of
been extract solutions containing coliphages gave very low recoveries
(less than 5%) and were very difficult to handle due to the high
viscosity of the concentrated material.
Ultrafiltration works on a principle similar to hydroextraction.
Solutions are passed through membranes designed to permit the passage of
water and low molecular weight substances, while larger macromolecules,
such as viruses are retained and concentrated in liquid left behind
(64). Logan et al. (113) used ultrafiltration as a reconcentration step
in the recovery of bacteriophages from large volumes of river water,
with final recoveries in the range of 50-60%. Reconcentration of
enteroviruses using ultrafiltration has been successful in their
recovery from samples of estuarine water (45).
Ultracentrifugation (180) has been used with limited success in the
concentration of viruses. One major problem with this method is the use
of expensive and non-portable equipment, thus its use in field studies
is impractical.
The early models of the Wallis-Melnick virus concentrator used
readsorption to and subsequent elution from a smaller diameter filter
similar to the one used in the first adsorption step of the procedure
(179). This readsorption was facilitated by dropping the pH of the
glycine eluate from 11.5 to 3.5 (178). Further modification of the
model included the addition of AlCl^ to 0.5 mM final concentration to the
eluate before readsorption to the second smaller filter (165) This
reconcentration procedure was efficient for smaller volumes of water.
However, difficulties were encountered in applying these procedures to
the detection of viruses in estuarine water (162) or in larger volumes
or tap water (44). Both organic compounds and viruses were adsorbed to


25
and eluted from the filters in the first stage. These organic compounds
and other impurities formed floes at the low pH values that were
required for the adsorption of viruses to negatively charged filters
(44, 46, 162). These floes clogged the filters and made a second stage
reconcentration with negatively charged filters impractical. Some of
these difficulties were overcome by the development of an inorganic
flocculation procedure (44) for a reconcentration step. Previous work
(182) had indicated that viruses readily adsorbed to floes of aluminum
or calcium salts. Farrah and coworkers used this as the basis for
development of an inorganic flocculation procedure using AlCl^ (44).
Initial eluates were conditioned by the addition of AlCl^ to 3 mM. This
led to a drop in pH to about 4 or 5. Eluates were then adjusted to pH
7.5, resulting in the formation of floes, which were collected by
centrifugation and resuspended in glycine. Virus recoveries averaged
40-50%.
Payment et al. (133) used FeCl^ at 3 mM concentration to form an
inorganic floe during the recovery of virus from estuarine water
samples. They recovered 53% overall of poliovirus-1 added to the water
samples. The authors noted that the efficiency of the reconcentration
step was nearly 100%, but it was found to be impractical for eluates
with a high concentration of organics because of the formation of a very
large floe (133) .
Protein solutions, such as beef extract, had been shown to be very
effective in the elution of viruses adsorbed to membrane filters (64,
107, 136, 138, 183), but this did not allow for reconcentration of the
eluate onto smaller filters. In 1976, Katzenelson and coworkers (92)
developed an organic concentration procedure for beef extract that


26
involved flocculating the beef extract by lowering the pH of the
solution to 3.5 (the isoelectric point of beef extract). Viruses in
solution adsorbed to these floes, which were recovered by
centrifugation. Viruses were recovered by solubilizing the floes in
small volumes of phosphate solution. Results in their laboratory
indicated that this method, used as a second step in conjunction with a
primary adsorption/elution step yielded a mean enterovirus recovery of
about 75%. Organic flocculation using non-fat dry milk and casein (16)
has also yielded efficient (over 70%) recoveries.
The organic flocculation step developed by Katzenelson (92) has
seen much use in the concentration and detection of enteroviruses from
natural samples (31, 69, 71, 76, 95). However, recent studies in
different laboratories (5, 84, 125, 132) have indicated that the source
and lot number of beef extract can effect its ability to flocculate,
thus lowering considerably the efficiency of this step. Payment et al.
(132) found that the addition of 2.5 mM ferric chloride to beef extract
solution which failed to flocculate at pH 3.5 cause the formation of
sufficient floe for efficient virus concentration (over 75% recovery).
In addition to flocculation variabilities, workers (87, 138) have
noted that beef extract concentrates have been shown to be toxic to cell
cultures. Despite these problems, organic flocculation continues to be
the main means of reconcentration of viruses in proteinaceous solutions.
Materials and Methods
A list of chemicals and their sources, and routine methods used in
animal and bacterial virus preparation and assay is presented in
appendix A. A complete list of media and solutions used in cell culture
work is presented in appendix B.


27
Filters
The filters used in this study, their characterization and sources,
are shown in Table 2.
Water Samples
The natural water samples used for indigenous phage concentration
and their concentration are described in Table 3.
Enterovirus Concentration from Tapwater
Tapwater (114 1) was dechlorinated by the addition of sodium
thiosulfate; absence of residual chlorine was ascertained by the
orthotolidine method (2). Following dechlorination, tapwater was
adjusted to a pH between 3.3 and 3.5 by the addition glacial of acetic
acid. Ten milliliters of the tapwater was removed and seeded with a
4 7
known amount of one of the enteroviruses tested. From 10 to 10
plaque-forming units (PFU) of virus were added in each trial. When low
numbers of viruses were added, the eluate from the Seitz filter was
assayed directly after adjustment of pH 7 to 7.5. When higher numbers
were added, viruses were assayed after dilution in phosphate-buffered
saline (PBS) with 2% fetal calf serum (FCS) (See appendix B). A 5-ml
portion of the viral suspension was added to the 114 liters and the
remaining 5 ml was assayed. The seeded tapwater was passed through a
10-in (ca. 25.4-cm), 0.2 ym Filterite filter. The adsorbed viruses were
eluted with 1 liter of 0.2 M trichloroacetic acid (TCA)-0.2 M lysine
that had been adjusted to pH 9 with sodium hydroxide. This eluate was
passed through a 47-mm Seitz S filter under positive pressure without pH
adjustment. Viruses adsorbed to the Seitz filter were recovered by one


TABLE 2. Filter characterization
FILTER
PORE SIZE
CONTACT
ANGLE3
MAJOR COMPONENTS
SOURCE
ELECTROPOSITIVE
Virosorb 1MDS
0.45y
151
Fiberglass/cellulose/
"surface-modified resin"
AMF Cuno, Inc.
Meriden, CN
Posidyne N66
0.45y
128
nvlnn 66 (polyamide)
Pall Trinity Micro
Corp.,
Cortland, NY
Zeta plus C-30
0.6 2.0u
1 0R
cellulose/diatomaceous
earth/"charge-modified
resin"
AMF Cuno, Inc.
Meriden, CN
Seitz S
0.5p
113
asbestos-cellulose
Republic Filters,
Milldale, CN
ELECTRONEGATIVE
Filterite
0.2y
NDb
epoxy-fiberglass
Filterite Corp.,
Timnium, MD
Millipore HA
0.4 5p
NDb
nitrocellulose
Millipore Corp.,
Bedford, MS
a Contact angle of carbon tetrachloride on water-saturated filters,
b ND: Not Determined.


TABLE 3.
Characterization of natural water samples
a
WATER SAMPLE
pH
TURBIDITY (NTUs)
CONDUCTIVITY (viMHOs)
ORGANICS
Cypress strand
6.9 -
7.2
46 61
350 500
0.48
-0.58
Secondary effluent
6.3 -
7.4
2.8 3.1
350 460
0.09
- 0.16
Holding pond
7.0 -
7.5
52 60
600 720
0.46
- 0.51
Land runoff
7.6 -
7.9
2.4 3.7
420 630
0.37
- 0.41
a Range of each measurement is indicated.
b Absorption at 254 nm, UV light (33).
N>


30
of two methods. In the first procedure, the filter was removed from the
holder and placed in a holder used for vacuum filtration (Gelman
Sciences, Inc., Ann Arbor, MI). Two 8 ml portions of 3% casitone were
pulled through the filter with a vacuum. After the first 2 to 3 ml of
each portion had passed through the filter, the vacuum was removed and
the solution was allowed to soak the filter for 5 minutes. After the
soaking period, the remaining part was pulled through the filter. The
two portions were pooled, neutralized and assayed. In the second
procedure, two 8 ml portions of FCS (pH 9) were pulled through the
filter with a 50 ml syringe. The two portions were pooled, neutralized
and assayed.
Adsorption/Elution of T7 using Electropositive Filters
Tapwater was dechlorinated as described above. After approximately
7
10 PFU of T7 phage were added, 10 ml of the tapwater was passed through
the filters, which were held in 25 mm holders, at a rate of 1 ml/s.
Viruses in the initial tapwater solution and in the filter effluent were
assayed to confirm viral retention by the filters. Next, 10 ml of a
test eluent (see Tables) was passed through the filters the eluted
viruses were assayed. Finally, 10 ml of 3% beef extract was passed
through the filter to elute residual virus. The data obtained from beef
extract controls indicated that phage was not being inactivated or
aggregated by test solutions (data not shown). The virus eluted was
expressed as a percentage of the virus present in the initial tapwater
solution. Values were obtained in 2-3 trials and represent the means
and standard deviations of 4 to 6 determinations.


31
Contact Angle Measurements
A cubic cell (3 cm x 3 cm x 3 cm) was filled with distilled water
and a filter was placed in the cell and allowed to stand for 1 hr in
order to be saturated with water. A small drop of carbon tetrachloride
was injected in the water just above the filter surface using a 50 yl
syringe (Hamilton Co., Reno, NV). The contact angle between the filter
and droplet was measured using a NRL Gonimeter model A-100, Rame-Hart,
Inc. (129).
Capillary Rise Measurements
Filters were cut in strips measuring 20 cm long and one cm wide.
These strips were then dipped in distilled water or other liquid one cm
deep. The time at which the strip was dipped in the liquid was taken as
the zero reference time. The rise of liquid on the filter was measured
at one minute intervals for 10 to 12 minutes. The height of the liquid
on the filter (or difference of liquid and water) versus time was
plotted using the mean values of at least three trials (77).
Concentration of Indigenous Bacteriophage using Filters
Samples of water (see Table 3) were collected in pressure vessels
and brought back to the laboratory for immediate processing. A five ml
aliquot was removed to determine the input concentration of
bacteriophage. For the one-step procedure, samples were passed through
an AP20 prefilter (Millipore Corp., Bedford, MS) and 3 layers of
Virosorb 1MDS filters without pH adjustment. The filtrates were
collected and assayed to confirm bacteriophage adsorption. Next, 1.0 M
NaCl in 0.05 M imidazole buffered at pH 7 was used to elute the


32
adsorbed phage. The eluate volume was 5 ml when filters were held in 25
mm holders, 10 ml for 47 mm filters and 40 ml for 90 mm filters. The
two-step concentration procedure started as the one-step procedure
described above, with 90 mm filters used. The 40 ml sodium chloride
eluate was pulled through a 47 mm Seitz S filter using vacuum filtration
without pH adjustment and collected to check for phage adsorption.
Next, 10 ml of a 4% beef extract and 0.1% Tween 80 solution at pH 7 was
placed on the filter and 2 to 3 ml was pulled through using a vacuum to
displace residual fluid and ensure that the beef extract/detergent
eluate completely soaked the filter. The remaining 7 to 8 ml of the
eluate remained on the filter, soaking for 5 minutes, then was pulled
through and assayed for phage elution.
Flocculation of Seeded Beef Extract
Forty ml of 10% beef extract was seeded with 107 PFU of either
bacteriophage or animal virus. One half of this beef extract solution
was flocculated by lowering the pH to 3.5. The floe was collected by
centrifugation at 3000 x g for 10 minutes, and resuspended in two ml of
0.15 M sodium phosphate solution, pH 9, according to the method
described by Katzenelson et al. (92). The other half was mixed with 2
volumes of a saturated ammonium sulfate solution. Floe immediately
formed when these solutions were mixed, and the pH remained buffered
between 7 and 7.5. The beef extract/ammonium sulfate was centrifuged at
14,500 x g for 20 minutes at 5 10C and the floe was solubilized in 2
ml sterile distilled water.


33
Concentration of Phage and Enterovirus from Sewage Effluents
Unchlorinated secondary effluent from the University of Florida
treatment plant was collected in a sterile container and brought back to
the laboratory for immediate processing. One liter was seeded with 10^
PFU enterovirus (see Table 9) and 5 ml was removed to assay for initial
input of enterovirus and indigenous bacteriophage. E. coli C (ATCC
13706) was used as host bacterium for indigenous bacteriophage assay
(75). The one liter sample was passed through three layers of Virosorb
1MDS filters with an AP20 prefilter encased in a 47 mm filter housing
(Millipore) without pH adjustment. Twenty ml of 10% beef extract, pH 9,
was used to elute adsorbed virus. The beef extract was adjusted to pH
by the addition of 1 N HC1, and divided into two equal portions. One
half of the eluate was reconcentrated using organic flocculation at pH
3.5, while the other half was reconcentrated by ammonium sulfate
flocculation, as described above. The final volume with both procedures
was 2 ml. The procedure was the same when other protein solutions (10%
tryptose phosphate broth and 10% brain heart infusion) were used in the
elution step, except that only one volume of saturated ammonium sulfate
was needed for flocculation.
Recovery of Low Levels of Indigenous Bacteriophage
Samples of water were passed through Seitz S filter with an AP20
prefilter, held in a 90 mm stainless steel holder. Direct assay of
these water samples indicated that no bacteriophage was present. Next,
10% beef extract, pH 9, was used to elute any phage that may have been
adsorbed. Beef extract was dropped to pH 7 and flocculated with
saturated ammonium sulfate as described above.


34
Results
Concentration of Enteroviruses from Large Volumes of Tapwater
We developed a modified method to concentrate enteroviruses from
large volumes of tapwater (Table 4). We used acetic acid to adjust the
pH of the tapwater to 3.5, in lieu of HC1 (44) to aid in virus
adsorption to Filterite filters. A chaotropic salt, trichloroacetic
acid was used to elute viruses from the filter. Virus was efficiently
eluted, and readsorption of the virus to a second dissimilar filter
(Seitz S) was accomplished without any modification of this eluting
solution. Viruses adsorbed to the Seitz filters were eluted with pH 9
solutions of either 3% casitone or fetal calf serum (FCS). Casitone is
less expensive than FCS, but a soaking period was required when casitone
was used as the eluate. The mean recovery of enteroviruses with
casitone was 54%, and that with FCS was 43%.
Differential Elution of Phage from Positively Charged Filters
Initial studies were conducted to determine the adsorption of
bacteriophage T7 to four positively charged membrane filters: Virosorb
1MDS, Zeta plus C-30, Posidyne N66, and Seitz S. In these experiments,
tapwater was dechlorinated and adjusted to three different pH values
(4, 7 and 9), and in all cases, adsorption was nearly complete (>99%
adsorbed, data not shown). Based on these results, dechlorinated
tapwater was used as the adsorbing solution in subsequent studies with
T7 and electropositive filters.
The results of preliminary studies on the ability of salts and
detergents to elute T7 phage adsorbed to these filters are shown in
Table 5. A solution of the buffer alone (50 mM imidazole) eluted less
than 10% of the adsorbed phage from all of the filters tested. A


35
TABLE 4. Concentration of viruses from tapwater.
Virus
Number of
trials
Percent of added
virus recovered Eluent
mean
SDb
Poliovirus-1
3
67
8
Coxsackievirus B3
3
+1
KD
18
Coxsackievirus B4
2
43
20 3% casitone, pH 9
Echovirus-1
2
63
12
Total
10
54
18
Poliovirus-1
4
35
7
Coxsackievirus B3
2
+1
o
7
Coxsackievirus B5
2
+1
4 Fetal calf serum, pH '
Echovirus-1
2
+i
00
in
9
Total
10
43
11
a Tap water was dechlorinated, adjusted to pH 3.3 to 3.5 by the
addition of acetic acid, and seeded with the indicated virus.
The sample was passed through a 10", 0.25-ym porosity Filterite
filter at 3 to 5 gal/min. Next, 1,000 ml of 0.2 M NaTCA + 0.2 M
lysine, pH 9, was passed through the filter. This solution was
then passed through a 47-mm Seitz S filter. Virus adsorbed to the
Seitz filter was recovered by treating the filters with two 8-ml
volumes of 3% casitone or FCS, pH 9, as described in the text.
b SD: Standard
deviation.


a
TABLE 5. Elution of T7 adsorbed to positively charged membrane filters by solutions at pH 7.
% of adsorbed
virus eluted by:
FILTER
BUFFER ALONE
0.1% TWEEN 80
0.5 M EDTA
0.1% TWEEN 80
+0.5 M EDTA
Virosorb 1MDS
7 6
8 8
1+
105
+
20
Zeta plus C-30
0
49 9
0
72
+
2
Posidyne N66
8 3
37 + 9
5 1
78
+
14
Seitz S
0
0
0
22

8
a All solutions contained 0.05 M imidazole and were adjusted to pH 7.0.
LO


37
solution of detergent (Tween 80) at pH 7 did not elute any T7 adsorbed
to the Seitz filters and less than 10% of the phage adsorbed to the
Virosorb 1MDS filters. This detergent solution eluted greater then 35%
of the T7 adsorbed to the Zeta plus C-30 or Posidyne N66 filters. A
solution of EDTA eluted 74% of the virus adsorbed to the Virosorb IMDS
filters but less than 5% of the virus adsorbed to the other filters. It
had previously been shown that solutions containing salts and detergents
were very efficient at eluting viruses adsorbed to electronegative
filters (156). When such a solution was used with the positively
charged filters, greater than 70% of the T7 adsorbed to the Zeta plus
C-30, Posidyne N66 and Virosorb IMDS filters was eluted. However, only
22% of the T7 adsorbed to the Seitz S filters was eluted.
Preliminary results indicated that the ionic strength of the
eluting solution affected the amount of virus eluted from these filters.
Figure 1 shows the influence of ionic strength in solutions of detergent
at pH 7 on the elution of T7 adsorbed to membrane filters. In the
presence of detergent, solutions with ionic strengths of 0.4 or above
efficiently eluted the bacteriophage adsorbed to the Zeta plus C-30 and
the Posidyne N66 filters, while solutions with ionic strengths below 0.4
eluted the virus adsorbed to the Virosorb IMDS filters. Again little or
no virus adsorbed to the Seitz S filters was eluted.
The effect of detergent concentration in the presence of 1 M NaCl
on viral elution was determined (Figure 2). Again, a solution of buffer
alone at pH 7 eluted less than 10% of the T7 adsorbed to all filters
tested. A solution of 1 M NaCl at pH 7 did not elute any virus adsorbed
to either the Zeta plus C-30 or Seitz S filters, while less than 20% of
the adsorbed T7 was eluted from the Posidyne N66 filter. However,


FIGURE 1. Elution of bacteriophage T7 adsorbed to various positively
charged filters by solutions of 0.1 % Tween 80 and NaCl or
Na S0 at pH7.
2 4


%VIRUS ELUTED
39
IONIC STRENGTH


FIGURE 2. Elution of bacteriophage T7 adsorbed to various positively charged filters by
solutions of NaCl and Tween 80 at pH 7. V = Virosorb 1MDS, Z = Zeta plus C-30,
P = Posidyne N66, S = Seitz S.


p
0.001 % Tween 80 0.1% Tween 80


42
greater than 75% of the T7 adsorbed to the Virosorb 1MDS was eluted
using the salt alone. Tween 80 concentrations of 0.001% in the presence
of NaCl greatly increased the amount of phage eluted from the Posidyne
N66 filters, yet had no effect on the elution of phage adsorbed to Zeta
plus C-30 or Seitz S filters. A solution of 0.1% Tween 80 in presence
of salt eluted greated than 75% of the T7 adsorbed to the Virosorb 1MDS,
Posidyne N66 and Zeta plus C-30 filters, yet had no effect on elution of
T7 adsorbed to the Seitz S filters. Tween 80 was not soluble in the
salt solution in higher concentrations.
Indigenous bacteriophage from several water sources (Table 3) were
concentrated using a one step adsorption/elution procedure using
Virosorb 1MDS filters as the adsorbent and 1 M NaCl as the eluting
solution. Table 6 shows the percent recovery of indigenous
bacteriophages using filters of various sizes and input volumes of
water. In general, recovery of virus was better when the smaller filter
holders were used. In addition, there was no significant difference in
viral recovery using E. coli B or E. coli C-3000 as the host bacteria.
The average recover of the 41 samples from 4 different sources was 55%
using E. coli B as the host and 61% using E. coli C-3000 as the host.
In samples with relatively high numbers of phages, a one-stage
concentration procedure may be sufficient to detect viruses. However,
where low levels are found, a two-stage procedure may be required to
provide a sufficient concentration factor to detect indigenous phages.
The results of this study have shown that some solutions that permit
virus adsorption to one filter are capable of eluting virus adsorbed to
another filter. This raises the possibility that a simple two-step
virus concentration procedure can be developed in which virus adsorbed
to one filter can be eluted using a solution permits virus adsorption to


TABLE 6. One-step concentration procedure for the recovery of indigenous bacteriophage from
natural water samples.
% recovery
E. coli B
E. coli
C-3000
WATER SOURCE
FILTER SIZE (d)3
VOLUME FILTERED
mean
SD"
mean
SD
REPLICATIONS
Cypress strand
25
50
_
100
133
23
105
19
4
47
100
-
500
84
38
66
12
6
90
500
-
1500
63
33
53
16
4
Secondary effluent
25
250
-
400
47
22
60
11
3
47
500
-
1000
26
1
58
10
4
90
2000
-
7000
26
5
68
23
3
Holding pond
25
50
-
75
40
3
89
16
3
47
375
475
32
7
61
30
4
Land runoff
25
50
-
100
82
19
59
9
3
47
300
-
700
30
7
34
16
5
90
2000
13
4
19
1
2
TOTALS
55
40
61
27
41
a diameter in mm.
b in milliliters.
c SD: standard deviation


44
a second smaller filter. The elution-adsorption step can be
accomplished with little or no modification of the solution. Based on
the results of this study, several two step procedures using two
different filters in series are possible. One such procedure is
outlined in Figure 3. We have used this procedure to concentrate phage
from several secondary effluent samples (Table 7). Using this
procedure, the average recovery of bacteriophages was 26% using E. coli
B as the host, and 63% E. coli C-3000 as the host. In two samples,
bacteriophage was detected in the final concentrates when none detected
in the initial sample (data no shown).
Characterization of Virus-Adsorbing Filters
In order to determine the relative hydrophobic-hydrophilic nature
of filters used for virus adsorption, the contact angle of carbon
tetrachloride on membrane filters submerged under water was measured
(Table 2). The contact angle of carbon tetrachloride on the Seitz S
filter was the smallest (113), the contact angle on the Virosorb 1MDS
was the largest (151), while the contact angles on the Zeta plus C-30
(128) and Posidyne N66 (128) were intermediate. Under the conditions
used to measure the contact angle, carbon tetrachloride was spread
immediately on the two negatively charged filters tested (Millipore HA
and Filterite), therefore, measurement was not possible.
The filters were further characterized using the capillary rise
method. Figure 4 shows the rise of deionized water on positively
charged membrane filters. The most rapid raise of water was observed in
the Virosorb 1MDS filters, while the smallest rate of water rise was


FIGURE 3. One possible two-step procedure for the concentration of bacteriophages from water.


STEP I
STEP 2
Adsorption of phage to three layers of
Vrosorb IMDS filters
I
Elution of adsorbed phage using I M NaCI, pH 7
I
Adsorption of phage in I M NaCI, pH 7,
to Seitz S filters
I
Elution of phage adsorbed to Seitz S filters using
4% beef extract and O. I % Tween 80, pH 7


TABLE 7. Recovery of bacteriophages from treatment plant effluents
SOURCE, TYPE
VOLUME
Initial
PFU/1 using:
% of bacteriophages
recovered using:
OF EFFLUENT
SAMPLED (1)
E. <
coli B
E. coli
C-3000
E. coli B
E. coli C-3000
Tallahassee, FL
chlorinated effluent
66.3
6.7
x 104
4.3 x
104
43
39
U. of Florida
unchlorinated effluent
3
6.0
x 103
6.9 x
104
15
54
U. of Florida
unchlorinated effluent
4
6.7
x 104
6.9 x
104
21
59
U. of Florida
unchlorinated effluent
4
8.3
x 104
9.1 x
104
24
100
a
Total Mean SD
26 10
63 23
a Standard deviation


FIGURE 4. The rise of water on various positively charged filters.


HEIGHT (cm)
49


50
observed for the Posidyne N66 filters. Zeta plus C-30 and Seitz S
filters were intermediate in nature. Figure 5 shows the results of the
same test on negatively charged filters. The rise of deionized water on
Filterite filters was more rapid than that of Millipore HA filters.
Figures 6 through 8 show the change in rise (solvent water) of
the positively charged filters. In all cases, the change in rise was
the most positive for the Seitz S filters, while the difference was most
negative for the Virosorb 1MDS filters. As with contact angle
measurements, Zeta plus C-30 and Posidyne N66 filters were intermediate
in nature. The change in rise (chloroform water) was plotted for
negatively charged filters (Figure 9). In this instance, there was
little or no change in rise for Millipore HA filters, while this change
was slightly negative for the Filterite filters.
Ammonium Sulfate Flocculation as a Reconcentration Step
The ability of certain salt solutions, such as sodium phosphate,
sodium citrate, ammonium sulfate or sodium sulfate, to precipitate
proteins has been well documented (73, 93, 146). Preliminary
experiments in our laboratory indicated that optimum precipitation
occurred when beef extract was mixed with saturated ammonium sulfate.
The efficiency of this flocculation step was compared to the organic
flocculation procedure as described by Katzenelson et al. (92) and the
results are shown in Table 8. Bacteriophage recovery using organic
flocculation was less than 3% for all three phage tested, while
virtually all was recovered using ammonium sulfate flocculation. In
addition, animal virus recovery was high (greater than 70% for all
three tested) using the ammonium sulfate method as compared to organic
flocculation, which gave the highest recovery with poliovirus-1 at 53%.


FIGURE 5. The rise of water on various negatively charged filters.


52


FIGURE 6
The change in capillary rise on various positively charged
filters: carbon tetrachloride water.


DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
54
CARBON TETRACHLORIDE-WATER


FIGURE 7.
The change in capillary rise on various positively charged
filters: butanol water.


I MDS
DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
Ln


FIGURE 8. The change in capillary rise on various positively charged
filters: chloroform water.


I MDS
DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
.*>
1
CHLOROFORM-WATER


FIGURE 9. The change in capillary rise on various negatively charged
filters: chloroform water.


DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
60
CHLOROFORM-WATER


61
TABLE 8. Virus recovery from seeded 10% beef extract, pH 7, by ammonium
sulfate flocculation versus organic flocculation.
% Recovery
Ammonium Sulfate Organic
Virus Flocculation Flocculation
a
MS 2
101
+
8
1
+
0
X174
100
+
17
1
+
1
T3
107
+
8
2
+
1
Poliovirus-1
97
+
11
53
+
8
Echovirus-5
84
+
12
22
+
5
Coxsackievirus B5
72
+
8
10

1
a standard deviation


62
It should be noted that during the course of these experiments, three
lots of beef extract were tested before one was found that flocculated
at low pH. Both in our laboratory and in others (71, 92, 132), higher
recoveries using the organic flocculation procedure have been obtained
with some lots of beef extract.
This ammonium sulfate flocculation procedure was compared with the
organic flocculation procedure for recovering viruses in natural water
samples (Table 9). The overall mean recovery of indigenous
bacteriophage using the ammonium sulfate flocculation was 85% as
compared to 12% for the organic flocculation method. It was found that
the mean recovery of seeded enteroviruses using the ammonium sulfate
flocculation method was 73% as compared to 42% for organic flocculation.
Other proteinaceous solutions were tested for enterovirus and
indigenous bacteriophage recovery using this ammonium sulfate
precipitation method, and these results are shown in Table 10. For the
recovery of indigenous bacteriophage, there was no difference for the
solutions used. Ten percent tryptose phosphate broth (TPB), 10% brain
heart infusion (BHI) and 10% beef extract all recovered 71% of the phage
when used as a primary eluting solution followed by ammonium sulfate
flocculation. For three enteric viruses tested, the mean recovery was
76% when beef extract was used as the primary eluting solution, 62% for
BHI and 73% for TPB.
Finally, this procedure was also used to recover indigenous
bacteriophage in less heavily contaminated waters (Table 11). Direct
assay of these waters indicated that no phage was present. After
reconcentration of the beef extract eluate using ammonium sulfate


63
TABLE 9. Recovery of indigenous bacteriphage and seeded enterovirus
from sewage effluent by ammonium sulfate flocculation versus
organic flocculation.
ANIMAL VIRUS
% recovery
INDIGENOUS BACTERIOPHAGE
% recovery
Trial
Virus added
ASFb
O
o
ASFb
OFC
1
Poliovirus-1
47
33
115
15
2
Poliovirus-1
80
71
86
15
3
Echovirus-5
91
39
61
11
4
Echovirus-5
73
23
76
5
Mean
d
SD
7316
4218
8520
124
a. Unchlorinated effluent was seeded with 10 PFU of enterovirus
listed and assayed for initial enterovirus and indigenous
bacteriophage. One liter of sample was passed through a 47mm, 3
layers of Virosorb 1MDS filters. Adsorbed virus was eluted with
20ml 10% beef extract, pH 9. Beef extract was adjusted to pH 7 and
split into equal halves (see text).
b. ASF ammonium sulfate flocculation. One half of beef extract
eluate was mixed with two volumes of saturated ammonium sulfate,
floe was centrifuged and resuspended in 2 ml distilled water (see
text).
c. OF organic flocculation. One half of beef extract eluate was
adjusted to pH 3.5, floe was centrifuged and resuspended in 2 ml
0.15 M Na^HPO^ (see text).
d. SD standard deviation.


TABLE 10.
Recovery of indigenous bacteriophage and seeded enterovirus from sewage effluent by
ammonium sulfate flocculation.
ANIMAL VIRUS
% recovery
INDIGENOUS PHAGE
% recovery
Trial
Virus added
BEb
c
BHI
d
TPB
BEb
c
BHI
d
TPB
1
Poliovirus-1
476
52
109
1156
59
77
2
Poliovirus-1
806
74
94
86e
76
71
3
Echovirus-5
9ie
69
65
6ie
77
83
4
Echovirus-5
73e
41
70
76e
103
69
5
Coxsackievirus
B5
109
73
55
42
63
65
6
Coxsackievirus
B5
55
60
44
48
49
62
mean SD'*'
76 21
62 12
73 22
71 25
71 17
71 7
a -
Unchlorinated effluent was
enterovirus and indigenous
7
seed with 10
bacteriophage
PFU of enterovirus listed
. One liter of sample was
and assayed for
passed through a
initial
47 mm,
3 layers of Virosorb 1MDS filters. Adsorbed virus was eluted with 10% solution of protein
listed. Eluate was mixed with saturated ammonium sulfate, floe was collected by
centrifugation and resuspended in distilled water as described in text.
b BE = beef extract.
c BHI = brain heart infusion.
d TPB = tryptose phosphate broth.
e Values from Table 9.
f SD standard deviation.


65
TABLE 11. Recovery of low levels of indigenous bacteriophage by
ammonium sulfate flocculation.
SAMPLE
INITIAL
Volume (ml) PFU/ml
BEEF EXTRACT
Volume (ml)
ELUATE
PFU/ml
FINAL CONCENTRATE
Volume (ml) PFU/ml
Chlorinated
effluent
3200 <1
42
<1
5
12.5
Lake water
2000 <1
34
1400
5
8000
Lake water
2500 <1
50
234
3
4350
a. Sample
was passed through
Seitz S filter
with AP
20 Prefilter
held
in 90 mm stainless filter holder. Next, 10% beef extract, pH 9,
was used to elute adsorbed virus. Beef extract was dropped to pH
7, and mixed with two volumes saturated ammonium sulfate to form
floe. Floe obtained by centrifugation was resuspended in sterile
distilled water (see text).


66
precipitation, phage was detected in all three samples, and there
appeared to be no probelms, such as toxicity, when the final concentrate
was plated directly.
Discussion
Concentration of Enteroviruses from Large Volumes of Tapwater
Several concentration procedures to detect viruses in tapwater have
been developed and subsequently modified. Wallis et al. (178) and
Sobsey et al. (165) described two-stage concentration procedures in
which the same type (but different sizes) of filters were used in both
stages. The use of the same filter in a reconcentration procedure
involved reconditioning of the primary eluting solution to allow for
readsorption. Problems with clogging and co-concentration of organic
compounds (44, 162) and other impurities made a second stage
concentration procedure with negatively charged filters impractical.
This problem was overcome by the use of an inorganic flocculation step
for reconcentration (44), yet viral recovery was low. Katzenelson et
al. (92) increased the amount of viral recovery by using a second-stage
organic flocculation procedure with beef extract. While this procedure
has proven to be useful, problems such as large final volumes and
variability in the flocculation of beef extract supplies led us to
investigate possible modifications of currently used methods.
Our investigations led to three modifications of existing
procedures for concentrating enteroviruses from large volumes of
tapwater. The first modification is the use of acetic acid in place of
hydrochloric acid to adjust the pH of the tap water to pH 3.5.
Adsorption of viruses to negatively charged filters such as Filterite
filters is facilitated at a low pH (165). Hydrochloric acid has been


67
used to adjust water samples to the low pH values required for viral
adsorption in many studies. We have found that acidification of
tapwater with either hydrochloric or acetic acid permits the adsorption
of greater than 99% of the enteroviruses studied to Filterite filters.
However, the acidification of water under filed conditions with Dema
injectors (134) is easier when acetic acid is used in place of HC1.
Changes in the flow rate or pressure may cause fluctuations in the
amount of acid injected when Dema or pressure injectors are used to
adjust water pH. With HC1, these changes may cause wide fluctuations in
the pH unless the injection rate is carefully controlled. We have found
that these fluctuations are minimized when 1 or 2 M acetic acid is used
for acidification. Acetic acid (pH = 4.75) will buffer more closely to
the desired pH range than will HC1, which dissociates completely.
The second modification was the use of sodium trichloroacetate
(NaTCA) as the primary eluate. The association of viruses with solids
such as membrane filters has been shown to be influenced by both
hydrophobic and electrostatic interactions (40, 49, 50, 53, 156).
Previous studies have shown that detergents, which disrupt hydrophobic
interactions, can be used to elute viruses adsorbed to membrane filters
(49, 156). In addition, recent studies have shown that certain salts
may disrupt hydrophobic interactions (49). These salts, called
chaotropic salts, are relatively large, singly charged ions such as TCA
and thiocyanate. It has been suggested that these compounds disrupt
hydrophobic interactions by decreasing the structure of water and making
aqueous solutions more lipophilic. Chaotropic agents have been used to
elute viruses adsorbed to various solids by disrupting hydrophobic
interactions between the viruses and the solids (48, 49, 177). A


68
solution of the chaotropic salt, NaTCA, eluted 77% of the enteroviruses
used in this study that were adsorbed to Filterite filters.
Reconcentration of the viruses in the primary eluate with a second,
smaller electronegative filter requires acidification of the high-pH
eluent used to recover viruses adsorbed to the primary virus-adsorbing
filter (165, 178). It was previously shown (44) that such a drop in pH
may be accompanied by the precipitation of organic compounds that may
have been concentrated along with the viruses. These organic compounds
may interfere with the reconcentration process by reducing the
adsorption of viruses to the filters or by clogging the filters (44).
Therefore, readsorption of viruses to a second filter at the same pH
that was used for the elution of viruses from the first filter was
studied.
Sobsey and Jones (164) studied the adsorption of poliovirus-1 to
positively charged filters at several pH values. They found that both
Zeta plus filters and Seitz filters efficiently adsorbed polioviruses at
a relatively high pH. The last modification of our procedure introduced
was the use of Seitz filters for the second-stage concentration. When
the NaTCA-lysine eluate from the Filterite filters was passed through
the Seitz filters, greater than 95% of the viruses were adsorbed.
Viruses adsorbed to the Seitz filters were eluted efficiently with pH 9
solutions of either 3% casitone or fetal calf serum.
These three modifications make the pH adjustment step easier, and
permit detection of viruses in water by a two-stage concentration
procedure in which different types of membrane filters are used in each
stage. This use of different types of filters allows readsorption
without any modification of the primary eluate, simplifying the overall
procedure.


69
Differential Elution of Phage from Positively Charged Filters
The use of negatively charged filters, such as the Filterite
filters used in the primary adsorption step discussed above, requires
the lowering of the pH of the water sample and/or the addition of
cations (178, 181) to facilitate viral adsorption. Since it had been
previously shown that these concentration procedures are unsuitable for
bacteriophage recovery (149, 150), investigators have explored the
possibility of the use of electropositive filters for the concentration
of bacteriophages (67, 150, 157). These filters are more positively
charged at the pH range of most natural water samples (pH range 6.5 8)
than are the electronegative filters, and so adsorption to the
electropositive filters can be accomplished with little or no
manipulation of the water sample.
We have examined four filters, Virosorb 1MDS, Zeta plus C-30,
Posidyne N66 and Seitz S filters. All four are more electropositive at
pH 5 8 than are negatively charged filters and adsorb virus in water
at these pH values. However, their composition is quite varied (Table
2). We have attempted to determine what effects these differences in
composition would have on the association of bacteriophage with the
filters.
Although all of the filters adsorbed T7 in tapwater at pH values
between 4 and 9, differences in the ability of certain solutions to
elute the adsorbed phage were noted. Previous studies have shown that
certain solutions of a neutral detergent, such as Tween 80, can disrupt
hydrophobic interactions between viruses and membrane filters (40, 50,
181). Once the hydrophobic interactions have been disrupted, the
addition of certain salts to this solution will disrupt electrostatic


70
interactions and result in the elution of adsorbed viruses (50, 156).
When hydrophobic interactions were disrupted at pH 7 by the addition of
detergent, only T7 adsorbed to Posidyne N66 and Zeta plus C-30 filters
was eluted to any appreciable extent. If salt was added to the
detergent solution, thus disrupting both hydrophobic and electrostatic
interactions, most of the adsorbed phage was eluted from all filters
tested except the Seitz S filter. These findings indicated that there
were distinct differences in the relative strength of hydrophobic and
electrostatic interactions with the phage among these four filters.
It was found that the ionic strength of the eluting solution, in
the presence of a detergent, affected the amount of adsorbed phage that
was eluted. Solutions of detergent with sodium chloride or sodium
sulfate with ionic strengths greater than 0.4 eluted most of the
adsorbed virus from all filters tested except the Seitz S. It is
assumed that increasing the ionic strength decreases the strength of the
electrostatic interactions between the virus and the filter (156).
Therefore, the filters that form relatively strong electrostatic
associations with viruses require solutions with relatively high ionic
strength to elute adsorbed viruses.
A titration of the amount of detergent necessary to elute adsorbed
T7 in the presence of 1 M sodium chloride provided the most striking
results. A solution of sodium chloride alone eluted most of the T7
adsorbed to the Virosorb 1MDS filter. Since no detergent was added, it
can be assumed that no hydrophobic interactions were disrupted. Since
salts can disrupt electrostatic interactions but promote hydrophobic
interactions (40, 49), this result can be explained by assuming that
hydrophobic interactions are not important in the association of phage


71
to the Virosorb 1MDS filter. Similarly, since a solution of a salt
alone did not elute viruses adsorbed to the other three positively
charged filters, some hydrophobic interactions are probably involved in
maintaining viral association to these filters. Since less detergent in
the presence of salt was necessary to elute phage adsorbed to the
Posidyne N66 filter than was required to elute phage adsorbed to the
Zeta plus C-30 filter, it can be assumed that hydrophobic interactions
are relatively stronger for the Zeta plus C-30 filters than for the
Posidyne N66 filters. Again, these specific solutions eluted little or
no phage adsorbed to the Seitz S filters.
Characterization of Virus-Adsorbing Filters
These results led us to characterize further these four
electropositive filters, as well as two electronegative filters often
used in virus adsorption schemes (Filterite and Millipore filters). We
used two means of characterizing the surface of these filters, which can
be done without destruction of the filter surface. One such test was
contact angle measurement. The contact angle determines the affinity of
a liquid (in this case carbon tetrachloride) for a surface submerged
under water. If carbon tetrachloride preferentially wets the submerged
filter, the resulting contact angle between the filter surface and
carbon tetrachloride droplet will be small (129). This would, in this
case, be an indication of the preferential hydrophobicity of the filter.
It was found that Virosorb 1MDS filters had the largest contact angle,
indicating that it was the least hydrophobic of the filters tested.
This was consistent with elution studies which had previously shown that
hydrophobic interactions were not important in phage association with
the Virosorb 1MDS filters. The Seitz S filters had the smallest contact


72
angle, and therefore were found to be the most hydrophobic. This may be
one reason why elution from these filters using defined solutions was so
poor. Indeed, in our laboratory and others (70, 155, 163), the eluting
solutions found to be most effective for virus adsorbed to Seitz S
filters are undefined protein solutions, such as beef extract.
Consistent with elution studies, Zeta plus C-30 and Posidyne N66 filters
were found to be intermediate in the hydrophobic nature of their
filters. Due to the nature of the experimental setup, contact angles on
the two negatively charged filters were not able to be determined.
The second method used for characterization of the filters was the
capillary rise method. In this case, the movement of a liquid through
the filters is measured (77). If the liquid is water, the rate of rise
is related to the hydrophilicity of a filter. Those filters which are
more hydrophilic will have a correspondingly higher rate of rise of
water. The results of this test were also in agreement with elution
studies. The Virosorb 1MDS filters, previously shown to be least
hydrophobic were found to be most hydrophilic, while Zeta plus C-30,
Seitz S and Posidyne N66, respectively, were found to be less
hydrophilic.
The rate of rise of some hydrophobic solvents, such as carbon
tetrachloride, chloroform, or butanol, when measured and plotted as the
difference between the solvents and water, gives another indication of
the relative hydrophobicity of these filters. These capillary rise
measurements were in complete agreement with both elution studies and
contact angle measurements. In all cases, the Virosorb 1MDS filters
were once again shown to be the least hydrophobic, while the Seitz S


73
filters were found to be the most hydrophobic of the positively charged
filters tested.
These capillary rise measurements were also done with two
negatively charged filters. Although both Millipore and Filterite
filters are electronegative, their composition is different (Table 2).
It was found that Filterite filters were more hydrophilic than the
Millipore filters, while the hydrophobic nature of both these filters
appeared to be slight, with Millipore being slightly more hydrophobic as
evidence by little of no change in capillary rise for chloroform -
water. Differences in the elution patterns of these two filters have
been documented by researchers (42). It was found that elution of
poliovirus-1 adsorbed to Filterite filters could be accomplished using
casamino acids or individual amino acids such as glycine or lysine,
while these solutions had little effect on the elution of viruses
adsorbed to Millipore filters. Since hydrolyzed proteins, such as
casamino acids have been increased number of free carboxyl and amine
groups, the ionic strength of the solution is also increased (42). It
is likely that this effect of increased ionic strength promoting elution
of adsorbed viruses from Filterite filters is related to the relative
hydrophilicity of these filters or the relatively more hydrophobic
nature of the Millipore filters.
The availability of Virosorb 1MDS filters in a pleated-sheet
cartridge allows for large volumes of water to be processed. Sobsey and
Glass (163) found that the concentration of poliovirus-1 from seeded
tapwater was equally efficient using Virosorb 1MDS filter cartridges or
the highly electronegative Filterite filters, yet the Virosorb 1MDS
filters were much simpler to use, due to little preconditioning of water


74
necessary for adsorption. The Virosorb 1MDS filters have been used to
concentrate indigenous virus from sewage, well water and chlorinated
tapwater collected during an outbreak of gastroenteritis (76). The use
of Virosorb 1MDS filters in the concentration of animal viruses led us
to develop a method for concentrating bacteriophages with them. These
filters were found to adsorb indigenous phage from the four natural
water samples tested without any modification or treatment of the
sample, and concentration of the indigenous phage was accomplished by
using sodium chloride by pH 7 for elution. The results of this study
indicated that sodium chloride, which could be used to elute virus
adsorbed to the Virosorb 1MDS filters, was ineffective for eluting
viruses adsorbed to any of the other three electropositive filters
examined. This raises the possibility of using two different filters in
series for a simple two-step concentration procedure for bacteriophages
based on differences in the elution characteristics of the filters used.
This method has the advantage of maintaining a constant pH value
throughout the concentration procedure, thus minimizing inactivation of
the bacteriophage due to pH extremes. In addition, the method is
relatively simple and requires no costly instrumentation, and,
therefore, it is suitable for field use. One possible combination of
filters was used successfully in this study to concentration indigenous
bacteriophages. It is likely that other filters and eluting solutions
may be used in improved procedures.


75
Ammonium Sulfate Flocculation as a Reconcentration Step
The development of the organic flocculation method by Katzenelson
et al. (92) greatly simplified the reconcentration of viruses in beef
extract solutions, and has been incorporated into many concentration
schemes (44, 64, 70). A major difficulty with this procedure has
developed recently, that is, the variability of different lots and
sources of beef extract with regard to flocculation capacity (5, 84,
125, 132). Another problem with organic flocculation is that the use of
low pH prohibits its use for the reconcentration of bacteriophage, which
have been shown to be sensitive to extremes in pH (149, 150). Our use
of a saturated ammonium sulfate solutions to cause flocculation
eliminates both of these problems. Ammonium sulfate flocculation of
beef extract containing bacteriophage was found to be very superior to
organic flocculation. Moreover, animal virus recovery was also improved
by ammonium sulfate floccuation.
We incorporated this ammonium sulfate flocculation step into a
procedure to recover seeded animal viruses and indigenous bacteriophage
from wastewater effluent samples. The results using this method were
consistently higher than the recoveries using organic flocculation.
The recoveries of bacteriophage were not unexpected, as the pH drop to
3.5 necessary for organic flocculation probably inactivated many
indigenous phages in the beef extract eluate.
The availability of organic flocculation as a reconcentration step
has made beef extract the eluting solution of choice in many
concentration procedures (44, 64, 70). However, other protein solutions
may work as well as, if not better than beef extract, yet
reconcentration becomes a problem (70). Ammonium sulfate flocculation


76
has been found to work efficiently with other protein solutions, such as
typtose phosphate broth and brain heart infusion.
This procedure was also used to recover very low levels of
indigenous bacteriophage. When bacteriophages are serving as models for
enteric viruses, or as indicator organisms, the use of a concentration
method suitable for both animal and bacterial viruses would simplify
assay procedures considerably. It appears that ammonium sulfate
flocculation works equally as well with animal viruses as with
bacteriophages.
Ammonium sulfate flocculation appears to be an efficient method for
reconcentration of beef extract eluates when a fluctuation in pH is
undesirable, or when a beef extract lot fails to flocculate.


CHAPTER III
VIRUS ASSOCIATION WITH OTHER SOLIDS
Review of the Literature
Enteric viruses can be excreted in concentrations as high as one
million viruses per gram of feces (52, 68, 158), and enter domestic
wastewater. Although large numbers of viruses may be present in
wastewater, conventional wastewater treatment plants have essentially
been designed to reduce the amount of organic material and suspended
material discharged to natural waters, and not to reduce pathogens. In
fact, the only step intentionally included for the reduction of
microbial pathogens is disinfection, usually via chlorination (166).
However, several of the steps in wastewater treatment facilities do
reduce the population of microorganisms in raw sewage.
Raw sewage may go through several treatment processes before
discharge from a treatment plant (Figure 10). This process usually
involves primary treatment followed by secondary treatment. In some
cases, wastewater is further conditioned by tertiary treatment.
Primary treatment is the first major process in conventional
wastewater treatment facilities. This is essentially a physical process
designed to remove heavy solids and other materials. The removal of
viruses during this stage of treatment is minimal at best (68, 166).
Effluent from primary treatment often passes on for secondary treatment.
Secondary treatment is a biological process that will metabolize and
flocculate soluble organics in the sewage. One type of secondary
treatment is the activated sludge process. In this system, air is


FIGURE 10. The processing of raw sewage (adapted from 12 and 47).


raw
iiewaj-.o
*
S|
VO


80
added, creating an aerobic environment to ensure adequate mixing of the
sludge. This "activated sludge" contains organisms that are thought to
be the main agents responsible for production of various polymers from
the oxidation of soluble organic matter that aid in flocculation (12).
After settling, the liquid portion is removed (possibly for further
treatment) and sludge floes are separated into two portions. One is
called "return sludge", and is used as an inoculum for primary effluent
entering the aeration tanks. The other part is called "wasted sludge",
which will undergo further treatment, then disposal. The activated
sludge treatment is very effect at removal of viruses from the liquid
portion of wastewater (68, 166).
After final treatment, both sludges (115, 170, 186) and wastewater
effluents (68, 135, 185) have been demonstrated to still contain viable
virus particles. Disposal of these "treated" liquids and solids is,
therefore, of major public health importance. Some methods used for
their disposal are shown in Table 12. As can be seen, in one form or
another, treated sewage is likely to be disposed of on land. Land
spreading of wastewater sludges and effluents has many advantages,
(Table 12) such as crop and soil renovation due to the addition of
nutrients, and water conservation and aquifer recharge. A major problem
with land disposal methods is groundwater and surface water
contamination as a result of virus transport through the soil (174).
With this in mind, it is important to look at the factors involved
in the association of viruses with solids, such as sludges, soils and
clays.


a
TABLE 12. Land disposal methods for wastewater effluents and sludges, and potential benefits and risks.
APPLICATION METHOD
DESCRIPTION/RATE
OVERALL BENEFITS
POTENTIAL VIRUS RISKS
Overland flow
Effluents flow along slope
and are collected in a
a ditch
Rate: 5-14 cm/wk
Additional wastewater
treatment
Soil renovation
Crop production
Virus contamination of food corps
Virus penetration of soil-
groundwater contamination
Surface water contamination via
runoff
Rapid infiltration
Effluent applied via
surface application
Rate: >50 cm/wk
Rapid wastewater
disposal
Recharge of groundwater
aquifer
Moderate level of
addition wastewater
treatment
Viral contamination of
groundwater
Spray irrigation
Effluent applied via
sprinkling or surface
application
Rate: 1.5-10 cm/wk
Speedy disposal of
wastewater
Recharge of groundwater
aquifer
Crop production
Additional wastewater
treatment
Aerosolization of virus
Contamination of crops
Groundwater contamination
Contamination of surface water
via runoff
Sludge surface
spreading
Sludge is spread on
surface and mixed with
soil
Rate: variable
Soil renovation
Crop production
Crop contamination
Groundwater contamination
Contamination of surface water
via runoff
Sludge sub-surface
injection
Sludge injection at 10-20
cm below soil surface
Rate: up to 3000 1/min
Rapid wastewater
disposal
Little contamination of
soil surface
Groundwater contamination
a Adapted from 12 and 174.


82
Association of Viruses with Sludge Floes
Researchers in various laboratories have shown that virus
adsorption to sludge floes occurs rapidly (13, 24, 118). Using
coxsackievirus A9 and poliovirus-1 (Mahoney), Clarke et al. (24) found
that within 4 hours, 99% and 90%, respectively, of these viruses were
associated with the sludge floes, and that the removal of the viruses
from the liquid to solid fraction conformed to the Freundlich isotherm.
Using a laboratory-scale activated sludge unit, Malina et al. (118)
found that over 95% of poliovirus-1 was adsorbed to the sludge in 10
minutes. Both of these studies indicated that viruses were inactivated
over time once adsorbed.
Recent work (131) has indicated that the type of sludge can
influence the amount of adsorption seen. After one hour, only 57% of
seeded poliovirus-1 was found to be associated to mixed liquor, 70% to
anaerobically digested sludge, and 95% to aerobically digested sludge.
The authors suggested that the lower pH values found in aerobically
digested sludge may explain the difference in adsorption.
Due to the complex and variable nature of sludge, little or no work
has been done on the determination of viruses to sludge. Recent work in
this laboratory (32) explored the factors involved in bacteriophage
association to sludge floes. Using various salt and detergent
solutions, it was found that hydrophobic interactions do not appear to
be a major factor in promoting viral association to sludge floes.
Eluting solutions of increasing ionic strength were found to increase
the percent elution of indigenous bacteriophage adsorbed to sludge. The
author hypothesized that electrostatic interactions appeared to be the
major force involved in the association of bacteriophages with sludge
floes.


83
Methods for the Recovery of Viruses from Sludge
Because viruses have been shown to retain their infectivity when
adsorbed to solids (122, 143), it is important to monitor their fate
during and after treatment. To follow the fate of viruses during
treatment and disposal of sludge, methods have been developed to detect,
and in some cases, concentrate viruses adsorbed to sludge floes.
An early study by Clarke et al. indicated that virus appeared to
be inactivated once adsorbed to the sludge floes (24). However, the
solution used to elute the adsorbed viruses (versene and buffer
solutions) has been shown to be a poor eluting solution (49), and the
conclusion drawn by the investigators may be inaccurate. In addition to
the choice of eluting solution used, studies have shown that viruses may
be embedded in, as well as adsorbed to, sludge and therefore some
mechanical means is necessary to agitate the sludge-eluting solutions
mixture (47, 85, 186).
Hurst et al. (85) found that the method of agitation and the
eluting solution used affected the amount of poliovirus-1 recovered from
sludge floes. When mixing with a magnetic stirrer was used, recovery
was greater if 0.05 M glycine was the eluting solution. If sonication
was used, then 3% beef extract as the primary eluate gave higher
recoveries. In the final procedure, elution was accomplished using
glycine at pH 11 and stirring the glycine-sludge mixture, followed by
concentration of the viruses using inorganic flocculation or adsorption
to filters. Poliovirus recovery from anaerobically digested sludge was
55%, while recovery from aerobically digested sludge was 25%.
In another study, three techniques were used to free embedded
virus: sonication, mechanical stirring and freon extraction (186). It


84
was found that no one treatment gave consistent results, but a larger
number of isolates were recovered from samples treated by sonication and
mechanical stirring, although stirring required more time.
The method developed by Hurst (85) was used by Farrah et al. with
limited success (48). This method resulted in overall volumes that were
quite large. Additionally this method did not effectively recover virus
adsorbed to aerobically digested sludge. In order to overcome these
difficulties, they developed a urea-lysine method for the recovery of
viruses adsorbed to sludge (48). Briefly, 4 M urea 1% lysine, pH 9
was mixed for five minutes with the sludge, resulting in 70% elution of
poliovirus-1 adsorbed to sludge floes. Once eluted, the virus was
separated from the sludge floes by centrifugation and concentrated by
aluminum hydroxide flocculation (44). The virus were eluted from these
floes by with 0.1 M EDTA 3% beef extract, and further reconcentrated
by organic flocculation. This resulted in a very low final volume with
approximately 40% recovery of poliovirus-1 and coxsackievirus B3 (48).
However, problems, such as inactivation of virus by urea over time, and
length of time necessary to process samples proclude field use of this
method.
Recently, two methods were compared for their ability to recover
viruses from different sludge types under the auspices of the American
Society for Testing Materials (ASTM) in the hopes of establishing a
standard method (66). One method tested was the Environmental
Protection Agency (EPA) method, developed by Donald Berman of EPA (6,
10). Briefly, this method involves mixing sludge on a magnetic stirrer,
followed by the addition of AlCl^ to a final concentration of 0.5 mM
and pH adjustment to 3.5. Elution was accomplished by mixing the sludge


85
for 30 minutes with 10% buffered beef extract. After centrifugation to
separate viruses in beef extract from sludge floes, the beef extract
solution was filtered through a Filterite series to remove contaminants.
The filtrate was diluted with sterile distilled water at a ratio of 7 ml
of water for every 3 ml of beef extract, and reconcentrated using
organic flocculation. The second method was the Glass method, developed
by Steven Glass of New Mexico State University (62). In this method,
sludge was placed in a blender and dry beef extract (final concentration
ca. 4%) and antifoam were added. This was blended for 3 minutes,
adjusted to pH 9 and stirred for 25 minutes longer. The viruses were
separated from the sludge floes by centrifugation and concentrated by
organic flocculation. The final concentrate was detoxified by a
dithizone-chloroform mixture. The results of this comparison indicated
that the EPA procedure was slightly more sensitive than the Glass method
for recovering viruses from all sludge types but on (66).
All methods detailed here require extremes in pH, and are therefore
unsuitable for the recovery of bacteriophages from these samples.
Association of Viruses with Soils
The application of sewage treatment solids and liquids onto land
can lead to the adsorption of any surviving viruses to the soil. The
desorption of virus, and its subsequent transport through the soil
matrix can lead to groundwater contamination (142, 187). The
dissociation of viruses from soils is due to changes in the
physicochemical properties within the soil. Because the potential of
waterborne outbreaks of viral diseases is great, it is important to
study the mechanisms of and the factors associated with the adsorption of
viruses to soils.


86
The adsorption of viruses to soils is a reversible process that can
be described in terms of an equilibrium isotherm. Two such isotherms
have been used to examine the kinetics of virus adsorption to soils, the
Langmuir and Freundlich isotherms (53, 174). When the relationship
between the amount of adsorbed and unadsorbed virus is found to be
linear, a Freundlich isotherm is used. It has been proposed that
conformance to the Freundlich iostherm indicates that the surface
contains adsorption sites heterogeneous with regard to the strength of
bond formation (21). The Langmuir isotherm indicates that there are a
large but finite number of adsorption sites on a solid, thus equilibrium
Langmuir isotherms would indicate saturation-limited adsorption (174).
There is considerable debate whether viral adsorption to soils is
best described by either of these two isotherms. The linear plot of the
Freundlich isotherm has been used to describe a variety of virus
adsorption phenomenon, such as the adsorption of poliovirus-1 to loamy
sand (58) the adsorption of T2 and F2 to silty loam (34) <{>xl74
adsorption to clay and silt loans (21) and poliovirus-2 adsorption to
over 30 soils and minerals (124). However, it has been hypothesized
(176) that most adsorption processes were most likely saturation-
limited, and therefore conformed to the Langmuir isotherm. The author
suggested that in most adsorption studies, insufficient concentrations
of viruses were used, and as such, the saturation region of a Langmuir
plot was never seen, and the linear section of such an isotherm give the
appearance of conformance to the linear Freundlich isotherm. Several
studies have dictated that this indeed may be the case. Moore et al.
(124) studied the adsorption of radioactive poliovirus-2 to 34 minerals
and soils and found that at low virus concentrations, data conformed to


87
the Freundlich isotherm. However, when saturated conditions were
examined, their data conformed well to the Langmuir equation. The
authors, however, also indicated that in systems where saturation
appears to be occurring, it may likely be due to a coating of soil
adsorption sites with competing material. In addition, the Langmuir
isotherm has been used to describe MS2 adsorption to Indian soils (99),
T4 adsorption to activated carbon (27) and the adsorption of <¡>xl74 to a
silt loam (21).
As these data above indicate, adsorption of viruses to soils is a
complicated process. The adsorption, and subsequent desorption of
viruses from various soil types can be affected by many factors. These
factors are listed in Table 13 and are examined below.
When viruses dispersed in liquid are applied to soils, they must
come into close contact with one another in order for adsorption to
occur. This is directly related to the infiltration or flow rate of the
applied liquid (61, 174). Lance and coworkers (101, 103, 104, 105) have
studied the effect of various infiltration rates of sewage effluent
waters on the adsorption and elution of several enteroviruses. Using a
250 cm coarse sand column, and poliovirus-1 (101) or echovirus-1 (105),
they found that increased flow rates decreased the amount of virus
retained at the top portions of the column. At flow rates greater than
1.2 m/d, both poliovirus (101) and echovirus-1 (105) were leached
through the column. While the percentage of virus found in the column
effluent was low (<1%), the fact that virus will percolate through soil
at higher flow rates indicated the importance of contact time in viral
retention by soils.


TABLE 13.
Factors that may influence viral adsorption to soils.
a
FACTOR
REMARKS
REFERENCES
Flow rate
Low flow rates result in very efficient removal of
viruses (>99%) in clean waters. As flow rate
increases, virus adsorption decreases proportionally.
61, 101, 103, 104
105, 174
PH
Generally, a low pH favors virus adsorption while a
high pH results in elution of adsorbed virus.
21, 171
Cations and ionic
Cations, especially divalent cations, can act to
neutralize or reduce repulsive electrostatic forces
between virus and soil particles, allowing adsorption
to proceed. Solutions with low ionic strength
(i.e. rainwater) promote elution of adsorbed viruses.
36, 102, 104, 106
108, 123, 144,
171, 185, 187
Soluble organics
Soluble organic matter has been shown to compete with
viruses for adsorption sites on soil, resulting in
decreased adsorption or elution of an already adsorbed
virus.
17, 55, 61
Chemical composition
of soil
Soils with high organic content adsorb viruses less
efficiently. The presence of iron oxides increases
virus adsorption.
11, 61, 123, 124,
144
Nature of virus
The optimum pH for virus adsorption is expected to occur
at or below its isoelectric point, where the virus
possesses no charge or a positive charge. Different
strains of the same virus adsorb differently.
20, 55, 56, 106
Clay content
This is the active fraction of the soil. High virus
retention by clays results from their high ion
exchange capacity and large surface area per volume.
21, 22, 34, 36,
61, 99, 112, 143,
169, 174
a Adapted from 61.


89
More recent work in the same laboratory (108) compared the movement
of poliovirus-1 in a 250 cm long soil column during saturated and
unsaturated flow of sewage water. In was found that during unsaturated
flow, poliovirus-1 was not detected below the 40 cm mark. However,
during maximum flow rates, poliovirus-1 penetrated to 160 cm, and in
some cases, past the 200 cm level. These data indicated the importance
of low infiltration rates to maximize virus retention by soils.
In natural systems, both viruses and most soils are negatively
charged. As such, pH and salt content can have a tremendous impact on
virus adsorption to and subsequent elution from the soil matrix. Taylor
et al. (171) studied the interaction of radiolabelled poliovirus-2 with
various soils, sands and clay. They found that at the pH values above
9, virus adsorption to these substrates was limited. At pH values near
neutrality, adsorption of poliovirus-2 to soils was greatly facilitated
by the addition of 10 mM CaCl^ or Na^SO^, while adsorption was limited
in solutions which did not contain these ions. These authors found that
for each substrate, there was a characteristic pH region of transaction
from strong to weak attraction for virus uptake, and in order for
adsorption to occur, these electrostatic repulsive forces must be
overcome. In a study comparing the adsorption of 4>Xl74 to five
different soils (21) it was found that the low adsorption capacity of a
loamy sand was related to its high pH.
The importance of cation addition in the promotion of virus
adsorption to soils has been well documented. Retention of f2 and
poliovirus-1 by sand was greatly increased by the presence of 10 mM
CaCl2 or MgCl^ (108). Scheuerman et al. (144) found the adsorption of
poliovirus-1 to soils with high organic content was less than 20%. The


90
addition of 10 mM CaCl^ increased its adsorption to greater than 98%.
Moore et al. (123) found that adsorption of reovirus-3 to various sands
and silts was enhanced by the addition of 1 mM CaCl^ or MgCl^ to the
suspending medium. Using soil columns, Lance and Gerba (102)
demonstrated that the addition of 0.2 mM AlCl^ prevented the leaching of
poliovirus-1 in sewage effluents from the soil column. They suggested
that the increase in virus adsorption associated with increasing ionic
strength of the suspending solution was probably due to a decrease in
the thickness of the layers of charged ions around soil particles and
viruses. This allowed for short-range attractive forces, such as van
der Waals interactions, to take over.
The desorption of adsorbed viruses by low ionic strength solutions
such as rainwater has been well documented (36, 104, 106, 185, 187).
Duboise et al. (36) found that poliovirus-1 adsorbed to soil columns in
sewage effluents were easily desorbed by the addition of deionized water
to simulate rainfall. Studies by Wellings et al. (185, 187)
demonstrated the presence of viruses in wells 10 to 20 feet below soil
that had been treated with sewage effluents. These viruses appeared
after a period of heavy rainfall, and the authors hypothesized that the
increase in water/soil ratio resulting from the rainfall led to
desorption of the attached virus. Lance and coworkers (104) found that
the viral desorption and movement caused by simulated rainwater was
greatly reduced or eliminated by the addition of salts, again
implicating the importance of ionic strength in the association of
viruses to soils.


91
The presence of soluble organic matter in suspending solutions can
interfere with viral adsorption to soils by competing with viruses for
adsorption sites (61). Gerba and Goyal (55) found that virus suspended
in secondarily treated sewage, which has a high content of soluble
organics, adsorbed less to soils than virus suspended in deionized water
adsorbed to the same soil. Work by Bitton et al. (17) indicated that
humic substances interfered with the adsorption of coliphage T2 and
poliovirus-1 to a sandy soil.
The organic content of the soil itself may also play a role in the
association of viruses to soils. In studies examining adsorption of
virus to over 30 different soils and minerals, Moore and coworkers found
that both poliovirus-2 (124) and reovirus-3 (123) were adsorbed poorly
in soils with higher organic content. Burge and Enkiri found that 4>xl74
adsorbed well to four of five soils tested, the exception being a clay
loam high in organic content (21). Soils with high amount of humic and
fulvic acids were shown to be poor adsorbers of poliovirus-1 (144).
In addition to organic components, other constituents of the soils
will influence the association of viruses with them. The adsorption of
Xl74 to soils was found to be correlated with the cation exchange
capacity and specific surface area, as well as organic content (21).
The natural pH of a soil has been found to effect virus adsorption (21,
34, 55). Generally, soils with lower pH's tended to adsorb virus more
effectively. The presence of iron oxides, such as magnetite and
hematite, in soil increases the adsorption capacity for viruses (11, 61,
124). Clay content has been shown to be very important component in
soils that promotes virus adsorption (61, 174) and will be discussed
later.


92
The nature of the virus itself will play a paramount role in
adsorption of viruses to soil surfaces. Burge and Enkiri (20) found
that <(>xl74 purified by density gradient centrifugation was still
heterogenous. They found 2 fractions with different adsorption rates.
Landry et al. (106) compared lab strains and wild type strains of
poliovirus-1 and discovered that field strains were eluted from soils by
rainwater to a greater extent (>33%) than were lab strains (<3% eluted).
Gerba and Goyal (55) examined the adsorption of 27 different
reference strains of enteroviruses to a loamy sand. They found that
most viruses adsorbed very well (>90%) but there were some exceptions
including echoviruses-1 (55%), -12 (78%) and -29 (14%). They further
examined different strains of the same virus and found that adsorption
to loamy sand was very strain dependent. For six different strains of
echovirus-1, adsorption varied from 0 to 99%. This same group
statistically analyzed their data to determine variables associated with
virus-soil interactions (56). They found that different types and
strains of viruses could be grouped by their ability to similarly
effected by certain soil characteristics. Group I, which included
echovirus-1, coxsackievirus B4, <¡>xl74 and MS2, was found to be
influenced by pH, organic matter and exchangable iron content of the
soil. Group II (poliovirus-1, echovirus-7, coxsackievirus B3, T2 and
T4) did not appear to be influenced significantly by any soil factor.
The authors concluded that viruses in group I were more likely to be
influenced by changes in soil characteristics than group II as far as
adsorption/elution of these viruses was concerned.


93
Association of Viruses with Clays
Clay is perhaps the most important constituent of soils that
governs the adsorption of viruses (61, 174). Studies have shown that
the higher the clay content of the soil, the higher the viral adsorption
to that soil (34, 169, 174). The adsorption of MS2 to Indian soils was
shown to be depending on clay content (99). Lateritic soil, with a clay
content of 32%, was found to adsorb more MS2 than Black Cotton soil or
Kampur soil, with clay contents of 28% and 10% respectively. Sobsey et
al. (161) found that reovirus-3 and poliovirus-1 adsorption to different
soils could be related to clay content. Gerba and Goyal compared
adsorption of selected enteroviruses to nine different soils, and found
increased clay content in the soil allowed for more virus adsorption
(55). Work by Drewry and Eliassen (34) indicated that there was a
direct relationship between the clay content of nine soils from Arkansas
and California and their retention of bacteriophage.
High virus retention by clays has been related to their ion
exchange capacity (21, 112, 169) and their large surface area available
(21, 36, 61, 143) for adsorption. The research group headed by Stotzky
has examined the adsorption of various viruses to pure clay minerals to
determine the factors involved in this association (110, 111, 112, 145,
169) .
The adsorption of coliphages T1 and T7 to kaolin, a two-layer non
expanding clay, and bentonite, a three layer expanding clay, was
examined (145). The adsorption of T7 to these clays was found to be
related to the cation exchange capacity (CEC) of these clays, indicating
that T7 adsorbed to the negative sites on the clay surface. T1
adsorption, however, was not correlated with CEC. Pretreatment of clays
with 1% sodium metaphosphate, which blocked positive sites on the clay,


Full Text
FACTORS INFLUENCING VIRUS
ADSORPTION TO SOLIDS
by
PATRICIA ANN SHIELDS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986

Mom and Dad, and Tom, Sharon, Carrie and Jennifer

ACKNOWLEDGMENTS
I gratefully acknowledge the help and guidance given me by the
members of my committee, Dr. Samuel R. Farrah, Dr. Lonnie 0. Ingram, Dr.
Stephen G. Zam, Dr. Gabriel Bitton and Dr. Dinesh 0. Shah. I am forever
indebted to my chairman, Dr. Farrah, who has been my friend as well as
my boss. The example of scientific excellence he has set will never be
forgotten.
In addition, I extend thanks to those individuals who have helped,
in many ways, to make this journey enjoyable, especially Dr. Phillip
Scheuerman, Orlando Lanni, Sharon Berenfeld, Kathi Moody, Vivienne
Thompson, Dave Preston, Lena Dingier, Jane Strandberg and Gail Waldman.
In particular, I am beholden to Lena Dingier, and Jane Strandberg, both
of whom have shown me the true meaning of friendship.
Finally, I extend thanks to my family for the love and support they
have given me in this and all other endeavors. This dissertation is
dedicated to them.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS i ii
ABSTRACT v
CHAPTERS
I INTRODUCTION 1
II VIRUS ASSOCIATION WITH MEMBRANE FILTERS 7
Review of the Literature
Materials and Methods 25
Results 33
Discussion 65
III VIRUS ASSOCIATION WITH OTHER SOLIDS 76
Review of the Literature 76
Materials and Methods 95
Results 99
Discussion 123
IV CHARACTERIZATION OF VIRUSES 133
Review of the Literature 133
Materials and Methods 141
Results 143
Discussion 156
V SUMMARY 166
APPENDICES
A ROUTINE METHODS USED IN ANIMAL AND BACTERIAL VIRUS
PROPAGATION AND ASSAY 168
B COMPOSITION OF MEDIA AND SOLUTIONS USED IN CELL
CULTURE WORK 172
BIBLIOGRAPHY 175
BIOGRAPHICAL SKETCH 190
iv

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FACTORS INFLUENCING VIRUS
ADSORPTION TO SOLIDS
By
Patricia Ann Shields
May 1986
Chairman: Samuel R. Farrah
Major Department: Microbiology and Cell Science
Several factors influence the association of viruses with solids.
The natures of the adsorbent and virus studied are among the most
important factors that influence this association. Differences in virus
adsorptive behavior have been related to the relative strength of
hydrophobic and electrostatic interactions between the virus and solid.
The main objective of this study was to determine the relative
hydrophobic and electrostatic nature of viruses and various virus-
adsorbing solids.
Membrane filters used in virus concentration procedures were
characterized by contact angle and capillary rise measurements. Among
electropositive filters tested, Seitz S filters were found to be the
most hydrophobic, while Virosorb 1MDS filters were found to be the most
hydrophilic. Among electronegative filters tested, Millipore HA filters
were found to be the most hydrophobic, while Filterite filters were
found to be the most hydrophilic. These physical measurements were
found to correlate well with the ability of certain solutions to elute
v

viruses adsorbed to the filters. Based on these differences, several
two-step concentration procedures for viruses in water were developed
that used dissimilar filters for each stage.
Beef extract solutions are often used for the elution of viruses
adsorbed to membrane filters. The use of ammonium sulfate flocculation
to concentrate viruses in these solutions was compared to organic
flocculation. The recovery of seeded enteroviruses from sludge was 62%
using ammonium sulfate flocculation as compared to 34% when organic
flocculation was used as a concentration step. Ammonium sulfate
flocculation was also found to improve recoveries of viruses from
sewage effluents and soils.
The relative hydrophobic and electrostatic nature of viruses was
determined. Hydrophobic interaction chromatography and hydrocarbon
adherence tests were used to assess the relative hydrophobicity of the
viruses. Adsorption to DEAE-sepharose was used to assess the relative
electrostatic nature of the viruses. Based on these tests, echovirus-5
and bacteriophage MS2 were found to have the highest relative
hydrophobicity. Bacteriophages T2, T4 and MS2 were found to adsorb very
strongly to DEAE-sepharose, indicating these viruses had the strongest
relative electrostatic interactions of the viruses tested.
vi

CHAPTER I
INTRODUCTION
Viruses that are shed in fecal matter are collectively referred to
as enteric viruses. More than 100 different enteric viruses are known
to be excreted in the human feces (60, 68, 158). Examples of these
viruses and the diseases they cause are shown in Table 1. A human
showing no symptoms of disease can excrete up to a million infectious
virus particles per gram of feces and this number may be higher in
individuals manifesting disease (52, 68, 158).
Theoretically, any virus excreted in feces and capable of producing
infection when ingested could be transmissible by inefficiently treated
water (68, 88). However, epidemiological evidence for water-borne
transmission of human viruses is limited to Hepatitis A virus, Norwalk
virus and adenovirus infections associated with swimming pools (88).
For most enteric viruses, demonstration of water-borne transmission is
hampered by several factors. Many of these enteric viruses cause
asymptomatic infections (52) . As such, the role of short-term carriers
of viruses who do not develop disease is not easily assessable (88,
158). In addition, the possibility that other secondary transmission
sources may exist makes accurate epidemiological studies virtually
impossible (68, 88) .
Studies have shown that some human enteric viruses can survive
sewage treatment and persist in wastewater sludges and effluents (89,
115, 122, 172, 186). At the end of the treatment cycle, wastewater

2
Table 1. Human
enteric viruses
and associated diseases.3
Virus Group
Number
of Types
Disease
Poliovirus
3
Paralytic poliomyelitis, aseptic
meningitis
Coxsackievirus
Group A
24
Herpangina, aseptic meningitis,
paralysis, fever
Group B
6
Pleurodynia (Bornholm disease),
aseptic meningitis, acute
infantile myocarditis, rash,
fever, respiratory disease
Echovirus
34
Aseptic meningitis, fever,
rash, diarrheal disease,
respiratory disease
Hepatitis A
1
Infectious hepatitis
Reovirus
3
Fever, respiratory disease,
diarrhea
Rotavirus
4
Severe diarrhea, vomiting,
low grade fever, dehydration
Adenovirus
31
Respiratory and eye infections
Norwalk virus
3 (?)
Diarrhea, vomiting
a - Adapted from 59, 60 and 68.

3
sludges and effluents are often disposed of on land (174). Once
disposed of in this manner, viruses associated with sludge floes or in
effluents interact with soils can lead to viral contamination of crops,
soils, and surface and groundwaters (65, 68, 172, 174).
While the number of viruses in raw sewage may be quite high, the
concentration of viruses after treatment, and in subsequently
contaminated solids and water supplies is low. Even at these low
numbers, enteric viruses are capable of causing disease and thus are
cause for concern. Because the numbers of viruses are low, it is not
always practical to screen samples for the presence of these pathogens.
Therefore, it is logical to screen for the presence of an indicator
organism whose presence may indicate fecal pollution of the sample. The
presence of coliform organisms, such as E. coli, has been used
extensively as a measurement of fecal contamination (2). However,
studies have shown that enteric viruses are more resistent to
environmental conditions and sewage treatment processes than are the
coliform organisms (7, 8, 88). Enteric viruses have been isolated from
water supplies which have met coliform limits (7, 31, 57, 76, 187).
Therefore, there appears to be a need to find another, more suitable
organism to serve as an indicator for enteric viruses.
The ideal microbial indicator should have the following
characteristics: i) It should be present when pathogens are present and
absent when pathogens are absent. ii) The growth characteristics and
persistence of the indicator should be similar to the pathogen, iii)
Both the pathogen and the indicator should occur in a constant ratio so
that counts of the indicator will estimate the amount of pathogen

4
present, iv) The indicator should be found in greater numbers than the
pathogen, v) The indicator should be at least as resistant to
environmental factors as is the pathogen, vi) The indicator should be
nonpathogenic, easily quantifiable, and be able to be tested for in a
variety of environments (63, 167). Several investigators have advanced
the idea of using bacteriophage as indicators for the presence of
enteric viruses in fecally contaminated water (35, 78, 98, 167).
Recently, Stetler monitored coliphages in conjunction with indicator
bacteria and enteroviruses in a drinking water plant (167). Statistical
analysis of the data indicated that coliphages were much better
indicators of enteroviruses than were coliforms or fecal streptococci.
On the other hand, other investigators have noted that bacteriophage
levels do not correlate well with the presence of enteric viruses in
field samples (91, 175). If bacteriophages are to serve as indicator
organisms, then simple and reliable methods must be developed to detect
and concentrate them from natural samples. Procedures used to
concentrate animal viruses often required extremes in pH, which are
unsuitable methods for phage concentration (149, 150). Investigators
have developed methods to concentrate phage using materials which do not
require these fluctuations in pH (67, 113, 150, 157). The development
of a method which could be used to detect both bacterial and animal
viruses in the same sample would simplify and speed up assay procedures
considerably.
Virus interactions with solid surfaces play a major role in their
removal by sewage treatment processes, as well as their response to,
transportation in and persistence in their environment. The phenomenon
of virus adsorption is also important in many methods used for the
detection and concentration of viruses in the environment (53) . Thus,

5
it is important to study the mechanism of and factors influencing virus
adsorption to solids.
Several factors may influence the association of viruses with
solids. These factors include pH (21, 121, 171, 183), the concentration
and nature of salt in solution (102, 123, 177, 178, 180), the presence
of organic compounds (17, 55, 183), the flow rate of virus through the
adsorbent (61, 103, 147, 174), the nature of the virus (53, 55, 56, 117,
189) and the nature of the adsorbent surface (11, 123, 124, 164, 174).
Accordingly, these factors have been studies to better understand
virus-solid interactions and determine the mechanism of viral
adsorption.
The adsorption of viruses to solids was though to be mainly
electrostatic in nature (94, 173). Since viruses are essentially
biocolloids, theories which describe colloidal behavior have been used
to describe viral interaction with solids.
If the charges on both the virus particle and adsorbing surface are
opposite, electrostatic attraction results in viral adsorption (94, 117,
190). Conversely, if both are of the same charge, electrostatic
repulsion occurs. In most natural water systems, virus particles are
above their isoelectric point and assume a negative charge. By altering
such factors as salt concentration and pH, adsorption to electronegative
surfaces can occur. This phenomenon can be explained by the double
layer theory (52, 53, 173). Essentially, this theory says that ions
associated with both surfaces will attract counterions from the medium
into a compact layer (called the stern layer) around the surfaces. This
layer of counterions will partially neutralize the charge on the
surface. In order for the system to maintain electrical neutrality,

6
there also exists a diffuse layer containing a lower number of
counterions some distance away. If the bulk solution of counterions
increases by altering the pH or the addition of cationic salts, the
thickness of this diffuse layer decreases. This is because less volume
is needed to contain enough counterions to neutralize the surface
charge. As the size of this layer is decreased, the two surfaces can
come closer to each other and short range attractive forces may take
over. Thus interactions between two disperse particles are thought to
result from a balance between repulsive double layer interactions and
attractive short-range forces, such as van der Waals forces.
Accordingly, altering the pH or addition of cations allows for increased
adsorption to occur (52, 53).
Many studies have confirmed that electrostatic interactions are a
primary mechanism for adsorption of viruses to solids (53, 183, 190).
However, such complex theories such as the double layer theory or simple
theories such as salt bridging fail to explain some of the experimental
results. For example, at high pH, viral adsorption to negatively charge
filters is maintained by the presence of multivalent metal ions.
However, the addition of a chelating agent, which should effectively
remove such ions and prevent salt bridge formation, does not promote
viral elution (40, 42). Furthermore, elution of adsorbed viruses at
high pH has been accomplished using neutral solutions such as urea or
nonionic detergents (42, 48, 180, 183). These variable effects of
different solutions on the elution of viruses adsorbed to solids cannot
be explained in purely electrostatic terms, indicating that additional
forces are involved in viral adsorption to solids. These forces
include hydrogen bonding and more importantly, hydrophobic interactions.

7
The role of hydrophobic interactions in virus adsorption phenomena
has been examined in several studies and results have shown that these
interactions are influenced by salts, and the nature of the virus and
adsorbent studies (32, 40, 49, 50, 154, 156, 177).
The overall objective of this study was to examine the
contributions of electrostatic and hydrophobic interactions in the
association of viruses with solids, in the hope of better understanding
the factors involved in viral adsorption.
Chapter II of this dissertation examines the association of viruses
to membrane filters. These filters are characterized by several
methods, and the differences in the nature of these filters are used to
explain the differences in viral elution patterns seen. In addition,
several concentration procedures are developed for the detection of
animal and bacterial viruses in water samples. Finally, a new
concentration procedure is shown that allows for simultaneous
concentration of animal and bacterial viruses from the same sample.
Chapter III examines the association of viruses to solids such as
clay, soil and wastewater sludges. The extent of hydrophobic
interactions involved in virus association to clays is examined.
Additionally, methods are developed to recover viruses adsorbed to soils
and sludges.
Chapter IV describes the characterization of viruses themselves.
Viruses are examined by adsorption to or elution from ion exchange gels
and hydrophobic gels. Viruses are further characterized by adherence to
hydrocarbons. These results are then used to explain differences in the
adsorption/elution patterns of different viruses.

CHAPTER II
VIRUS ASSOCIATION WITH MEMBRANE FILTERS
Review of the Literature
Concentration of Viruses on Membrane Filters
A wide variety of methods are available for the detection and
concentration of viruses in water samples, however, methods which employ
adsorption to and subsequent elution from membrane filters are still
considered to be among the best (53, 64, 136). In an early study,
Ellford (37) noted a decrease in bacteriophage titer when salt solutions
containing viruses were passed through nitrate cellulose filters. He
found that dilution of the phage into protein solution prior to passage
through the filter avoided loss of viruses onto the filter by
adsorption. In 1967, Cliver (25) found that some enteroviruses were
adsorbed to cellulose nitrate and cellulose acetate filters. In these
experiments, Cliver was attempting to rid solutions of bacteria so as to
assay these solutions for the presence of virus, and therefore this
adsorption was not desirable. He found, much the same as Ellford, that
proteins (in this case serum or gelatin) blocked this viral adsorption.
Later, work in the same laboratory indicated that 99% of enteroviruses
seeded into deionized water were adsorbed when passed through Millipore
(cellulose nitrate) filters (26). Over 80% of these viruses were
subsequently recovered by soaking the membrane in phosphate-buffered
saline containing 30-50% agamma chicken serum.

9
In 1967, Wallis and Melnick reported the first applied use of
membrane filters to concentrate viruses from water samples (180). This
report detailed several important discoveries. First, they showed in
this correspondence that the addition of salts (0.05 M MgCl2) greatly
enhanced virus adsorption to Millipore filters. The adsorbed virus was
recovered by grinding the filter with a mortar and pestle in the
presence of Melnick B media supplemented with 10% fetal bovine serum.
Over the course of several months, using this procedure, 2795 virus
isolates from sewage water were detected in the concentrate, while only
4 isolates were found in unconcentrated samples from the same period
(180). In addition, during the course of this study, Wallis and Melnick
noted interference with adsorption after the passage of about 100 ml of
sewage through the membrane filter. They suggested that this was due to
the high amount of organic material in the sewage. When the sewage was
passed through anion resins, the interfering material was removed and
virus adsorption was enhanced.
In an subsequent paper (181), Wallis and Melnick reported that
protein and other materials present in crude virus harvests interfered
with virus adsorption to Millipore filters. They called these materials
"membrane coating components" or MCC. These MCC could be removed by
adsorption to anion resins. Once the crude cell harvest was treated to
remove MCC, viruses in these harvests were efficiently adsorbed to
Millipore filters in the presence of salts at pH 5.0. Adsorbed virus
was eluted with a small volume of fetal bovine serum, and in virtually
all cases, 80 - to 100-fold concentrations were achieved.
A study by Rao and Labzoffsky (138) confirmed and expanded on the
work of Wallis and Melnick. They found that low levels of calcium (200
ppm) facilitated virus adsorption to Millipore filters. In addition,

10
this was the first report of using a prefilter (Millipore AP25) in
series with the adsorbing filter. They suggested that in some water
samples, viruses attached to small solids were lost if the prefiltering
stage was separate from the adsorption stage. By using a filter series,
virus retained on the prefilter was eluted along with virus adsorbed to
the cellulose nitrate filter by treatment with 3% beef extract.
Since different eluting solutions had been used to recover viruses
adsorbed to membrane filters, a comparative study of these eluates was
conducted by Konawalchuk and Speirs (97). Coxsackievirus B5 and
poliovirus-1 adsorbed to Millipore filters were eluted using different
concentrations of beef extract, yeast extract and fetal bovine serum.
It was found that elution improved with increases in concentration of
eluting agent and the volume of the eluent. These researchers found
that the best eluate was undiluted fetal bovine serum, which recovered
over 80% of the adsorbed enteroviruses tested, and did not result in any
toxicity problems during viral assay procedures.
The ability of salts other than MgCl2 and CaCl2 to promote viral
adsorption was examined by Wallis et al. (178). They found that 0.5 mM
AlCl^ was as effective in promoting poliovirus adsorption to Millipore
HA filters as 50 mM MgCl^, and was therefore more cost-effective when
large volumes of water were processed.
The processing of large volumes of water generally requires larger
filters, and subsequently larger volumes of the eluting solution are
necessary for efficient viral elution. In order to reduce the amount of
sample to be assayed, there is a need for a second or reconcentration
step. Since the protein solutions commonly used for viral elution

11
interfered with viral readsorption (25, 37, 180, 181), Wallis et al.
(178) sought another eluate. They found that a high pH (pH= 11.5)
glycine buffer solution eluted viruses adsorbed to Millipore filters
effectively and these eluted viruses were easily readsorbed to a smaller
diameter filter of the same type by lowering the pH and adding AlCl^.
This study laid the groundwork for the development of an apparatus for
concentrating viruses from large volumes of water.
Wallis and coworkers (179) developed an apparatus for separating
virus from water contaminants so that the virus contained in the
clarified water could be readily concentrated on adsorbents at high flow
rates. In its final form, the apparatus consisted of five non-viral
adsorbing texile fibers in series, pretreated with Tween 80 to aid in
removal of contaminants without removal of virus. After passage of the
water through this filter series, the water was treated by passage
through an anion-exchange resin to remove organics which may interfere
with virus adsorption. Prior to virus adsorption onto cellulose nitrate
filters, the water was conditioned with MgCl^ to a final concentration
of 0.05 M to facilitate adsorption. Elution was accomplished using a
glycine buffer at pH 11.5, and virus was reconcentrated by adsorption to
and elution from a smaller diameter filter (178). Poliovirus-1 was
seeded at high levels into 150 gallons of tapwater, and using this
apparatus, 84% of the virus was recovered. WHen very low levels of
virus were added to the tapwater, virus recovery was still over 60%.
This original model was modified by Homma and coworkers (83) and
this prototype was developed for commercial use by the Carborundum
Company (Niagara Falls,NY) as the "Aquella Virus Concentrator" (64,
136). In this system, the water was passed through a series of

12
prefilters consisting of 5 ym and 1 ym polyester depth cartridge filters
followed by a Tween 80 treated, 1 ym cotton cartridge. After passage
through these filters, the pH of the water was adjusted to 3.5 and AlCl^
was added instead of MgCl^ since previous studies had shown the use of
AlCl^ to be more cost effective than MgCl^ (178). Viruses were adsorbed
to a 1 ym fiberglass or cellulose acetate cartridge filter and eluted
with one liter of 0.5 M glycine buffer, pH 11.5, and reconcentrated onto
smaller diameter cellulose nitrate membrane filters.
Sobsey and colleagues (165) found that further modifications of the
Wallis-Melnick concentrator were in order. After adjustment of water to
pH 3.5, it was passed through a fiberglass depth prefilter (K-27)
followed by a 142 mm epoxy-fiberglass-asbestos Cox filter in series.
Elution of adsorbed virus was accomplished using one liter of high pH
glycine buffer solution and virus was reconcentrated to a 10 ml volume
by adsorption onto a smaller diameter (47 mm) Cox filter series. Using
this modified system, they were able to recover over 75% of poliovirus-1
seeded into 100 gallons of tapwater. Problems developed with this
system when it was scaled up to process larger volumes of tapwater (44,
46). Farrah et al. (44) found that there was clogging of the initial
virus adsorbent when large volumes of water were processed. This lead
to a reduced flow rate and organics, such as humic acids (46) were
concentrated along with the viruses and interfered with the
reconcentration of the viruses by adsorption to a smaller diameter
filter. The initial clogging problem appeared to be due to the use of a
flat filter at the primary adsorption step. This problem was eliminated
by using a pleated membrane filter cartridge (epoxy-fiberglass Filterite
filter). This cartridge filter provided a larger surface area for virus

13
adsorption and was capable of adsorbing virus in tapwater at
considerably higher flow rates. The adsorbed virus was eluted with 2 1
of 0.05M glycine, pH 10.5. To overcome the problem of organics
concentrated along with viruses interfering with readsorption to smaller
filters, this step was eliminated and replaced by an aluminum
flocculation procedure (44). THe initial eluate was neutralized and
AlCl^ was added to a 0.003 M concentration. This lowered the pH of the
water to 4. The solution was neutralized and a floe formed, which was
collected by centrifugation. Virus in the floe was eluted by mixing
the floe with an equal volume of 1.0 M glycine, pH 11.5. The complete
process could be done in 3 hours and virus recovery from 1900 1 of tap
water averaged between 40 and 50%.
Up to this time, most work done on the development of concentration
procedures to detect viruses had used tapwater seeded with virus. With
the development and subsequent modification of the Wallis-Melnick virus
concentrator, researchers began to examine its use with more polluted
water supplies. Using this concentration system, enteroviruses were
efficiently recovered from estuarine water (45, 120, 133) , raw sewage
(54, 83, 151), and seawater (54).
Prior to 1978, work done on the concentration of viruses from water
supplies using membrane chromatography centered around the use of
negatively-charged filters such as Filterite, Millipore and Cox filters.
Efficient viral adsorption was accomplished by conditioning of the water
sample by addition of salts and acidification. In 1979, Sobsey and
Jones (164) reported the first use of electropositive filters in the
concentration of viruses from water samples. They tested two positively
charged filters: Seitz S, composed of cellulose-asbestos, and Zeta plus

14
grade S, composed of cellulose-diatomaceous earth-"charged-modified"
resin. These filters were compared with two negatively charged filters
used in virus concentration, Filterite and Cox filters. At pH values
near neutrality (pH 7.5), less than 20% of poliovirus-1 in unmodified
tapwater was adsorbed by either the Filterite or Cox filters, while
greater than 99% was adsorbed by the Zeta plus and Seitz S filters.
Zeta plus filters were used to concentrate poliovirus-1 from 100 gallons
of tapwater by adsorption to a 267 mm diameter 60S filter, and followed
by elution with 1 1 glycine buffer, pH 10. Virus in the eluate was
further concentrated by dropping the pH to approximately 7 and adsorbing
to a similar, smaller diameter filter followed by elution in a smaller
volume. Using this procedure, recovery of the poliovirus-1 equaled 75%.
Because these electropositive filters did not require acidification
of water samples, researchers began exploring the use of these filters
for the concentration of viruses sensitive to low pH, such as
bacteriophages (67, 113, 136, 149). While bacteriophages are of no
health concern to humans, this work has been done for several reasons.
Several investigators have advanced the idea of using bacteriophages as
indicators of fecally contaminated water (35, 78, 98, 167). Others have
indicated that concentration of naturally occurring phages is necessary
in order to learn more about bacteriophage ecology (136, 150).
Additionally, bacteriophages have been used as models to study the fate
of animal viruses in sewage treatment plants (140).
Logan et al. (113) evaluated the ability of positively charged
filters to adsorb coliphages at different pH values. They found that
greater than 90% of phages MS2 and T2 were adsorbed at pH 7 by the Zeta
plus filters. Efficient elution of virus was accomplished using a

15
solution of 1% beef extract in 50 mM arginine at pH 9.0. Next, they
seeded 65 1 of prefiltered pond water adjusted to pH 6.0 with various
coliphages. The pond water was passed through a 273 mm Zeta plus 60S
filter and adsorbed phage was eluted with 500 ml of the beef extract
solution. Recoveries of 87% for T2 and 93% of MS2 were obtained using
this method, while only 25% of 'í’Xl’M was recovered. Use of this method
with river water resulted in 50-60% recovery of naturally occurring
bacteriophage.
Goyal and coworkers (67) also evaluated the use of Zeta plus
filters for the concentration of phage. They found that MS2, T2 and T4
were all efficiently adsorbed when suspended in tapwater, sewage and
lake water. Again, xl74 was found to adsorb less efficiently than the
other phage tested. Different eluting solutions for viruses adsorbed to
positively charged filters were tested and it was found that 4% beef
extract plus 0.5 M NaCl gave the best recoveries. To determine the
efficiency of this method, 500 ml of sewage water was passed through a
90 mm diameter Zeta plus 50S filter. Adsorbed indigenous bacteriophages
were eluted with 40-50 ml of the beef extract-NaCl solution with an
average efficiency of recovery of 56.5%.
The search group headed by Seeley and Primrose developed (148) and
modified (136) a portable concentrator using positively charged filters.
In this system, water is passed through a series of three prefilters,
precoated with 0.1% Tween 80 to prevent virus adsorption. The clarified
water was adjusted to pH 6.0 and pumped through a 500 mm diameter Zeta
plus filter. Adsorbed virus eluted with a solution of 1.0% beef extract
in 50 mM arginine. Recovery of naturally occurring bacteriophage in
pond water was greater than 60% (136).

16
The use of positively charged membrane filters for concentrating
viruses from very large volumes of water was hindered due to the lack of
availability of these filters in cartridge form. Previous work with
negatively charged filters had shown that these pleated cartridge
filters were capable of processing several thousand liters of tap water
without clogging (44, 46), while flat disc filters tended to clog
rapidly (44, 165). Sobsey and Glass (163) reported the use of a new
positively charged filter, Virosorb 1MDS, composed of fiberglass-
cellulose-"surface-modified resin" that was available as a double layer,
pleated-sheet cartridge. They compared these filters to the
electronegative Filterite pleated cartridge filters for the recovery of
poliovirus-1 seeded into 1000 1 of tapwater. Using beef extract as the
eluate and an organic flocculation procedure (92) for reconcentration,
they found virtually no difference in virus recovery between the
Filterite and Virosorb 1 MDS filter (33% and 30% respectively). However,
the Virosorb 1MDS filters were much easier for field use as no
conditioning of the water prior to adsorption was necessary.
Currently, both positively charged and negatively charged filters
are being used for the processing of large volumes of natural water
samples to detect low levels of viruses. Zeta plus 30S filters were
used to detect indigenous enteroviruses in activated sludge effluents
(23). Virus types isolated included poliovirus-1,-2,-3 and
coxsackievirus B3. During an outbreak of gastroenteritis and hepatitis
in Texas (76), concentration of water supplies by both Filterite and
Virosorb 1MDS filters resulted in the detection of several enteroviruses
such as coxsackieviruses B2 and B3, and Hepatitis A virus.

17
Factors Influencing Viral Adsorption to Filters
Several factors influence the adsorption of viruses to membrane
filters. These factors include the nature of the virus, the composition
of the filter, and the nature of the solution, including pH, the
concentration and type of salt in solution, the presence of organic
compounds such as proteins and humic acids, and the flow rate through
the filter.
Adsorption of viruses to membrane filters is greatly dependent on
the virus being studied (53). Viruses have protein coats composed of
many amino acids. These amino acids may be acidic, basic or
hydrophobic. The ionization of these amino acids is determined by the
pH values of the viral suspension. At the virus isoelectric point,
there is a net charge of zero. While determination of an isoelectric
point provides some insight to the charged nature of a virus, there are
many problems in drawing any conclusions based on this data alone.
First, studies have shown that the isoelectric point is not only type
dependent, but strain dependent (117, 189). In addition, isoelectric
point data tell us nothing about the charge density of the virus (53).
Little work has been done to elucidate data concerning the charge
density of viruses.
In addition to electrostatic forces effecting the adsorption of
viruses to filters, the hydrophobic nature of the virus may also play a
role. An examination of the amino acid sequence of the coat protein of
MS2 phage has indicated that most spans of amino acids along the
sequence are hydrophobic in nature (188). The relevance of this data

18
has yet to be determined. It is possible that an understanding of the
relative degree of hydrophobicity of viruses may be useful in predicting
the adsorption behavior of different viruses to membrane filters.
The type and composition of a filter can influence the adsorption
of viruses to the surface. Early work by several investigators (25, 26,
180, 181) found that viruses adsorb efficiently to filters composed of
cellulose acetate, cellulose nitrate and cellulose triacetate. Oliver
(26) found that filters composed of nylon did not adsorb virus as
efficiently as others. This was explained as being due to the greater
hydrophilic nature of nylon compared to the other filters examined
(121). Mix examined various filters used in virus concentration (121)
and concluded that the relative contributions of ionic and hydrophobic
interactions by the filter determined the extent of virus adsorption.
Kessick and Wagner (94) evaluated the electrophoretic mobilities of
virus adsorbing filters as a function of pH, ionic strength, and salt
type. The filters studies were Millipore (cellulose nitrate), Filflo
W10A-7 (cellulose acetate) and Filterite (epoxy-fiberglass).
Electrophoretic mobility measurements through a pH range of 2.0 to 7.0
indicated that all three filters were negatively charged in this pH
range. They also found that increased concentration and valency of
salts worked to decrease the net negative charge of the filters tested.
This provided an explanation of why salt addition helped to promote
virus adsorption to membrane filters (178, 181).
Sobsey and Jones evaluated some positively charged filters using
electrophoretic mobility (164). They found that the electrophoretic
mobility of Zeta plus filters (composed of cellulose-diatomaceous earth-
"charge-modified" resin) and Seitz S (composed of cellulose-asbestos)

19
filters become more electropositive with a decrease in pH. The Zeta
plus filters had an isoelectric point between 5 and 6, while the Seitz S
filter was found to have an isoelectric point near 7.
Methods such as electrophoretic mobility measurements have limited
application. These measurements are done on very small particles of the
filter material obtained by shredding of the filter material (94). As
such, these measurements may not be indicative of conditions existing on
the intact filter surface. Indeed, Sobsey and Jones (164) data on the
isoelectric point of Seitz S filters was found to be considerably lower
than expected. They indicated that in the shearing process, fibers from
the inner layers of the filter become exposed, possibly lowering the
isoelectric point of the filter.
Despite the known effect of detergents (83, 179) and proteins (25,
37, 94) on viral adsorption to membrane filters, little or no work has
been done to measure the hydrophobic nature of these virus adsorbing
filters.
As discussed earlier, the pH of a solution can have a tremendous
effect on the adsorption of viruses to filters. At low pH values,
viruses are often below their isoelectric point and are thus positively
charged, and electronegative filters possess a net negative charge. The
resultant electrostatic attraction is likely a factor in promoting virus
adsorption (94, 117). The converse is true at high pH values, where
viruses are above their isoelectric point and are negatively charged.
Adsorption to electropositive filters is promoted by this electrostatic
attractive force (94).
Studies of virus adsorption to membrane filters have shown that
adsorption is enhanced in the presence of cations (181, 183). Moreover,

20
trivalent cations, such as aluminum, have been found to be as effective
in this promotion as divalent ions, such as magnesium, at a lower
concentration (178). Three possible mechanisms for the observed
enhancement of virus adsorption in the presence of cations have been
suggested. It has been proposed that these cations act as bridges
between the negative charges on the viruses and the filters (94). A
second suggestion is that the cations are adsorbed by the filters and
reverse the net charge of the filter (117). Electrophoretic mobility
measurements in the presence of cations (94, 163) tend to support this
theory. Another possibility is that the addition of cations (and
therefore an increase in ionic strength) reduces the diffuse layer of
ions surrounding the virus and the filter. This permits short-range
attractive forces to overcome the electrostatic barriers and permits
adsorption of the viruses (173). All of these proposals indicate that
salts influence electrostatic interactions between viruses and membrane
filters.
Recent work by Farrah and his research group (40, 49, 156) has
indicated that salts may also effect adsorption of viruses by
influencing hydrophobic interactions between filters and viruses.
Chaotropic salts are able to increase the solubility of certain proteins
(73). These chaotropic ions are relatively large, singly charged ions
such as trichloroacetate (TCA), thiocyanate, and iodide (74). It has
been suggested that hydrophobic interactions are a result of the
unfavorable interactions of apolar groups with water, and accordingly,
chaotropic ions have been viewed as decreasing the structure of water
and therefore making aqueous solutions more lipophilic. In contrast,
antichaotropic ions are generally small, singly charged ions such as

21
floride, or multivalent ions such as citrate, calcium or magnesium ions.
These antichaotropic ions have been found to promote hydrophobic
interactions, presumably by increasing water structure (73, 74). Farrah
et al. (49) found that at high pH, solutions of chaotropic salts, such
as TCA, eluted over 95% of poliovirus-1 adsorbed to Millipore filters,
while an antichaotropic salt, sodium phosphate, eluted little adsorbed
virus. The authors hypothesized that the ability of chaotropic salts to
elute adsorbed virus indicated that hydrophobic interactions were a
major factor in maintaining virus associations to filters at high pH.
In a further study (40), antichaotropic salts, such as magnesium
sulfate, efficiently promoted adsorption of MS2 to Zeta plus filters at
pH 6.0, while TCA effectively blocked adsorption, again indicating that
hydrophobic interactions were involved. The use of agents involved in
promoting or weakening hydrophobic interactions has yet to be
incorporated in a concentration method for the recovery of viruses from
water samples.
The presence of organic materials, such as humic acids, serum, and
other proteins can effectively reduce virus adsorption to filters. In
early work, Ellford (37) and Cliver (25) both prevented virus adsorption
to filters by pretreating the filters with protein solutions. Wallis
and Melnick found that materials in crude virus harvest fluids (MCC)
interfered with viral adsorption to Millipore filters (181).
Later, the research group headed by Melnick (83, 178, 179) observed
that organics in water samples were adsorbed to membrane filters and
influenced viral adsorption. Elution of viruses from these filters also
resulted in elution of these organic impurities, which interfered with
readsorption of viruses to a smaller filter (44, 45). These impurities,

22
humic acids and other organic compounds, were characterized in a
subsequent publication (46). They found that these organics adsorbed at
low pH, were eluted at high pH, formed floes at low pH and could be
removed by anion-exchange resins.
Recently, humic and fulvic acids were tested for their ability to
interfere with adsorption to some positively charged filters (69). It
was discovered that fulvic acid had little or no effect on the
adsorption of poliovirus-1 to Zeta plus or Seitz S filters. Humic acid,
while interfering with viral adsorption to these filters to some extent,
still allowed up to 40% of the virus to be recovered.
Organic compounds are believed to interfere with viral adsorption
to membrane filters by competing for membrane adsorption sites (53, 64).
Since organics can compete with viruses for adsorption sites, they have
been used to elute viruses from filter surfaces. Some such solutions
used for viral elution include beef extract (64, 107, 136, 138, 183),
yeast extract (97), tryptose phosphate broth (70, 159), casein (16, 42),
Tween 80 (154, 156), and fetal bovine serum (138, 180, 181).
The final factor that can effect virus adsorption to membrane
filters is the flow rate of water through the adsorbing filters, Scutt
(147) examined the adsorption of poliovirus-1 and reovirus-1 to
Millipore filters at different flow rates. While reovirus-1 was
adsorbed at all flow rates tested, breakthrough was observed with
poliovirus-1 as the flow rate was increased. These results indicate
that a minimum contact time is required for an electrostatic bond to
form between the virus and the adsorption site (64, 147).
The flow rate at which virus adsorption becomes diminished depends
on factors influencing the strength of the electrostatic forces between

23
viruses and filters. During the development of the Wallis-Melnick virus
concentrator, it was discovered that the flow rate of sewage through
glass filters effected virus adsorption (83). At low flow rates (1.9
1/min), 96% of virus as adsorbed compared to only 68% adsorption when
the flow rate was increased to 9.5 1/min.
Reconcentration Procedures
In concentration procedures, as the volume of water processed is
increased, larger filters, and subsequently larger primary eluates, are
used. As such, a second, or reconcentration step is often necessary.
This second step may lower virus recovery, but it reduces the amount of
eluate to a manageable volume for subsequent assays (64). Some
procedures used to reconcentrate viruses after initial adsorption to and
elution from membrane filters include: hydroextraction,
ultrafiltration, ultracentrifugation, readsorption to and subsequent
elution from smaller filters of the same type, and inorganic and organic
flocculation.
One means of reducing the volume of the initial eluate and thus
reconcentrate viruses is hydroextraction. The principle of
hydroextraction is simple. An aqueous solution containing virus is
placed in a semipermeable membrane, often dialysis tubing, and the
tubing is placed into a hygroscopic material overnight. Water passes
through the membranes while large macromolecules, such as viruses are
retained and concentrated (43). Farrah et al. (45) used hydroextraction
with polyethylene glycol as a reconcentration step in the recovery of
enteroviruses from estuarine water. Using this procedure, 80% of seeded

24
poliovirus-1 was recovered. Others (149) found that hydroextraction of
been extract solutions containing coliphages gave very low recoveries
(less than 5%) and were very difficult to handle due to the high
viscosity of the concentrated material.
Ultrafiltration works on a principle similar to hydroextraction.
Solutions are passed through membranes designed to permit the passage of
water and low molecular weight substances, while larger macromolecules,
such as viruses are retained and concentrated in liquid left behind
(64). Logan et al. (113) used ultrafiltration as a reconcentration step
in the recovery of bacteriophages from large volumes of river water,
with final recoveries in the range of 50-60%. Reconcentration of
enteroviruses using ultrafiltration has been successful in their
recovery from samples of estuarine water (45).
Ultracentrifugation (180) has been used with limited success in the
concentration of viruses. One major problem with this method is the use
of expensive and non-portable equipment, thus its use in field studies
is impractical.
The early models of the Wallis-Melnick virus concentrator used
readsorption to and subsequent elution from a smaller diameter filter
similar to the one used in the first adsorption step of the procedure
(179). This readsorption was facilitated by dropping the pH of the
glycine eluate from 11.5 to 3.5 (178). Further modification of the
model included the addition of AlCl^ to 0.5 mM final concentration to the
eluate before readsorption to the second smaller filter (165) . This
reconcentration procedure was efficient for smaller volumes of water.
However, difficulties were encountered in applying these procedures to
the detection of viruses in estuarine water (162) or in larger volumes
or tap water (44). Both organic compounds and viruses were adsorbed to

25
and eluted from the filters in the first stage. These organic compounds
and other impurities formed floes at the low pH values that were
required for the adsorption of viruses to negatively charged filters
(44, 46, 162). These floes clogged the filters and made a second stage
reconcentration with negatively charged filters impractical. Some of
these difficulties were overcome by the development of an inorganic
flocculation procedure (44) for a reconcentration step. Previous work
(182) had indicated that viruses readily adsorbed to floes of aluminum
or calcium salts. Farrah and coworkers used this as the basis for
development of an inorganic flocculation procedure using AlCl^ (44).
Initial eluates were conditioned by the addition of AlCl^ to 3 mM. This
led to a drop in pH to about 4 or 5. Eluates were then adjusted to pH
7.5, resulting in the formation of floes, which were collected by
centrifugation and resuspended in glycine. Virus recoveries averaged
40-50%.
Payment et al. (133) used FeCl^ at 3 mM concentration to form an
inorganic floe during the recovery of virus from estuarine water
samples. They recovered 53% overall of poliovirus-1 added to the water
samples. The authors noted that the efficiency of the reconcentration
step was nearly 100%, but it was found to be impractical for eluates
with a high concentration of organics because of the formation of a very
large floe (133) .
Protein solutions, such as beef extract, had been shown to be very
effective in the elution of viruses adsorbed to membrane filters (64,
107, 136, 138, 183), but this did not allow for reconcentration of the
eluate onto smaller filters. In 1976, Katzenelson and coworkers (92)
developed an organic concentration procedure for beef extract that

26
involved flocculating the beef extract by lowering the pH of the
solution to 3.5 (the isoelectric point of beef extract). Viruses in
solution adsorbed to these floes, which were recovered by
centrifugation. Viruses were recovered by solubilizing the floes in
small volumes of phosphate solution. Results in their laboratory
indicated that this method, used as a second step in conjunction with a
primary adsorption/elution step yielded a mean enterovirus recovery of
about 75%. Organic flocculation using non-fat dry milk and casein (16)
has also yielded efficient (over 70%) recoveries.
The organic flocculation step developed by Katzenelson (92) has
seen much use in the concentration and detection of enteroviruses from
natural samples (31, 69, 71, 76, 95). However, recent studies in
different laboratories (5, 84, 125, 132) have indicated that the source
and lot number of beef extract can effect its ability to flocculate,
thus lowering considerably the efficiency of this step. Payment et al.
(132) found that the addition of 2.5 mM ferric chloride to beef extract
solution which failed to flocculate at pH 3.5 cause the formation of
sufficient floe for efficient virus concentration (over 75% recovery).
In addition to flocculation variabilities, workers (87, 138) have
noted that beef extract concentrates have been shown to be toxic to cell
cultures. Despite these problems, organic flocculation continues to be
the main means of reconcentration of viruses in proteinaceous solutions.
Materials and Methods
A list of chemicals and their sources, and routine methods used in
animal and bacterial virus preparation and assay is presented in
appendix A. A complete list of media and solutions used in cell culture
work is presented in appendix B.

27
Filters
The filters used in this study, their characterization and sources,
are shown in Table 2.
Water Samples
The natural water samples used for indigenous phage concentration
and their concentration are described in Table 3.
Enterovirus Concentration from Tapwater
Tapwater (114 1) was dechlorinated by the addition of sodium
thiosulfate; absence of residual chlorine was ascertained by the
orthotolidine method (2). Following dechlorination, tapwater was
adjusted to a pH between 3.3 and 3.5 by the addition glacial of acetic
acid. Ten milliliters of the tapwater was removed and seeded with a
4 7
known amount of one of the enteroviruses tested. From 10 to 10
plaque-forming units (PFU) of virus were added in each trial. When low
numbers of viruses were added, the eluate from the Seitz filter was
assayed directly after adjustment of pH 7 to 7.5. When higher numbers
were added, viruses were assayed after dilution in phosphate-buffered
saline (PBS) with 2% fetal calf serum (FCS) (See appendix B). A 5-ml
portion of the viral suspension was added to the 114 liters and the
remaining 5 ml was assayed. The seeded tapwater was passed through a
10-in (ca. 25.4-cm), 0.2 ym Filterite filter. The adsorbed viruses were
eluted with 1 liter of 0.2 M trichloroacetic acid (TCA)-0.2 M lysine
that had been adjusted to pH 9 with sodium hydroxide. This eluate was
passed through a 47-mm Seitz S filter under positive pressure without pH
adjustment. Viruses adsorbed to the Seitz filter were recovered by one

TABLE 2. Filter characterization
FILTER
PORE SIZE
CONTACT
ANGLE3
MAJOR COMPONENTS
SOURCE
ELECTROPOSITIVE
Virosorb 1MDS
0.45y
151°
Fiberglass/cellulose/
"surface-modified resin"
AMF Cuno, Inc.
Meriden, CN
Posidyne N66
0.45y
128°
nvlnn 66 (polyamide)
Pall Trinity Micro
Corp.,
Cortland, NY
Zeta plus C-30
0.6 - 2.0u
1 *>R°
cellulose/diatomaceous
earth/"charge-modified
resin"
AMF Cuno, Inc.
Meriden, CN
Seitz S
0.5p
113°
asbestos-cellulose
Republic Filters,
Milldale, CN
ELECTRONEGATIVE
Filterite
0.2y
NDb
epoxy-fiberglass
Filterite Corp.,
Timónium, MD
Millipore HA
0.4 5pi
NDb
nitrocellulose
Millipore Corp.,
Bedford, MS
a - Contact angle of carbon tetrachloride on water-saturated filters,
b - ND: Not Determined.

TABLE 3.
Characterization of natural water samples
a
WATER SAMPLE
pH
TURBIDITY (NTUs)
CONDUCTIVITY (viMHOs)
ORGANICS
Cypress strand
6.9 -
7.2
46 - 61
350 - 500
0.48
-0.58
Secondary effluent
6.3 -
7.4
2.8 - 3.1
350 - 460
0.09
- 0.16
Holding pond
7.0 -
7.5
52 - 60
600 - 720
0.46
- 0.51
Land runoff
7.6 -
7.9
2.4 - 3.7
420 - 630
0.37
- 0.41
a - Range of each measurement is indicated.
b - Absorption at 254 nm, UV light (33).
N>

30
of two methods. In the first procedure, the filter was removed from the
holder and placed in a holder used for vacuum filtration (Gelman
Sciences, Inc., Ann Arbor, MI). Two 8 ml portions of 3% casitone were
pulled through the filter with a vacuum. After the first 2 to 3 ml of
each portion had passed through the filter, the vacuum was removed and
the solution was allowed to soak the filter for 5 minutes. After the
soaking period, the remaining part was pulled through the filter. The
two portions were pooled, neutralized and assayed. In the second
procedure, two 8 ml portions of FCS (pH 9) were pulled through the
filter with a 50 ml syringe. The two portions were pooled, neutralized
and assayed.
Adsorption/Elution of T7 using Electropositive Filters
Tapwater was dechlorinated as described above. After approximately
7
10 PFU of T7 phage were added, 10 ml of the tapwater was passed through
the filters, which were held in 25 mm holders, at a rate of 1 ml/s.
Viruses in the initial tapwater solution and in the filter effluent were
assayed to confirm viral retention by the filters. Next, 10 ml of a
test eluent (see Tables) was passed through the filters the eluted
viruses were assayed. Finally, 10 ml of 3% beef extract was passed
through the filter to elute residual virus. The data obtained from beef
extract controls indicated that phage was not being inactivated or
aggregated by test solutions (data not shown). The virus eluted was
expressed as a percentage of the virus present in the initial tapwater
solution. Values were obtained in 2-3 trials and represent the means
and standard deviations of 4 to 6 determinations.

31
Contact Angle Measurements
A cubic cell (3 cm x 3 cm x 3 cm) was filled with distilled water
and a filter was placed in the cell and allowed to stand for 1 hr in
order to be saturated with water. A small drop of carbon tetrachloride
was injected in the water just above the filter surface using a 50 yl
syringe (Hamilton Co., Reno, NV). The contact angle between the filter
and droplet was measured using a NRL Gonimeter model A-100, Rame-Hart,
Inc. (129).
Capillary Rise Measurements
Filters were cut in strips measuring 20 cm long and one cm wide.
These strips were then dipped in distilled water or other liquid one cm
deep. The time at which the strip was dipped in the liquid was taken as
the zero reference time. The rise of liquid on the filter was measured
at one minute intervals for 10 to 12 minutes. The height of the liquid
on the filter (or difference of liquid and water) versus time was
plotted using the mean values of at least three trials (77).
Concentration of Indigenous Bacteriophage using Filters
Samples of water (see Table 3) were collected in pressure vessels
and brought back to the laboratory for immediate processing. A five ml
aliquot was removed to determine the input concentration of
bacteriophage. For the one-step procedure, samples were passed through
an AP20 prefilter (Millipore Corp., Bedford, MS) and 3 layers of
Virosorb 1MDS filters without pH adjustment. The filtrates were
collected and assayed to confirm bacteriophage adsorption. Next, 1.0 M
NaCl in 0.05 M imidazole buffered at pH 7 was used to elute the

32
adsorbed phage. The eluate volume was 5 ml when filters were held in 25
mm holders, 10 ml for 47 mm filters and 40 ml for 90 mm filters. The
two-step concentration procedure started as the one-step procedure
described above, with 90 mm filters used. The 40 ml sodium chloride
eluate was pulled through a 47 mm Seitz S filter using vacuum filtration
without pH adjustment and collected to check for phage adsorption.
Next, 10 ml of a 4% beef extract and 0.1% Tween 80 solution at pH 7 was
placed on the filter and 2 to 3 ml was pulled through using a vacuum to
displace residual fluid and ensure that the beef extract/detergent
eluate completely soaked the filter. The remaining 7 to 8 ml of the
eluate remained on the filter, soaking for 5 minutes, then was pulled
through and assayed for phage elution.
Flocculation of Seeded Beef Extract
Forty ml of 10% beef extract was seeded with 107 PFU of either
bacteriophage or animal virus. One half of this beef extract solution
was flocculated by lowering the pH to 3.5. The floe was collected by
centrifugation at 3000 x g for 10 minutes, and resuspended in two ml of
0.15 M sodium phosphate solution, pH 9, according to the method
described by Katzenelson et al. (92). The other half was mixed with 2
volumes of a saturated ammonium sulfate solution. Floe immediately
formed when these solutions were mixed, and the pH remained buffered
between 7 and 7.5. The beef extract/ammonium sulfate was centrifuged at
14,500 x g for 20 minutes at 5 - 10°C and the floe was solubilized in 2
ml sterile distilled water.

33
Concentration of Phage and Enterovirus from Sewage Effluents
Unchlorinated secondary effluent from the University of Florida
treatment plant was collected in a sterile container and brought back to
the laboratory for immediate processing. One liter was seeded with 10^
PFU enterovirus (see Table 9) and 5 ml was removed to assay for initial
input of enterovirus and indigenous bacteriophage. E. coli C (ATCC
13706) was used as host bacterium for indigenous bacteriophage assay
(75). The one liter sample was passed through three layers of Virosorb
1MDS filters with an AP20 prefilter encased in a 47 mm filter housing
(Millipore) without pH adjustment. Twenty ml of 10% beef extract, pH 9,
was used to elute adsorbed virus. The beef extract was adjusted to pH
by the addition of 1 N HC1, and divided into two equal portions. One
half of the eluate was reconcentrated using organic flocculation at pH
3.5, while the other half was reconcentrated by ammonium sulfate
flocculation, as described above. The final volume with both procedures
was 2 ml. The procedure was the same when other protein solutions (10%
tryptose phosphate broth and 10% brain heart infusion) were used in the
elution step, except that only one volume of saturated ammonium sulfate
was needed for flocculation.
Recovery of Low Levels of Indigenous Bacteriophage
Samples of water were passed through Seitz S filter with an AP20
prefilter, held in a 90 mm stainless steel holder. Direct assay of
these water samples indicated that no bacteriophage was present. Next,
10% beef extract, pH 9, was used to elute any phage that may have been
adsorbed. Beef extract was dropped to pH 7 and flocculated with
saturated ammonium sulfate as described above.

34
Results
Concentration of Enteroviruses from Large Volumes of Tapwater
We developed a modified method to concentrate enteroviruses from
large volumes of tapwater (Table 4). We used acetic acid to adjust the
pH of the tapwater to 3.5, in lieu of HC1 (44) to aid in virus
adsorption to Filterite filters. A chaotropic salt, trichloroacetic
acid was used to elute viruses from the filter. Virus was efficiently
eluted, and readsorption of the virus to a second dissimilar filter
(Seitz S) was accomplished without any modification of this eluting
solution. Viruses adsorbed to the Seitz filters were eluted with pH 9
solutions of either 3% casitone or fetal calf serum (FCS). Casitone is
less expensive than FCS, but a soaking period was required when casitone
was used as the eluate. The mean recovery of enteroviruses with
casitone was 54%, and that with FCS was 43%.
Differential Elution of Phage from Positively Charged Filters
Initial studies were conducted to determine the adsorption of
bacteriophage T7 to four positively charged membrane filters: Virosorb
1MDS, Zeta plus C-30, Posidyne N66, and Seitz S. In these experiments,
tapwater was dechlorinated and adjusted to three different pH values
(4, 7 and 9), and in all cases, adsorption was nearly complete (>99%
adsorbed, data not shown). Based on these results, dechlorinated
tapwater was used as the adsorbing solution in subsequent studies with
T7 and electropositive filters.
The results of preliminary studies on the ability of salts and
detergents to elute T7 phage adsorbed to these filters are shown in
Table 5. A solution of the buffer alone (50 mM imidazole) eluted less
than 10% of the adsorbed phage from all of the filters tested. A

35
TABLE 4. Concentration of viruses from tapwater.
Virus
Number of
trials
Percent of added
virus recovered Eluent
mean
SDb
Poliovirus-1
3
67 ±
8
Coxsackievirus B3
3
+1
KD
18
Coxsackievirus B4
2
43 ±
20 3% casitone, pH 9
Echovirus-1
2
63 ±
12
Total
10
54 ±
18
Poliovirus-1
4
35 ±
7
Coxsackievirus B3
2
+1
o
7
Coxsackievirus B5
2
+1
4 Fetal calf serum, pH '
Echovirus-1
2
+i
00
in
9
Total
10
43 ±
11
a - Tap water was dechlorinated, adjusted to pH 3.3 to 3.5 by the
addition of acetic acid, and seeded with the indicated virus.
The sample was passed through a 10", 0.25-ym porosity Filterite
filter at 3 to 5 gal/min. Next, 1,000 ml of 0.2 M NaTCA + 0.2 M
lysine, pH 9, was passed through the filter. This solution was
then passed through a 47-mm Seitz S filter. Virus adsorbed to the
Seitz filter was recovered by treating the filters with two 8-ml
volumes of 3% casitone or FCS, pH 9, as described in the text.
b - SD: Standard
deviation.

a
TABLE 5. Elution of T7 adsorbed to positively charged membrane filters by solutions at pH 7.
% of adsorbed
virus eluted by:
FILTER
BUFFER ALONE
0.1% TWEEN 80
0.5 M EDTA
0.1% TWEEN 80
+0.5 M EDTA
Virosorb 1MDS
7 ± 6
8 ± 8
74 ± 7
105
+
20
Zeta plus C-30
0
49 ± 9
0
72
+
2
Posidyne N66
8 ± 3
37 + 9
5 ± 1
78
+
14
Seitz S
0
0
0
22
±
8
a - All solutions contained 0.05 M imidazole and were adjusted to pH 7.0.
LO

37
solution of detergent (Tween 80) at pH 7 did not elute any T7 adsorbed
to the Seitz filters and less than 10% of the phage adsorbed to the
Virosorb 1MDS filters. This detergent solution eluted greater then 35%
of the T7 adsorbed to the Zeta plus C-30 or Posidyne N66 filters. A
solution of EDTA eluted 74% of the virus adsorbed to the Virosorb IMDS
filters but less than 5% of the virus adsorbed to the other filters. It
had previously been shown that solutions containing salts and detergents
were very efficient at eluting viruses adsorbed to electronegative
filters (156). When such a solution was used with the positively
charged filters, greater than 70% of the T7 adsorbed to the Zeta plus
C-30, Posidyne N66 and Virosorb IMDS filters was eluted. However, only
22% of the T7 adsorbed to the Seitz S filters was eluted.
Preliminary results indicated that the ionic strength of the
eluting solution affected the amount of virus eluted from these filters.
Figure 1 shows the influence of ionic strength in solutions of detergent
at pH 7 on the elution of T7 adsorbed to membrane filters. In the
presence of detergent, solutions with ionic strengths of 0.4 or above
efficiently eluted the bacteriophage adsorbed to the Zeta plus C-30 and
the Posidyne N66 filters, while solutions with ionic strengths below 0.4
eluted the virus adsorbed to the Virosorb IMDS filters. Again little or
no virus adsorbed to the Seitz S filters was eluted.
The effect of detergent concentration in the presence of 1 M NaCl
on viral elution was determined (Figure 2). Again, a solution of buffer
alone at pH 7 eluted less than 10% of the T7 adsorbed to all filters
tested. A solution of 1 M NaCl at pH 7 did not elute any virus adsorbed
to either the Zeta plus C-30 or Seitz S filters, while less than 20% of
the adsorbed T7 was eluted from the Posidyne N66 filter. However,

FIGURE 1. Elution of bacteriophage T7 adsorbed to various positively
charged filters by solutions of 0.1 % Tween 80 and NaCl or
Na S0„ at pH7.
2 4

%VIRUS ELUTED
39
IONIC STRENGTH

FIGURE 2. Elution of bacteriophage T7 adsorbed to various positively charged filters by
solutions of NaCl and Tween 80 at pH 7. V = Virosorb 1MDS, Z = Zeta plus C-30,
P = Posidyne N66, S = Seitz S.

80
Q
Lü
H
jj 60
LU
CO
g 40-
>
^ 20»
V
0
Buffer
1.0 M NaCI
P
0.001 % Tween 80 0.1% Tween 80

42
greater than 75% of the T7 adsorbed to the Virosorb 1MDS was eluted
using the salt alone. Tween 80 concentrations of 0.001% in the presence
of NaCl greatly increased the amount of phage eluted from the Posidyne
N66 filters, yet had no effect on the elution of phage adsorbed to Zeta
plus C-30 or Seitz S filters. A solution of 0.1% Tween 80 in presence
of salt eluted greated than 75% of the T7 adsorbed to the Virosorb 1MDS,
Posidyne N66 and Zeta plus C-30 filters, yet had no effect on elution of
T7 adsorbed to the Seitz S filters. Tween 80 was not soluble in the
salt solution in higher concentrations.
Indigenous bacteriophage from several water sources (Table 3) were
concentrated using a one step adsorption/elution procedure using
Virosorb 1MDS filters as the adsorbent and 1 M NaCl as the eluting
solution. Table 6 shows the percent recovery of indigenous
bacteriophages using filters of various sizes and input volumes of
water. In general, recovery of virus was better when the smaller filter
holders were used. In addition, there was no significant difference in
viral recovery using E. coli B or E. coli C-3000 as the host bacteria.
The average recover of the 41 samples from 4 different sources was 55%
using E. coli B as the host and 61% using E. coli C-3000 as the host.
In samples with relatively high numbers of phages, a one-stage
concentration procedure may be sufficient to detect viruses. However,
where low levels are found, a two-stage procedure may be required to
provide a sufficient concentration factor to detect indigenous phages.
The results of this study have shown that some solutions that permit
virus adsorption to one filter are capable of eluting virus adsorbed to
another filter. This raises the possibility that a simple two-step
virus concentration procedure can be developed in which virus adsorbed
to one filter can be eluted using a solution permits virus adsorption to

TABLE 6. One-step concentration procedure for the recovery of indigenous bacteriophage from
natural water samples.
% recovery
E. coli B
E. coli
C-3000
WATER SOURCE
FILTER SIZE (d)3
VOLUME FILTERED
mean
SD"
mean
SD
REPLICATIONS
Cypress strand
25
50
_
100
133
23
105
19
4
47
100
-
500
84
38
66
12
6
90
500
-
1500
63
33
53
16
4
Secondary effluent
25
250
-
400
47
22
60
11
3
47
500
-
1000
26
1
58
10
4
90
2000
-
7000
26
5
68
23
3
Holding pond
25
50
-
75
40
3
89
16
3
47
375
475
32
7
61
30
4
Land runoff
25
50
-
100
82
19
59
9
3
47
300
-
700
30
7
34
16
5
90
2000
13
4
19
1
2
TOTALS
55
40
61
27
41
a - diameter in mm.
b - in milliliters.
c - SD: standard deviation

44
a second smaller filter. The elution-adsorption step can be
accomplished with little or no modification of the solution. Based on
the results of this study, several two step procedures using two
different filters in series are possible. One such procedure is
outlined in Figure 3. We have used this procedure to concentrate phage
from several secondary effluent samples (Table 7). Using this
procedure, the average recovery of bacteriophages was 26% using E. coli
B as the host, and 63% E. coli C-3000 as the host. In two samples,
bacteriophage was detected in the final concentrates when none detected
in the initial sample (data no shown).
Characterization of Virus-Adsorbing Filters
In order to determine the relative hydrophobic-hydrophilic nature
of filters used for virus adsorption, the contact angle of carbon
tetrachloride on membrane filters submerged under water was measured
(Table 2). The contact angle of carbon tetrachloride on the Seitz S
filter was the smallest (113°), the contact angle on the Virosorb 1MDS
was the largest (151°), while the contact angles on the Zeta plus C-30
(128°) and Posidyne N66 (128°) were intermediate. Under the conditions
used to measure the contact angle, carbon tetrachloride was spread
immediately on the two negatively charged filters tested (Millipore HA
and Filterite), therefore, measurement was not possible.
The filters were further characterized using the capillary rise
method. Figure 4 shows the rise of deionized water on positively
charged membrane filters. The most rapid raise of water was observed in
the Virosorb 1MDS filters, while the smallest rate of water rise was

FIGURE 3. One possible two-step procedure for the concentration of bacteriophages from water.

STEP I
STEP 2
Adsorption of phage to three layers of
Virosorb IMDS filters
I
Elution of adsorbed phage using I M NaCI, pH 7
I
Adsorption of phage in I M NaCI, pH 7,
to Seitz S filters
I
Elution of phage adsorbed to Seitz S filters using
4% beef extract and O. I % Tween 80, pH 7

TABLE 7. Recovery of bacteriophages from treatment plant effluents
SOURCE, TYPE
VOLUME
Initial
PFU/1 using:
% of bacteriophages
recovered using:
OF EFFLUENT
SAMPLED (1)
E. <
coli B
E. coli
C-3000
E. coli B
E. coli C-3000
Tallahassee, FL
chlorinated effluent
66.3
6.7
x 104
4.3 x
104
43
39
U. of Florida
unchlorinated effluent
3
6.0
x 103
6.9 x
104
15
54
U. of Florida
unchlorinated effluent
4
6.7
x 104
6.9 x
104
21
59
U. of Florida
unchlorinated effluent
4
8.3
x 104
9.1 x
104
24
100
a
Total Mean ± SD
26 ± 10
63 ± 23
a - Standard deviation

FIGURE 4. The rise of water on various positively charged filters.

HEIGHT (cm)
49

50
observed for the Posidyne N66 filters. Zeta plus C-30 and Seitz S
filters were intermediate in nature. Figure 5 shows the results of the
same test on negatively charged filters. The rise of deionized water on
Filterite filters was more rapid than that of Millipore HA filters.
Figures 6 through 8 show the change in rise (solvent - water) of
the positively charged filters. In all cases, the change in rise was
the most positive for the Seitz S filters, while the difference was most
negative for the Virosorb 1MDS filters. As with contact angle
measurements, Zeta plus C-30 and Posidyne N66 filters were intermediate
in nature. The change in rise (chloroform - water) was plotted for
negatively charged filters (Figure 9). In this instance, there was
little or no change in rise for Millipore HA filters, while this change
was slightly negative for the Filterite filters.
Ammonium Sulfate Flocculation as a Reconcentration Step
The ability of certain salt solutions, such as sodium phosphate,
sodium citrate, ammonium sulfate or sodium sulfate, to precipitate
proteins has been well documented (73, 93, 146). Preliminary
experiments in our laboratory indicated that optimum precipitation
occurred when beef extract was mixed with saturated ammonium sulfate.
The efficiency of this flocculation step was compared to the organic
flocculation procedure as described by Katzenelson et al. (92) and the
results are shown in Table 8. Bacteriophage recovery using organic
flocculation was less than 3% for all three phage tested, while
virtually all was recovered using ammonium sulfate flocculation. In
addition, animal virus recovery was high (greater than 70% for all
three tested) using the ammonium sulfate method as compared to organic
flocculation, which gave the highest recovery with poliovirus-1 at 53%.

FIGURE 5. The rise of water on various negatively charged filters.

52

FIGURE 6
The change in capillary rise on various positively charged
filters: carbon tetrachloride - water.

DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
54
CARBON TETRACHLORIDE-WATER

FIGURE 7.
The change in capillary rise on various positively charged
filters: butanol - water.

I MDS
DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
Ln
O

FIGURE 8. The change in capillary rise on various positively charged
filters: chloroform - water.

I MDS
DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
CHLOROFORM-WATER

FIGURE 9. The change in capillary rise on various negatively charged
filters: chloroform - water.

DIFFERENCE IN HEIGHT OF LIQUID ON FILTER (CM)
60
CHLOROFORM-WATER

61
TABLE 8. Virus recovery from seeded 10% beef extract, pH 7, by ammonium
sulfate flocculation versus organic flocculation.
% Recovery
Ammonium Sulfate Organic
Virus Flocculation Flocculation
a
MS 2
101
+
8
1
+
0
X174
100
+
17
1
+
1
T3
107
+
8
2
+
1
Poliovirus-1
97
±
11
53
±
8
Echovirus-5
84
±
12
22
±
5
Coxsackievirus B5
72
±
8
10
±
1
a - ± standard deviation

62
It should be noted that during the course of these experiments, three
lots of beef extract were tested before one was found that flocculated
at low pH. Both in our laboratory and in others (71, 92, 132), higher
recoveries using the organic flocculation procedure have been obtained
with some lots of beef extract.
This ammonium sulfate flocculation procedure was compared with the
organic flocculation procedure for recovering viruses in natural water
samples (Table 9). The overall mean recovery of indigenous
bacteriophage using the ammonium sulfate flocculation was 85% as
compared to 12% for the organic flocculation method. It was found that
the mean recovery of seeded enteroviruses using the ammonium sulfate
flocculation method was 73% as compared to 42% for organic flocculation.
Other proteinaceous solutions were tested for enterovirus and
indigenous bacteriophage recovery using this ammonium sulfate
precipitation method, and these results are shown in Table 10. For the
recovery of indigenous bacteriophage, there was no difference for the
solutions used. Ten percent tryptose phosphate broth (TPB), 10% brain
heart infusion (BHI) and 10% beef extract all recovered 71% of the phage
when used as a primary eluting solution followed by ammonium sulfate
flocculation. For three enteric viruses tested, the mean recovery was
76% when beef extract was used as the primary eluting solution, 62% for
BHI and 73% for TPB.
Finally, this procedure was also used to recover indigenous
bacteriophage in less heavily contaminated waters (Table 11). Direct
assay of these waters indicated that no phage was present. After
reconcentration of the beef extract eluate using ammonium sulfate

63
TABLE 9. Recovery of indigenous bacteriphage and seeded enterovirus
from sewage effluent by ammonium sulfate flocculation versus
organic flocculation.
ANIMAL VIRUS
% recovery
INDIGENOUS BACTERIOPHAGE
% recovery
Trial
Virus added
ASFb
O
o
ASFb
OFC
1
Poliovirus-1
47
33
115
15
2
Poliovirus-1
80
71
86
15
3
Echovirus-5
91
39
61
11
4
Echovirus-5
73
23
76
5
Mean ±
d
SD
73±16
42±18
85±20
12±4
a. Unchlorinated effluent was seeded with 10 PFU of enterovirus
listed and assayed for initial enterovirus and indigenous
bacteriophage. One liter of sample was passed through a 47mm, 3
layers of Virosorb 1MDS filters. Adsorbed virus was eluted with
20ml 10% beef extract, pH 9. Beef extract was adjusted to pH 7 and
split into equal halves (see text).
b. ASF - ammonium sulfate flocculation. One half of beef extract
eluate was mixed with two volumes of saturated ammonium sulfate,
floe was centrifuged and resuspended in 2 ml distilled water (see
text).
c. OF - organic flocculation. One half of beef extract eluate was
adjusted to pH 3.5, floe was centrifuged and resuspended in 2 ml
0.15 M Na^HPO^ (see text).
d. SD - standard deviation.

TABLE 10.
Recovery of indigenous bacteriophage and seeded enterovirus from sewage effluent by
ammonium sulfate flocculation.
ANIMAL VIRUS
% recovery
INDIGENOUS PHAGE
% recovery
Trial
Virus added
BEb
c
BHI
d
TPB
BEb
c
BHI
d
TPB
1
Poliovirus-1
476
52
109
1156
59
77
2
Poliovirus-1
806
74
94
86e
76
71
3
Echovirus-5
9ie
69
65
6ie
77
83
4
Echovirus-5
73e
41
70
76e
103
69
5
Coxsackievirus
B5
109
73
55
42
63
65
6
Coxsackievirus
B5
55
60
44
48
49
62
mean ± SD'*'
76 ± 21
62 ± 12
73 ± 22
71 ± 25
71 ± 17
71 ± 7
a -
Unchlorinated effluent was
enterovirus and indigenous
7
seed with 10
bacteriophage
PFU of enterovirus listed
. One liter of sample was
and assayed for
passed through a
initial
47 mm,
3 layers of Virosorb 1MDS filters. Adsorbed virus was eluted with 10% solution of protein
listed. Eluate was mixed with saturated ammonium sulfate, floe was collected by
centrifugation and resuspended in distilled water as described in text.
b - BE = beef extract.
c - BHI = brain heart infusion.
d - TPB = tryptose phosphate broth.
e - Values from Table 9.
f - SD - standard deviation.

65
TABLE 11. Recovery of low levels of indigenous bacteriophage by
ammonium sulfate flocculation.
SAMPLE
INITIAL
Volume (ml) PFU/ml
BEEF EXTRACT
Volume (ml)
ELUATE
PFU/ml
FINAL CONCENTRATE
Volume (ml) PFU/ml
Chlorinated
effluent
3200 <1
42
<1
5
12.5
Lake water
2000 <1
34
1400
5
8000
Lake water
2500 <1
50
234
3
4350
a. Sample
was passed through
Seitz S filter
with AP
20 Prefilter
held
in 90 mm stainless filter holder. Next, 10% beef extract, pH 9,
was used to elute adsorbed virus. Beef extract was dropped to pH
7, and mixed with two volumes saturated ammonium sulfate to form
floe. Floe obtained by centrifugation was resuspended in sterile
distilled water (see text).

66
precipitation, phage was detected in all three samples, and there
appeared to be no probelms, such as toxicity, when the final concentrate
was plated directly.
Discussion
Concentration of Enteroviruses from Large Volumes of Tapwater
Several concentration procedures to detect viruses in tapwater have
been developed and subsequently modified. Wallis et al. (178) and
Sobsey et al. (165) described two-stage concentration procedures in
which the same type (but different sizes) of filters were used in both
stages. The use of the same filter in a reconcentration procedure
involved reconditioning of the primary eluting solution to allow for
readsorption. Problems with clogging and co-concentration of organic
compounds (44, 162) and other impurities made a second stage
concentration procedure with negatively charged filters impractical.
This problem was overcome by the use of an inorganic flocculation step
for reconcentration (44), yet viral recovery was low. Katzenelson et
al. (92) increased the amount of viral recovery by using a second-stage
organic flocculation procedure with beef extract. While this procedure
has proven to be useful, problems such as large final volumes and
variability in the flocculation of beef extract supplies led us to
investigate possible modifications of currently used methods.
Our investigations led to three modifications of existing
procedures for concentrating enteroviruses from large volumes of
tapwater. The first modification is the use of acetic acid in place of
hydrochloric acid to adjust the pH of the tap water to pH 3.5.
Adsorption of viruses to negatively charged filters such as Filterite
filters is facilitated at a low pH (165). Hydrochloric acid has been

67
used to adjust water samples to the low pH values required for viral
adsorption in many studies. We have found that acidification of
tapwater with either hydrochloric or acetic acid permits the adsorption
of greater than 99% of the enteroviruses studied to Filterite filters.
However, the acidification of water under filed conditions with Dema
injectors (134) is easier when acetic acid is used in place of HC1.
Changes in the flow rate or pressure may cause fluctuations in the
amount of acid injected when Dema or pressure injectors are used to
adjust water pH. With HC1, these changes may cause wide fluctuations in
the pH unless the injection rate is carefully controlled. We have found
that these fluctuations are minimized when 1 or 2 M acetic acid is used
for acidification. Acetic acid (pH = 4.75) will buffer more closely to
the desired pH range than will HC1, which dissociates completely.
The second modification was the use of sodium trichloroacetate
(NaTCA) as the primary eluate. The association of viruses with solids
such as membrane filters has been shown to be influenced by both
hydrophobic and electrostatic interactions (40, 49, 50, 53, 156).
Previous studies have shown that detergents, which disrupt hydrophobic
interactions, can be used to elute viruses adsorbed to membrane filters
(49, 156). In addition, recent studies have shown that certain salts
may disrupt hydrophobic interactions (49). These salts, called
chaotropic salts, are relatively large, singly charged ions such as TCA
and thiocyanate. It has been suggested that these compounds disrupt
hydrophobic interactions by decreasing the structure of water and making
aqueous solutions more lipophilic. Chaotropic agents have been used to
elute viruses adsorbed to various solids by disrupting hydrophobic
interactions between the viruses and the solids (48, 49, 177). A

68
solution of the chaotropic salt, NaTCA, eluted 77% of the enteroviruses
used in this study that were adsorbed to Filterite filters.
Reconcentration of the viruses in the primary eluate with a second,
smaller electronegative filter requires acidification of the high-pH
eluent used to recover viruses adsorbed to the primary virus-adsorbing
filter (165, 178). It was previously shown (44) that such a drop in pH
may be accompanied by the precipitation of organic compounds that may
have been concentrated along with the viruses. These organic compounds
may interfere with the reconcentration process by reducing the
adsorption of viruses to the filters or by clogging the filters (44).
Therefore, readsorption of viruses to a second filter at the same pH
that was used for the elution of viruses from the first filter was
studied.
Sobsey and Jones (164) studied the adsorption of poliovirus-1 to
positively charged filters at several pH values. They found that both
Zeta plus filters and Seitz filters efficiently adsorbed polioviruses at
a relatively high pH. The last modification of our procedure introduced
was the use of Seitz filters for the second-stage concentration. When
the NaTCA-lysine eluate from the Filterite filters was passed through
the Seitz filters, greater than 95% of the viruses were adsorbed.
Viruses adsorbed to the Seitz filters were eluted efficiently with pH 9
solutions of either 3% casitone or fetal calf serum.
These three modifications make the pH adjustment step easier, and
permit detection of viruses in water by a two-stage concentration
procedure in which different types of membrane filters are used in each
stage. This use of different types of filters allows readsorption
without any modification of the primary eluate, simplifying the overall
procedure.

69
Differential Elution of Phage from Positively Charged Filters
The use of negatively charged filters, such as the Filterite
filters used in the primary adsorption step discussed above, requires
the lowering of the pH of the water sample and/or the addition of
cations (178, 181) to facilitate viral adsorption. Since it had been
previously shown that these concentration procedures are unsuitable for
bacteriophage recovery (149, 150), investigators have explored the
possibility of the use of electropositive filters for the concentration
of bacteriophages (67, 150, 157). These filters are more positively
charged at the pH range of most natural water samples (pH range 6.5 - 8)
than are the electronegative filters, and so adsorption to the
electropositive filters can be accomplished with little or no
manipulation of the water sample.
We have examined four filters, Virosorb 1MDS, Zeta plus C-30,
Posidyne N66 and Seitz S filters. All four are more electropositive at
pH 5 - 8 than are negatively charged filters and adsorb virus in water
at these pH values. However, their composition is quite varied (Table
2). We have attempted to determine what effects these differences in
composition would have on the association of bacteriophage with the
filters.
Although all of the filters adsorbed T7 in tapwater at pH values
between 4 and 9, differences in the ability of certain solutions to
elute the adsorbed phage were noted. Previous studies have shown that
certain solutions of a neutral detergent, such as Tween 80, can disrupt
hydrophobic interactions between viruses and membrane filters (40, 50,
181). Once the hydrophobic interactions have been disrupted, the
addition of certain salts to this solution will disrupt electrostatic

70
interactions and result in the elution of adsorbed viruses (50, 156).
When hydrophobic interactions were disrupted at pH 7 by the addition of
detergent, only T7 adsorbed to Posidyne N66 and Zeta plus C-30 filters
was eluted to any appreciable extent. If salt was added to the
detergent solution, thus disrupting both hydrophobic and electrostatic
interactions, most of the adsorbed phage was eluted from all filters
tested except the Seitz S filter. These findings indicated that there
were distinct differences in the relative strength of hydrophobic and
electrostatic interactions with the phage among these four filters.
It was found that the ionic strength of the eluting solution, in
the presence of a detergent, affected the amount of adsorbed phage that
was eluted. Solutions of detergent with sodium chloride or sodium
sulfate with ionic strengths greater than 0.4 eluted most of the
adsorbed virus from all filters tested except the Seitz S. It is
assumed that increasing the ionic strength decreases the strength of the
electrostatic interactions between the virus and the filter (156).
Therefore, the filters that form relatively strong electrostatic
associations with viruses require solutions with relatively high ionic
strength to elute adsorbed viruses.
A titration of the amount of detergent necessary to elute adsorbed
T7 in the presence of 1 M sodium chloride provided the most striking
results. A solution of sodium chloride alone eluted most of the T7
adsorbed to the Virosorb 1MDS filter. Since no detergent was added, it
can be assumed that no hydrophobic interactions were disrupted. Since
salts can disrupt electrostatic interactions but promote hydrophobic
interactions (40, 49), this result can be explained by assuming that
hydrophobic interactions are not important in the association of phage

71
to the Virosorb 1MDS filter. Similarly, since a solution of a salt
alone did not elute viruses adsorbed to the other three positively
charged filters, some hydrophobic interactions are probably involved in
maintaining viral association to these filters. Since less detergent in
the presence of salt was necessary to elute phage adsorbed to the
Posidyne N66 filter than was required to elute phage adsorbed to the
Zeta plus C-30 filter, it can be assumed that hydrophobic interactions
are relatively stronger for the Zeta plus C-30 filters than for the
Posidyne N66 filters. Again, these specific solutions eluted little or
no phage adsorbed to the Seitz S filters.
Characterization of Virus-Adsorbing Filters
These results led us to characterize further these four
electropositive filters, as well as two electronegative filters often
used in virus adsorption schemes (Filterite and Millipore filters). We
used two means of characterizing the surface of these filters, which can
be done without destruction of the filter surface. One such test was
contact angle measurement. The contact angle determines the affinity of
a liquid (in this case carbon tetrachloride) for a surface submerged
under water. If carbon tetrachloride preferentially wets the submerged
filter, the resulting contact angle between the filter surface and
carbon tetrachloride droplet will be small (129). This would, in this
case, be an indication of the preferential hydrophobicity of the filter.
It was found that Virosorb 1MDS filters had the largest contact angle,
indicating that it was the least hydrophobic of the filters tested.
This was consistent with elution studies which had previously shown that
hydrophobic interactions were not important in phage association with
the Virosorb 1MDS filters. The Seitz S filters had the smallest contact

72
angle, and therefore were found to be the most hydrophobic. This may be
one reason why elution from these filters using defined solutions was so
poor. Indeed, in our laboratory and others (70, 155, 163), the eluting
solutions found to be most effective for virus adsorbed to Seitz S
filters are undefined protein solutions, such as beef extract.
Consistent with elution studies, Zeta plus C-30 and Posidyne N66 filters
were found to be intermediate in the hydrophobic nature of their
filters. Due to the nature of the experimental setup, contact angles on
the two negatively charged filters were not able to be determined.
The second method used for characterization of the filters was the
capillary rise method. In this case, the movement of a liquid through
the filters is measured (77). If the liquid is water, the rate of rise
is related to the hydrophilicity of a filter. Those filters which are
more hydrophilic will have a correspondingly higher rate of rise of
water. The results of this test were also in agreement with elution
studies. The Virosorb 1MDS filters, previously shown to be least
hydrophobic were found to be most hydrophilic, while Zeta plus C-30,
Seitz S and Posidyne N66, respectively, were found to be less
hydrophilic.
The rate of rise of some hydrophobic solvents, such as carbon
tetrachloride, chloroform, or butanol, when measured and plotted as the
difference between the solvents and water, gives another indication of
the relative hydrophobicity of these filters. These capillary rise
measurements were in complete agreement with both elution studies and
contact angle measurements. In all cases, the Virosorb 1MDS filters
were once again shown to be the least hydrophobic, while the Seitz S

73
filters were found to be the most hydrophobic of the positively charged
filters tested.
These capillary rise measurements were also done with two
negatively charged filters. Although both Millipore and Filterite
filters are electronegative, their composition is different (Table 2).
It was found that Filterite filters were more hydrophilic than the
Millipore filters, while the hydrophobic nature of both these filters
appeared to be slight, with Millipore being slightly more hydrophobic as
evidence by little of no change in capillary rise for chloroform -
water. Differences in the elution patterns of these two filters have
been documented by researchers (42). It was found that elution of
poliovirus-1 adsorbed to Filterite filters could be accomplished using
casamino acids or individual amino acids such as glycine or lysine,
while these solutions had little effect on the elution of viruses
adsorbed to Millipore filters. Since hydrolyzed proteins, such as
casamino acids have been increased number of free carboxyl and amine
groups, the ionic strength of the solution is also increased (42). It
is likely that this effect of increased ionic strength promoting elution
of adsorbed viruses from Filterite filters is related to the relative
hydrophilicity of these filters or the relatively more hydrophobic
nature of the Millipore filters.
The availability of Virosorb 1MDS filters in a pleated-sheet
cartridge allows for large volumes of water to be processed. Sobsey and
Glass (163) found that the concentration of poliovirus-1 from seeded
tapwater was equally efficient using Virosorb 1MDS filter cartridges or
the highly electronegative Filterite filters, yet the Virosorb 1MDS
filters were much simpler to use, due to little preconditioning of water

74
necessary for adsorption. The Virosorb 1MDS filters have been used to
concentrate indigenous virus from sewage, well water and chlorinated
tapwater collected during an outbreak of gastroenteritis (76). The use
of Virosorb 1MDS filters in the concentration of animal viruses led us
to develop a method for concentrating bacteriophages with them. These
filters were found to adsorb indigenous phage from the four natural
water samples tested without any modification or treatment of the
sample, and concentration of the indigenous phage was accomplished by
using sodium chloride by pH 7 for elution. The results of this study
indicated that sodium chloride, which could be used to elute virus
adsorbed to the Virosorb 1MDS filters, was ineffective for eluting
viruses adsorbed to any of the other three electropositive filters
examined. This raises the possibility of using two different filters in
series for a simple two-step concentration procedure for bacteriophages
based on differences in the elution characteristics of the filters used.
This method has the advantage of maintaining a constant pH value
throughout the concentration procedure, thus minimizing inactivation of
the bacteriophage due to pH extremes. In addition, the method is
relatively simple and requires no costly instrumentation, and,
therefore, it is suitable for field use. One possible combination of
filters was used successfully in this study to concentration indigenous
bacteriophages. It is likely that other filters and eluting solutions
may be used in improved procedures.

75
Ammonium Sulfate Flocculation as a Reconcentration Step
The development of the organic flocculation method by Katzenelson
et al. (92) greatly simplified the reconcentration of viruses in beef
extract solutions, and has been incorporated into many concentration
schemes (44, 64, 70). A major difficulty with this procedure has
developed recently, that is, the variability of different lots and
sources of beef extract with regard to flocculation capacity (5, 84,
125, 132). Another problem with organic flocculation is that the use of
low pH prohibits its use for the reconcentration of bacteriophage, which
have been shown to be sensitive to extremes in pH (149, 150). Our use
of a saturated ammonium sulfate solutions to cause flocculation
eliminates both of these problems. Ammonium sulfate flocculation of
beef extract containing bacteriophage was found to be very superior to
organic flocculation. Moreover, animal virus recovery was also improved
by ammonium sulfate floccuation.
We incorporated this ammonium sulfate flocculation step into a
procedure to recover seeded animal viruses and indigenous bacteriophage
from wastewater effluent samples. The results using this method were
consistently higher than the recoveries using organic flocculation.
The recoveries of bacteriophage were not unexpected, as the pH drop to
3.5 necessary for organic flocculation probably inactivated many
indigenous phages in the beef extract eluate.
The availability of organic flocculation as a reconcentration step
has made beef extract the eluting solution of choice in many
concentration procedures (44, 64, 70). However, other protein solutions
may work as well as, if not better than beef extract, yet
reconcentration becomes a problem (70). Ammonium sulfate flocculation

76
has been found to work efficiently with other protein solutions, such as
typtose phosphate broth and brain heart infusion.
This procedure was also used to recover very low levels of
indigenous bacteriophage. When bacteriophages are serving as models for
enteric viruses, or as indicator organisms, the use of a concentration
method suitable for both animal and bacterial viruses would simplify
assay procedures considerably. It appears that ammonium sulfate
flocculation works equally as well with animal viruses as with
bacteriophages.
Ammonium sulfate flocculation appears to be an efficient method for
reconcentration of beef extract eluates when a fluctuation in pH is
undesirable, or when a beef extract lot fails to flocculate.

CHAPTER III
VIRUS ASSOCIATION WITH OTHER SOLIDS
Review of the Literature
Enteric viruses can be excreted in concentrations as high as one
million viruses per gram of feces (52, 68, 158), and enter domestic
wastewater. Although large numbers of viruses may be present in
wastewater, conventional wastewater treatment plants have essentially
been designed to reduce the amount of organic material and suspended
material discharged to natural waters, and not to reduce pathogens. In
fact, the only step intentionally included for the reduction of
microbial pathogens is disinfection, usually via chlorination (166).
However, several of the steps in wastewater treatment facilities do
reduce the population of microorganisms in raw sewage.
Raw sewage may go through several treatment processes before
discharge from a treatment plant (Figure 10). This process usually
involves primary treatment followed by secondary treatment. In some
cases, wastewater is further conditioned by tertiary treatment.
Primary treatment is the first major process in conventional
wastewater treatment facilities. This is essentially a physical process
designed to remove heavy solids and other materials. The removal of
viruses during this stage of treatment is minimal at best (68, 166).
Effluent from primary treatment often passes on for secondary treatment.
Secondary treatment is a biological process that will metabolize and
flocculate soluble organics in the sewage. One type of secondary
treatment is the activated sludge process. In this system, air is

FIGURE 10. The processing of raw sewage (adapted from 12 and 47).

raw
i;ewaj-,o
*
VO

80
added, creating an aerobic environment to ensure adequate mixing of the
sludge. This "activated sludge" contains organisms that are thought to
be the main agents responsible for production of various polymers from
the oxidation of soluble organic matter that aid in flocculation (12).
After settling, the liquid portion is removed (possibly for further
treatment) and sludge floes are separated into two portions. One is
called "return sludge", and is used as an inoculum for primary effluent
entering the aeration tanks. The other part is called "wasted sludge",
which will undergo further treatment, then disposal. The activated
sludge treatment is very effect at removal of viruses from the liquid
portion of wastewater (68, 166).
After final treatment, both sludges (115, 170, 186) and wastewater
effluents (68, 135, 185) have been demonstrated to still contain viable
virus particles. Disposal of these "treated" liquids and solids is,
therefore, of major public health importance. Some methods used for
their disposal are shown in Table 12. As can be seen, in one form or
another, treated sewage is likely to be disposed of on land. Land
spreading of wastewater sludges and effluents has many advantages,
(Table 12) such as crop and soil renovation due to the addition of
nutrients, and water conservation and aquifer recharge. A major problem
with land disposal methods is groundwater and surface water
contamination as a result of virus transport through the soil (174).
With this in mind, it is important to look at the factors involved
in the association of viruses with solids, such as sludges, soils and
clays.

a
TABLE 12. Land disposal methods for wastewater effluents and sludges, and potential benefits and risks.
APPLICATION METHOD
DESCRIPTION/RATE
OVERALL BENEFITS
POTENTIAL VIRUS RISKS
Overland flow
Effluents flow along slope
and are collected in a
a ditch
Rate: 5-14 cm/wk
Additional wastewater
treatment
Soil renovation
Crop production
Virus contamination of food corps
Virus penetration of soil-
groundwater contamination
Surface water contamination via
runoff
Rapid infiltration
Effluent applied via
surface application
Rate: >50 cm/wk
Rapid wastewater
disposal
Recharge of groundwater
aquifer
Moderate level of
addition wastewater
treatment
Viral contamination of
groundwater
Spray irrigation
Effluent applied via
sprinkling or surface
application
Rate: 1.5-10 cm/wk
Speedy disposal of
wastewater
Recharge of groundwater
aquifer
Crop production
Additional wastewater
treatment
Aerosolization of virus
Contamination of crops
Groundwater contamination
Contamination of surface water
via runoff
Sludge surface
spreading
Sludge is spread on
surface and mixed with
soil
Rate: variable
Soil renovation
Crop production
Crop contamination
Groundwater contamination
Contamination of surface water
via runoff
Sludge sub-surface
injection
Sludge injection at 10-20
cm below soil surface
Rate: up to 3000 1/min
Rapid wastewater
disposal
Little contamination of
soil surface
Groundwater contamination
a - Adapted from 12 and 174.

82
Association of Viruses with Sludge Floes
Researchers in various laboratories have shown that virus
adsorption to sludge floes occurs rapidly (13, 24, 118). Using
coxsackievirus A9 and poliovirus-1 (Mahoney), Clarke et al. (24) found
that within 4 hours, 99% and 90%, respectively, of these viruses were
associated with the sludge floes, and that the removal of the viruses
from the liquid to solid fraction conformed to the Freundlich isotherm.
Using a laboratory-scale activated sludge unit, Malina et al. (118)
found that over 95% of poliovirus-1 was adsorbed to the sludge in 10
minutes. Both of these studies indicated that viruses were inactivated
over time once adsorbed.
Recent work (131) has indicated that the type of sludge can
influence the amount of adsorption seen. After one hour, only 57% of
seeded poliovirus-1 was found to be associated to mixed liquor, 70% to
anaerobically digested sludge, and 95% to aerobically digested sludge.
The authors suggested that the lower pH values found in aerobically
digested sludge may explain the difference in adsorption.
Due to the complex and variable nature of sludge, little or no work
has been done on the determination of viruses to sludge. Recent work in
this laboratory (32) explored the factors involved in bacteriophage
association to sludge floes. Using various salt and detergent
solutions, it was found that hydrophobic interactions do not appear to
be a major factor in promoting viral association to sludge floes.
Eluting solutions of increasing ionic strength were found to increase
the percent elution of indigenous bacteriophage adsorbed to sludge. The
author hypothesized that electrostatic interactions appeared to be the
major force involved in the association of bacteriophages with sludge
floes.

83
Methods for the Recovery of Viruses from Sludge
Because viruses have been shown to retain their infe’ctivity when
adsorbed to solids (122, 143), it is important to monitor their fate
during and after treatment. To follow the fate of viruses during
treatment and disposal of sludge, methods have been developed to detect,
and in some cases, concentrate viruses adsorbed to sludge floes.
An early study by Clarke et al. indicated that virus appeared to
be inactivated once adsorbed to the sludge floes (24). However, the
solution used to elute the adsorbed viruses (versene and buffer
solutions) has been shown to be a poor eluting solution (49), and the
conclusion drawn by the investigators may be inaccurate. In addition to
the choice of eluting solution used, studies have shown that viruses may
be embedded in, as well as adsorbed to, sludge and therefore some
mechanical means is necessary to agitate the sludge-eluting solutions
mixture (47, 85, 186).
Hurst et al. (85) found that the method of agitation and the
eluting solution used affected the amount of poliovirus-1 recovered from
sludge floes. When mixing with a magnetic stirrer was used, recovery
was greater if 0.05 M glycine was the eluting solution. If sonication
was used, then 3% beef extract as the primary eluate gave higher
recoveries. In the final procedure, elution was accomplished using
glycine at pH 11 and stirring the glycine-sludge mixture, followed by
concentration of the viruses using inorganic flocculation or adsorption
to filters. Poliovirus recovery from anaerobically digested sludge was
55%, while recovery from aerobically digested sludge was 25%.
In another study, three techniques were used to free embedded
virus: sonication, mechanical stirring and freon extraction (186) . It

84
was found that no one treatment gave consistent results, but a larger
number of isolates were recovered from samples treated by sonication and
mechanical stirring, although stirring required more time.
The method developed by Hurst (85) was used by Farrah et al. with
limited success (48). This method resulted in overall volumes that were
quite large. Additionally this method did not effectively recover virus
adsorbed to aerobically digested sludge. In order to overcome these
difficulties, they developed a urea-lysine method for the recovery of
viruses adsorbed to sludge (48). Briefly, 4 M urea - 1% lysine, pH 9
was mixed for five minutes with the sludge, resulting in 70% elution of
poliovirus-1 adsorbed to sludge floes. Once eluted, the virus was
separated from the sludge floes by centrifugation and concentrated by
aluminum hydroxide flocculation (44). The virus were eluted from these
floes by with 0.1 M EDTA - 3% beef extract, and further reconcentrated
by organic flocculation. This resulted in a very low final volume with
approximately 40% recovery of poliovirus-1 and coxsackievirus B3 (48).
However, problems, such as inactivation of virus by urea over time, and
length of time necessary to process samples proclude field use of this
method.
Recently, two methods were compared for their ability to recover
viruses from different sludge types under the auspices of the American
Society for Testing Materials (ASTM) in the hopes of establishing a
standard method (66). One method tested was the Environmental
Protection Agency (EPA) method, developed by Donald Berman of EPA (6,
10). Briefly, this method involves mixing sludge on a magnetic stirrer,
followed by the addition of AlCl^ to a final concentration of 0.5 mM
and pH adjustment to 3.5. Elution was accomplished by mixing the sludge

85
for 30 minutes with 10% buffered beef extract. After centrifugation to
separate viruses in beef extract from sludge floes, the beef extract
solution was filtered through a Filterite series to remove contaminants.
The filtrate was diluted with sterile distilled water at a ratio of 7 ml
of water for every 3 ml of beef extract, and reconcentrated using
organic flocculation. The second method was the Glass method, developed
by Steven Glass of New Mexico State University (62). In this method,
sludge was placed in a blender and dry beef extract (final concentration
ca. 4%) and antifoam were added. This was blended for 3 minutes,
adjusted to pH 9 and stirred for 25 minutes longer. The viruses were
separated from the sludge floes by centrifugation and concentrated by
organic flocculation. The final concentrate was detoxified by a
dithizone-chloroform mixture. The results of this comparison indicated
that the EPA procedure was slightly more sensitive than the Glass method
for recovering viruses from all sludge types but on (66).
All methods detailed here require extremes in pH, and are therefore
unsuitable for the recovery of bacteriophages from these samples.
Association of Viruses with Soils
The application of sewage treatment solids and liquids onto land
can lead to the adsorption of any surviving viruses to the soil. The
desorption of virus, and its subsequent transport through the soil
matrix can lead to groundwater contamination (142, 187). The
dissociation of viruses from soils is due to changes in the
physicochemical properties within the soil. Because the potential of
waterborne outbreaks of viral diseases is great, it is important to
study the mechanisms of and the factors associated with the adsorption of
viruses to soils.

86
The adsorption of viruses to soils is a reversible process that can
be described in terms of an equilibrium isotherm. Two such isotherms
have been used to examine the kinetics of virus adsorption to soils, the
Langmuir and Freundlich isotherms (53, 174). When the relationship
between the amount of adsorbed and unadsorbed virus is found to be
linear, a Freundlich isotherm is used. It has been proposed that
conformance to the Freundlich iostherm indicates that the surface
contains adsorption sites heterogeneous with regard to the strength of
bond formation (21). The Langmuir isotherm indicates that there are a
large but finite number of adsorption sites on a solid, thus equilibrium
Langmuir isotherms would indicate saturation-limited adsorption (174).
There is considerable debate whether viral adsorption to soils is
best described by either of these two isotherms. The linear plot of the
Freundlich isotherm has been used to describe a variety of virus
adsorption phenomenon, such as the adsorption of poliovirus-1 to loamy
sand (58) , the adsorption of T2 and F2 to silty loam (34) , <{>xl74
adsorption to clay and silt loans (21) and poliovirus-2 adsorption to
over 30 soils and minerals (124). However, it has been hypothesized
(176) that most adsorption processes were most likely saturation-
limited, and therefore conformed to the Langmuir isotherm. The author
suggested that in most adsorption studies, insufficient concentrations
of viruses were used, and as such, the saturation region of a Langmuir
plot was never seen, and the linear section of such an isotherm give the
appearance of conformance to the linear Freundlich isotherm. Several
studies have dictated that this indeed may be the case. Moore et al.
(124) studied the adsorption of radioactive poliovirus-2 to 34 minerals
and soils and found that at low virus concentrations, data conformed to

87
the Freundlich isotherm. However, when saturated conditions were
examined, their data conformed well to the Langmuir equation. The
authors, however, also indicated that in systems where saturation
appears to be occurring, it may likely be due to a coating of soil
adsorption sites with competing material. In addition, the Langmuir
isotherm has been used to describe MS2 adsorption to Indian soils (99),
T4 adsorption to activated carbon (27) and the adsorption of <¡>xl74 to a
silt loam (21).
As these data above indicate, adsorption of viruses to soils is a
complicated process. The adsorption, and subsequent desorption of
viruses from various soil types can be affected by many factors. These
factors are listed in Table 13 and are examined below.
When viruses dispersed in liquid are applied to soils, they must
come into close contact with one another in order for adsorption to
occur. This is directly related to the infiltration or flow rate of the
applied liquid (61, 174). Lance and coworkers (101, 103, 104, 105) have
studied the effect of various infiltration rates of sewage effluent
waters on the adsorption and elution of several enteroviruses. Using a
250 cm coarse sand column, and poliovirus-1 (101) or echovirus-1 (105),
they found that increased flow rates decreased the amount of virus
retained at the top portions of the column. At flow rates greater than
1.2 m/d, both poliovirus (101) and echovirus-1 (105) were leached
through the column. While the percentage of virus found in the column
effluent was low (<1%), the fact that virus will percolate through soil
at higher flow rates indicated the importance of contact time in viral
retention by soils.

TABLE 13.
Factors that may influence viral adsorption to soils.
a
FACTOR
REMARKS
REFERENCES
Flow rate
Low flow rates result in very efficient removal of
viruses (>99%) in clean waters. As flow rate
increases, virus adsorption decreases proportionally.
61, 101, 103, 104
105, 174
PH
Generally, a low pH favors virus adsorption while a
high pH results in elution of adsorbed virus.
21, 171
Cations and ionic
Cations, especially divalent cations, can act to
neutralize or reduce repulsive electrostatic forces
between virus and soil particles, allowing adsorption
to proceed. Solutions with low ionic strength
(i.e. rainwater) promote elution of adsorbed viruses.
36, 102, 104, 106
108, 123, 144,
171, 185, 187
Soluble organics
Soluble organic matter has been shown to compete with
viruses for adsorption sites on soil, resulting in
decreased adsorption or elution of an already adsorbed
virus.
17, 55, 61
Chemical composition
of soil
Soils with high organic content adsorb viruses less
efficiently. The presence of iron oxides increases
virus adsorption.
11, 61, 123, 124,
144
Nature of virus
The optimum pH for virus adsorption is expected to occur
at or below its isoelectric point, where the virus
possesses no charge or a positive charge. Different
strains of the same virus adsorb differently.
20, 55, 56, 106
Clay content
This is the active fraction of the soil. High virus
retention by clays results from their high ion
exchange capacity and large surface area per volume.
21, 22, 34, 36,
61, 99, 112, 143,
169, 174
a - Adapted from 61.

89
More recent work in the same laboratory (108) compared the movement
of poliovirus-1 in a 250 cm long soil column during saturated and
unsaturated flow of sewage water. In was found that during unsaturated
flow, poliovirus-1 was not detected below the 40 cm mark. However,
during maximum flow rates, poliovirus-1 penetrated to 160 cm, and in
some cases, past the 200 cm level. These data indicated the importance
of low infiltration rates to maximize virus retention by soils.
In natural systems, both viruses and most soils are negatively
charged. As such, pH and salt content can have a tremendous impact on
virus adsorption to and subsequent elution from the soil matrix. Taylor
et al. (171) studied the interaction of radiolabelled poliovirus-2 with
various soils, sands and clay. They found that at the pH values above
9, virus adsorption to these substrates was limited. At pH values near
neutrality, adsorption of poliovirus-2 to soils was greatly facilitated
by the addition of 10 mM CaCl^ or Na^SO^, while adsorption was limited
in solutions which did not contain these ions. These authors found that
for each substrate, there was a characteristic pH region of transaction
from strong to weak attraction for virus uptake, and in order for
adsorption to occur, these electrostatic repulsive forces must be
overcome. In a study comparing the adsorption of 4>Xl74 to five
different soils (21) , it was found that the low adsorption capacity of a
loamy sand was related to its high pH.
The importance of cation addition in the promotion of virus
adsorption to soils has been well documented. Retention of f2 and
poliovirus-1 by sand was greatly increased by the presence of 10 mM
CaCl2 or MgCl^ (108). Scheuerman et al. (144) found the adsorption of
poliovirus-1 to soils with high organic content was less than 20%. The

90
addition of 10 mM CaCl^ increased its adsorption to greater than 98%.
Moore et al. (123) found that adsorption of reovirus-3 to various sands
and silts was enhanced by the addition of 1 mM CaCl^ or MgCl^ to the
suspending medium. Using soil columns, Lance and Gerba (102)
demonstrated that the addition of 0.2 mM AlCl^ prevented the leaching of
poliovirus-1 in sewage effluents from the soil column. They suggested
that the increase in virus adsorption associated with increasing ionic
strength of the suspending solution was probably due to a decrease in
the thickness of the layers of charged ions around soil particles and
viruses. This allowed for short-range attractive forces, such as van
der Waals interactions, to take over.
The desorption of adsorbed viruses by low ionic strength solutions
such as rainwater has been well documented (36, 104, 106, 185, 187).
Duboise et al. (36) found that poliovirus-1 adsorbed to soil columns in
sewage effluents were easily desorbed by the addition of deionized water
to simulate rainfall. Studies by Wellings et al. (185, 187)
demonstrated the presence of viruses in wells 10 to 20 feet below soil
that had been treated with sewage effluents. These viruses appeared
after a period of heavy rainfall, and the authors hypothesized that the
increase in water/soil ratio resulting from the rainfall led to
desorption of the attached virus. Lance and coworkers (104) found that
the viral desorption and movement caused by simulated rainwater was
greatly reduced or eliminated by the addition of salts, again
implicating the importance of ionic strength in the association of
viruses to soils.

91
The presence of soluble organic matter in suspending solutions can
interfere with viral adsorption to soils by competing with viruses for
adsorption sites (61). Gerba and Goyal (55) found that virus suspended
in secondarily treated sewage, which has a high content of soluble
organics, adsorbed less to soils than virus suspended in deionized water
adsorbed to the same soil. Work by Bitton et al. (17) indicated that
humic substances interfered with the adsorption of coliphage T2 and
poliovirus-1 to a sandy soil.
The organic content of the soil itself may also play a role in the
association of viruses to soils. In studies examining adsorption of
virus to over 30 different soils and minerals, Moore and coworkers found
that both poliovirus-2 (124) and reovirus-3 (123) were adsorbed poorly
in soils with higher organic content. Burge and Enkiri found that 4>xl74
adsorbed well to four of five soils tested, the exception being a clay
loam high in organic content (21). Soils with high amount of humic and
fulvic acids were shown to be poor adsorbers of poliovirus-1 (144).
In addition to organic components, other constituents of the soils
will influence the association of viruses with them. The adsorption of
Xl74 to soils was found to be correlated with the cation exchange
capacity and specific surface area, as well as organic content (21).
The natural pH of a soil has been found to effect virus adsorption (21,
34, 55). Generally, soils with lower pH's tended to adsorb virus more
effectively. The presence of iron oxides, such as magnetite and
hematite, in soil increases the adsorption capacity for viruses (11, 61,
124). Clay content has been shown to be very important component in
soils that promotes virus adsorption (61, 174) and will be discussed
later.

92
The nature of the virus itself will play a paramount role in
adsorption of viruses to soil surfaces. Burge and Enkiri (20) found
that <(>xl74 purified by density gradient centrifugation was still
heterogenous. They found 2 fractions with different adsorption rates.
Landry et al. (106) compared lab strains and wild type strains of
poliovirus-1 and discovered that field strains were eluted from soils by
rainwater to a greater extent (>33%) than were lab strains (<3% eluted).
Gerba and Goyal (55) examined the adsorption of 27 different
reference strains of enteroviruses to a loamy sand. They found that
most viruses adsorbed very well (>90%) but there were some exceptions
including echoviruses-1 (55%), -12 (78%) and -29 (14%). They further
examined different strains of the same virus and found that adsorption
to loamy sand was very strain dependent. For six different strains of
echovirus-1, adsorption varied from 0 to 99%. This same group
statistically analyzed their data to determine variables associated with
virus-soil interactions (56). They found that different types and
strains of viruses could be grouped by their ability to similarly
effected by certain soil characteristics. Group I, which included
echovirus-1, coxsackievirus B4, <¡>xl74 and MS2, was found to be
influenced by pH, organic matter and exchangable iron content of the
soil. Group II (poliovirus-1, echovirus-7, coxsackievirus B3, T2 and
T4) did not appear to be influenced significantly by any soil factor.
The authors concluded that viruses in group I were more likely to be
influenced by changes in soil characteristics than group II as far as
adsorption/elution of these viruses was concerned.

93
Association of Viruses with Clays
Clay is perhaps the most important constituent of soils that
governs the adsorption of viruses (61, 174). Studies have shown that
the higher the clay content of the soil, the higher the viral adsorption
to that soil (34, 169, 174). The adsorption of MS2 to Indian soils was
shown to be depending on clay content (99). Lateritic soil, with a clay
content of 32%, was found to adsorb more MS2 than Black Cotton soil or
Kampur soil, with clay contents of 28% and 10% respectively. Sobsey et
al. (161) found that reovirus-3 and poliovirus-1 adsorption to different
soils could be related to clay content. Gerba and Goyal compared
adsorption of selected enteroviruses to nine different soils, and found
increased clay content in the soil allowed for more virus adsorption
(55). Work by Drewry and Eliassen (34) indicated that there was a
direct relationship between the clay content of nine soils from Arkansas
and California and their retention of bacteriophage.
High virus retention by clays has been related to their ion
exchange capacity (21, 112, 169) and their large surface area available
(21, 36, 61, 143) for adsorption. The research group headed by Stotzky
has examined the adsorption of various viruses to pure clay minerals to
determine the factors involved in this association (110, 111, 112, 145,
169) .
The adsorption of coliphages T1 and T7 to kaolin, a two-layer non¬
expanding clay, and bentonite, a three layer expanding clay, was
examined (145). The adsorption of T7 to these clays was found to be
related to the cation exchange capacity (CEC) of these clays, indicating
that T7 adsorbed to the negative sites on the clay surface. T1
adsorption, however, was not correlated with CEC. Pretreatment of clays
with 1% sodium metaphosphate, which blocked positive sites on the clay,

94
reduced T1 adsorption to kaolin by 54% and to bentonite by 28%. The
authors surmized that T1 adsorption appeared to be related more to anion
exchange capacity (AEC) of these clays. Similar experiments with
reovirus-3 (110) indicated that CEC of these clays was important in
reovirus adsorption. The investigators also studied the adsorption of
reovirus-3 to bentonite which was made homoionic to various cations. It
was shown that adsorption was increased in the presence of bentonite made
homoionic to higher valency ions; that is, adsorption of reovirus-3 was
greater to bentonite homoionic to aluminum than bentonite homoionic to
calcium or magnesium. The authors suggested that this increase in
adsorption was due to reduction of the net negative charge on clays,
which allowed virus to come close enough to the clays to become
protonated and subsequently adsorb by cation exchange (110) .
The effect of various proteins on virus adsorption to clays has
been evaluated. Carlson et al. (22) found that egg albumin or bovine
albumin decrease the adsorption of poliovirus-1 or T2 to kaolin. Lipson
and Stotsky (111) Showed that chymotrypsin decreased reovirus-3
adsorption to kaolin by 26% and to bentonite by 66%. This reduction of
adsorption was also seen with the use of ovalbumin. It was suggested
that the blockage of negatively charged sites on the clay by these
proteins was the reason for this decrease. In subsequent study (112),
the adsorption of T1 to bentonite and kaolin was blocked completely by
pretreatment of the clay with minimal essential media (MEM) supplemented
with 10% fetal calf serum. In this case, the authors hypothesized that
the organics in the MEM blocked positively charged sites on the clay
that T1 appears to adsorb to. These authors also examined the
adsorption of a lipid-enveloped virus, herpesvirus hominis type 1

95
(HSV-1) to clays that had been pretreated with bovine serium albumin
(169). They found that this protein had little or no effect in blocking
the adsorption of HSV-1 to either kaolin or bentonite. No explanation
of these data were provided.
The results from Stotzky's group have indicated that the adsorption
of reovirus-3 and coliphages T1 and T7 to clay minerals is the results
primarily of electrostatic forces (110, 111, 145). However, hydrophobic
interactions may still be involved in the adsorption processes of these
viruses to these clay minerals, although these factors have yet to be
defined (112).
Method for the Recovery of Viruses from Soils
As represented by the vast number of reports sited above, the
association of viruses to soils and the factors governing this
association have been well studied. However, the study of the fate of
viruses which remain adsorbed to soils has been hampered, until
recently, by the lack of adequate methodology to detect the presence of
these viruses. Indeed, only a few reports on the development of
methodology for enteric virus detection in soil can be found in the
literature (13, 86).
In 1979, Hurst and Gerba (86) reported the development of
quantitative method for the detection of enteroviruses in soil. In the
final procedure, soil was mixed by stirring on a magnetic stirrer in the
presence of four volumes of 0.25 M glycine and 0.05 M EDTA at pH 11.5
for five minutes to elute virus. After centrifugation, the supernatant
was adjusted to 0.06 M AlCl^ and the pH was lowered to 3.5, causing
formation of a floe. The floe was collected by centrifugation and
resuspended in fetal calf serum. The supernatant was passed through a

96
Filterite series of filters to adsorb any virus still remaining in the
supernatant. Adsorbed virus was eluted with the glycine-EDTA solution,
MgCl2 was added to 0.12 M and the solution was neutralized. The
procedure took approximately 1.5 hours and gave an average recovery of
69% for four enteroviruses (poliovirus-1, coxsackievirus B3, echovirus-7
and coxsackievirus A9) adsorbed to loamy sand soil.
Bitton et al. (13) reported the use of isoelectric casein to
recover enteroviruses from soil. In this procedure, soil was mixed by
shaking in the presence of 3 volumes of 0.5% casein at pH 10 for 15
minutes. After centrifugation, the supernatent was organically flocced
by pH adjustment to 4.5 (the isoelectric point of casein). The floe was
collected by centrifugation and resuspended in 5 ml of 0.15 M phosphate
at pH 9. Using this procedure, the mean recovery of enteroviruses from
four sand soils tested was 50%.
Materials and Methods
A list of chemicals and their sources, and routine methods used in
animal and bacterial virus preparation and assay is presented in
appendix A. A complete list of-media and solutions used in cell culture
work is presented in appendix B.
Clays
Kaolin (Fisher Scientific Company, Fair Lawn, NJ) is a two-layer
(1:1, silica alumina) nonexpanding clay, with a CEC of 5.8 meq/100 g of
oven dried clay (168). Bentonite (Fisher Scientific Company, Fair Lawn,
NJ) is a three-layer (2:1, silica alumina) expanding clay, with a CEC of
97.7 meq/100 g of oven dried clay (168). Stock solutions of 1 g/100 ml
distilled water were autoclaved and diluted at needed.

97
Adsorption of Virus to Clay
Twenty-five ml of a 1 mg/ml solution of clay was placed in a 50 ml
tube and centrifuged at 15000 x g for 20 minutes to pellet the clay, and
supernatant was disgarded. Approximately 10^ PFU of virus was added to
test solution, and an aliquot was drawn to determine initial input of
virus. Next, 25 ml of this solution was placed in tube containing clay
the pellet was disrupted by vortexing for 2-3 minutes. The tube was
placed on a shaker and mixed at 1600 - 1800 rpm for 10 minutes. After
shaking, the clay suspension was centrifuged at 15000 x g for 20
minutes, and supernatant was assayed to determine amount of virus
adsorption. Control tubes containing virus in test solution without
clay were treated by shaking and centrifugation along with test samples
containing clay. Samples taken from control tubes at the beginning and
end of experiments indicated (except where noted) that virtually no loss
of virus occurred due to solutions or experimental protocol. For
elution tests, the supernatant for test adsorption was decanted and 25
ml of test eluate was added to the clay pellet. The clay suspension was
placed on shaker and centrifuged as above. After centrifugation, the
supernatant was assayed for virus eluted. Percent virus adsorbed was
determined by comparison to amount of virus in initial test solution and
represents the mean of 2 - 3 experiments and 4-6 determinations.
Riboflavin Solubility
To determine the solubility of riboflavin, test solutions (20 ml)
were placed in 50 ml tubes with 100 mg of riboflavin. The tubes were
placed on a shaker for 30 minutes. Undissolved riboflavin was removed
by centrifugation at 10000 x g for 10 minutes. The solutions were
diluted in distilled water and dissolved riboflavin was determined by

98
measuring the adsorbance at 446 ran. These measurements represent the
means of 3 determinations and are expressed as percentage relative to
the buffer solution alone.
Sludge and Sludge Preparation
Mixed liquor, obtained from the University of Florida Campus Plant,
was collected in a sterile container and brought back to the laboratory
for immediate processing. One hundred ml of sludge was placed in a 250
ml tube and centrifuged at 2500 x g for 15 minutes at 4°C to pellet
sludge floes. After supernatant was disgarded, 100 ml of dechlorinated
tapwater with 107 PFU enterovirus was added to the sludge pellet and
processed by EPA procedure (see below).
EPA Procedure for Virus Recovery from Sludge
One ml of a 0.05 M AlCl^ solution was added to virus-sludge
mixture, and mixture was placed on a magnetic stirrer. The pH of the
mixture was dropped to 3.5 by the addition of 5 N HC1, and allowed to
mix for 30 minutes. Sludge was centrifuged at 25000 x g for 15 minutes
at 4°C and supernatant was disgarded. Next, 100 ml of a buffered 10%
beef extract solution (10 g beef extract, 1.34 g Na2 HPO^. 7H20, and
0.12 g citric acid per 100 ml water) was added and mixed on a magnetic
stirrer for 30 minutes. This was centrifuged at 10000 x g for 30
minutes at 4°C. Supernatant was collected and filtered through a 47 mm
Filterite series filter. One half of beef extract solution was treated
by ammonium sulfate flocculation (see below). The other half of the
beef extract solution was diluted by the addition of 117 ml of sterile
deionized water (final concentration was 3%) and the pH was dropped to
3.5 by the addition of 1 N HC1 to form floe. The solution was mixed by

99
magnetic stirrer for 30 minutes, followed by centrifugation at 25000 x g
for 15 minutes at 4°C. The floe was resuspended by addition of 2.5 ml
of 0.15 M Na^HPO^, pH 9. This phosphate solution was neutralized and
assayed.
Soil and Soil Preparation
The soil used was a Eustis find sand (Psammentic Paleudult). It
has previously been characterized as sandy, siliceous and hyperthermic
(15). Fifty g of soil was placed in a 500 ml bottle. Virus was
adsrobed to soil by adding 10^ PFU enterovirus to 100 ml of a 0.01 M
CaCl^ solution in 0.01 M imidazole, adjusted to pH 6.5. The bottle was
capped and hand shaken for 2 to 3 minutes. The soil-virus mixture was
poured into 250 ml tubes and centrifuged at 7000 x g for 10 minutes at
4°C, and supernatant was disgarded. The soil pellet containing virus
was transferred back to the 500 ml bottle and processed as described
below.
Goyal Procedure for Virus Recovery from Soils
To the conditioned soil, 200 ml of 3% beef extract, pH 10.5, was
added and shaken vigorously by hand for 5 minutes. The soil-beef
extract mixture was placed in 250 ml tubes and centrifuged at 25000 x g
for 10 minutes at 4°C. Supernatant was collected and split in half.
One portion was processed by ammonium sulfate flocculation (see below).
The other half was transferred to a sterile beaker containing a stir
bar. The beaker was placed on a magnetic stir plate and mixed as the pH
of the solution was dropped to 3.5 by the addition of 1 N HC1. The
solution was stirred until a floe formed (usually 30 minutes), and then
was transferred to a 250 ml tube and centrifuged at 1000 x g for 5

100
minutes at 4°C. The supernatant was disgarded and the floe was
resuspended in 5 ml of a 0.005 M glycine solution (pH 11). The pH of
this solution was adjusted to 7.5 by the addition of 1 M HC1 and
assayed.
Ammonium Sulfate Flocculation for Virus Recovery from Solids
One half of the beef extract eluate from the soil or sludge was
mixed with 2 volumes of saturated ammonium sulfate, which resulted in
immediate floe formation. This solution was centrifuged at 15000 x g
for 30 minutes at 4°C and supernatant was disgarded. The floe was
resuspended in sterile distilled water and assayed. When 10% tryptose
phosphate broth or 10% brain heart infusion were used as eluting
solutions, only one volume of ammonium sulfate was used to form floe.
Results
Virus Association to Clays
Preliminary experiments were conducted to determine the effect of
solutions and pH on the adsorption of poliovirus-1 (Figure 11) and
bacteriophage T7 (Figure 12) to bentonite. At pH 4, greater than 99% of
both viruses were adsorbed to bentonite, regardless of whether the
suspending solution was a buffer consisting of 0.05 M potassium hydrogen
phthalate (KHP) and 0.05 M lysine, a salt solution of 1 M NaCl in buffer
or a detergent solution of 0.1% Nonidet P-40 (NP40) in buffer. An
increase in the pH of the suspending solution to 7 or 9 did not effect
the adsorption of either virus suspended in buffer or NaCl. However,
adsorption of poliovirus-1 suspended in the detergent solution was
decreased to 35% at pH 7 and 21% at pH 9 (Figure 11). Adsorption of T7

FIGURE 11.
The effect of pH, salt and detergent on the adsorptin of poliovirus-1 to bentonite.

pH 4 pH 7 pH 9
% POLIOVIRUS- I ADSORPTION TO BENTONITE
ro 4^ m oo o
o o o o o o
1 1 1 1 1—
[
jBuffer
|o â–¡ â–¡ â–¡â–¡ â–¡â–¡ â–¡ n d n,D,,0u a nnn 6 â–¡'"]
[â–¡â–¡â–¡â–¡PQDDDCO â–¡â–¡â–¡â–¡â–¡â–¡â–¡â–¡â–¡]
Nonidet P-40
¡Buffer
NaCI
loUnUc"pbnDn ^^onidet P-40
[
lBuffer
NaCI
[^7VÍZ]Nonidet P-40
301

FIGURE 12. The effect of pH, salt and detergent on the adsorption of bacteriophage T7 to bentonite.

pH 4 pH 7 pH 9
% T7 ADSORPTION TO BENTONITE
ro a o> CD o
o o o o o : c>
1 1 1 1 r
[
]Buffer
ImTTTnnmnm
â–¡ â–¡â–¡â–¡â–¡â–¡â–¡â–¡â–¡â–¡â–¡â–¡
□ UnUnUnUnUa°ndnUrjNonidet P~40
]Buffer
Pn^onidet P-40
NaCI
liUniNonidet P-40
^OT

105
in the detergent solution was less than 10% at both pH 7 and 9 (Figure
12). Based on these results, we chose to explore the adsorption of
viruses to clay in all subsequent experiments at pH 9.
The effect of varying nonionic detergent concentrations on virus
adsorption to two clays was examined. Figure 13 shows the adsorption of
poliovirus-1 and T7 in NP40 solutions to bentonite. In buffer alone or
0.001% NP40, greater than 99% of both viruses were adsorbed. Increasing
the concentration of NP40 to 0.01% had no effect on T7 adsorption, and
only a slight decrease in poliovirus-1 adsorption was noted. At
concentrations of 0.1% and 1.0% NP40, only 10% of T7 was associated with
the bentonite clay, while poliovirus-1 adsorption was decreased to 21
and 33% respectively.
This same experiment was done using kaolin clay as the adsorbent
and the results are shown in Figure 14. In buffer alone at pH 9, 80% of
poliovirus-1 and 94% of T7 was adosrbed to kaolin. Increasing
concentrations of NP40 resulted in a decrease of T7 adsorption to
kaolin, most notable at 0.1% NP40 (50% adsorption) and 1.0% NP40 (38%
adsorption). This increase in NP40 concentration had little or no
effect on poliovirus-1 adsorption to kaolin.
Since the effect of a nonionic detergent on the adsorption of virus
to kaolin was less dramatic than on virus adsorption to bentonite, the
effect of ionic detergents on poliovirus-1 and T7 adsorption to kaolin
at pH 9. Low concentrations (0.001%) of Triton QX-9, an anionic
detergent, did not effect the adsorption of either virus to kaolin.
Increasing Triton QS-9 concentration to 0.1% resulted in a decrease in
the adsorption of poliovirus-1 to 47% and that of T7 to 29%. Cetyl
trimethylammonium bromide (CTAB), a cationic detergent, had no effect on

FIGURE 13. The effect of varying detergent concentrations on the adsorption of poliovirus-1 and
bacteriophage T7 to bentonite at pH 9.

00
80
60
40
20
0
% NONIDET P-40
107

FIGURE 14. The effect of varying detergent concentrations on the adsorption of poliovirus-1 and
bacteriophage T7 to kaolin at pH 9.

% ADSORPTION TO KAOLIN
IOO
T
0 0.001 0.0 1
% NONIDET P
BT-7 Phage
Poliovirus- I
1.0
109

110
the adsorption of these viruses to kaolin at 0.001% concentration.
However, when the concentration of CTAB was increased to 0.1%, virtually
no poliovirus in this solution adsorbed to the kaolin. T7 was
inactivated by this high concentration of cationic detergent.
These results indicated that both hydrophobic and electrostatic
interactions were involved in virus adsorption to the clays. Since
previous results had indicated that salts can influence both hydrophobic
(40, 48) and electrostatic (50, 156) interactions between viruses and
solids, we examined the effect of salt and detergent solutions on the
adsorption of poliovirus-1 (Figure 16) to clays. The addition of NaCl to
0.1% NP40 decreased adsorption of poliovirus-1 to bentonite slightly,
while the adsorption of poliovirus-1 to kaolin was decreased greatly,
from 78% to 27%. A solution of detergent plus sodium sulfate or sodium
citrate increased poliovirus-1 to bentonite to 88% and 94%,
respectively. The addition of sodium sulfate to detergent allowed only
36% adsorption of poliovirus-1 to kaolin, while sodium citrate in the
detergent solution increased adsorption of poliovirus-1 to kaolin to
86%.
The addition of NaCl to detergent allowed less than 5% of T7 to
adsorb to both kaolin and bentonite (Figure 17). Detergent solutions
containing either sodium sulfate or citrate promoted greater than 90%
adsorption of T7 to both kaolin and bentonite.
Since it appeared that these salts varied in their ability to
effect the hydrophobicity of the detergent solution, we investigated the
relative ability of these salts to accommodate hydrophobic groups in
solution by measuring their ability to solubilize riboflavin (73).
These results are shown in Table 14. A 1.5 M NaCl solution increased

FIGURE 15. The effect of nonionic, anionic and cationic detergents on the adsorption of
poliovirus-1 and bacteriophage T7 to kaolin at pH 9.

NP40
.1%
NP40
.001 %
Triton QS-
BPoliovirue-1
Bacteriophage T7
.1%
Triton QS-9
.001%
CTAB
.1%
CTAB
112

FIGURE 16. The effect of salt and detergent solutions on the adsorption of poliovirus-1 to
bentonite and kaolin at pH 9.

hi
,I % NP40
1.5 M NaCI
BENTONITE
KAOLM
.I % NP40 .1 % NP40
I M Na^SO^ 1.5 M NaCitrate
114

FIGURE 17. The effect of salt and detergent solutions on the adsorption of bacteriophage T7 to
bentonite and kaolin at pH 9.

% T7 ADSORPTION
B BENTONITE
KAOLIN
100-
80-
60-
40-
20-
0
Buffer
[1H Úái
. I % NP40 .1 % NP40
1.5 M NaCI
T
. I % NP40
I M NagSC^ I
.1% NP40
.5 M NaCitrate
116

117
TABLE 14. The solubility of riboflavin in various salt solutions.3
. b
Solution
c
% Riboflavin solubility
1.5 M NaCl
121
Buffer
100
1 M Na SC)
2 4
49
1.5 M Na.Citrate
29
3
a - Solubility of riboflavin was determined by adsorption at 446
nm (see text).
b - All solutions were in buffer of 0.05 M lysine and 0.05 M KHP and
adjusted to pH 9.
c - Solubilities based on solubility of riboflavin relative to
buffer.

118
the solubility of buffer to 121%, while 1 M Na^SO^ decreased solubility
to 49% and 1.5 M NaCitrate decreased the solubility of riboflavin even
more (29%).
Control experiments were conducted to ensure that this decrease in
PFU of the virus was due to adsorption, and not caused by inactivation
of the viruses. After adsorption, protein solutions were used to elute
viruses that adsorbed to the clays. The results of one such experiment
are shown in Table 15. Poliovirus-1 and T7 were adsorbed to clay in a
solution of 0.1% NP40 plus 1.5 M citrate. Elution of T7 by three
different protein solutions listed was 65% or greater, while
poliovirus-1 elution ranged between 70 and 91%. These results confirmed
that viruses were neither inactivated nor aggregated by either the test
solution or adsorption to the clay particles under the conditions of
these experiments.
Recovery of Enteroviruses Adsorbed to Sludges and Soils
A recent report (66) detailed the results of a round robin
investigation initiated by the ASTM to select a tentative standard
method for the recovery of enteric virus from sludge. Of the two
procedures tested, one, the low pH - aluminum chloride method developed
by Berman (6, 10) appeared to work slightly better. This method was
chosen by the Environmental Protection Agency (EPA) as its standard
method (9). Organic flocculation of the beef extract used in the
primary eluting step is used as a reconcentration step in this method.
Results in Chapter II of this dissertation have indicated that ammonium
sulfate flocculation worked as good as if not better than organic
flocculation as a reconcentration step. Therefore, we compared the
standard EPA procedure for the recovery of viruses from sludge to our

119
TABLE 15. Elution of viruses adsorbed to clays by protein solutions at
pH 9.a
% virus recovered
Bacteriophage T7
adsorbed to
Poliovirus-1
adsorbed to
Solution
Kaolin
mean SD
b
Bentonite
mean SD
Kaolin
mean SD
Bentonite
mean SD
10%
c
BHI
72
11
91
10%
d
BE
67
2
78
10%
e
TPB
76
3
65
6
78
4
91
2
2
75
1
94
6
5
70
5
83
7
a - Virus was suspended in 0.1% NP40 plus 1.5 M sodium citrate at pH 9,
and then mixed with either bentonite or kaolin for 20 minutes to
adsorb virus.
b - SD - standard deviation,
c - BHI - brain heart infusion,
d - BE - beef extract
3 - TPB - tryptose phosphate broth.

120
ammonium sulfate flocculation procedure. The results of this comparison
for the recovery of seeded enteroviruses from sludge are shown in Table
16. For poliovirus-1, recovery using EPA method was 23% while 41% of
seeded poliovirus-1 was recovered using ammonium sulfate flocculation.
Results for echovirus-5 and coxsackievirus B5 exhibited the same trend,
with recovery using ammonium sulfate flocculation higher (73% and 72%,
respectively) than the recovery using the EPA method (46% and 33%,
respectively). The overall mean recovery of seeded enteroviruses from
sludge was 34% using the EPA method, compared to 62% using ammonium
sulfate flocculation in the recovery procedure.
We also tried the ammonium sulfate procedure using various other
protein solutions as eluents. Table 17 shows that the use of beef
extract as the primary eluting solution was more effective in the
recovery of enteroviruses from sludge (62% recovery) than was brain
heart infusion (33% recovery) or tryptose phosphate broth (21% recovery).
Our results with the sludge recoveries led us to investigate the use of
ammonium sulfate flocculation to recover viruses adsorbed to soils.
Currently, the ASTM is in the process of evaluating several procedures for
the recovery of enteroviruses from soils. One procedure to be tested was
developed by Sagar M. Goyal of the University of Minnesota (personal
communication) and is essentially a modification of a standard EPA method
(9). Again, organic flocculation of beef extract is the reconcentration
step used in this procedure. This method was compared to ammonium sulfate
flocculation for the recovery of seeded enteroviruses from a sandy soil
(Table 18). Both methods appeared to work well, with ammonium sulfate
flocculation giving a slightly higher mean recovery (79%) as compared
to the organic folcculation procedure (65%).

121
TABLE 16.
Recovery of seeded enteroviruses from sludge by the standard
EPA versus ammonium sulfate flocculation procedure.
b
EPA
c
ASF
Trial
Virus added
% recovery
% recovery
1
Poliovirus-1
25
48
2
Poliovirus-1
21
33
3
Echovirus-5
56
68
4
Echovirus-5
36
79
5
Coxsackievirus
B5
31
76
6
Coxsackievirus
B5
34
68
d
Mean recovery ± SD
34 ± 11
62 ± 16
a - One
hundred ml sludge was
mixed
with 100 ml
seeded dechlorinated
tapwater. One ml of 0.05 M AlCl^ was added and pH was dropped to
3.5 to facilitate virus adsorption. After centrifugation,
supernatant was disgarded and 100 ml 10% beef extract was added to
elute virus. After centrifugation, beef extract solution was
divided into equal halves.
b - EPA - One half of beef extract eluate was diluted to 3% and
adjusted to pH 3.5, floe was centrifuged and resuspended in 2.5 ml
0.15 M Na HPO (see text).
2 4
c - ASF - One half beef extract eluate was mixed with two volumes of
saturated ammonium sulfate, floe was centrifuged and resuspended in
2.5 ml distilled water (see text).
d - SD - standard deviation.

122
TABLE
17. Recovery of enteroviruses
solutions using ammonium
concentration.
adsorbed to sludge
sulfate flocculation
by protein
for
Trial
Virus added
BEb
% recovery
ELUTING SOLUTION
c
BHI
% recovery
d
TPB
% recovery
1
Poliovirus-1
48
15
1
2
Poliovirus-1
33
13
4
3
Echovirus-5
68
31
17
4
Echovirus-5
79
34
15
5
Coxsackievirus B5
76
46
41
6
Coxsackievirus B5
68
57
45
e
Mean recovery ± SD
62 ± 16
33 ± 16
21 ± 17
a -
See footnote a, Table
16.
b - BE - Beef extract, results from Table 16.
c - BHI - Brain Heart Infusion. 10% BHI was used to elute
adsorbed viruses and mixed with one volume of saturated
ammonium sulfate, floe was centrifuged and resuspended in 5 ml
distilled water (see text).
d - TPB - Tryptose Phosphate Broth. 10% TPB was used to elute
adsorbed viruses and mixed with one volume of saturated
ammonium sulfate, floe was centrifuged and resuspended in 5 ml
distilled water (see text).
e - SD - Standard deviation.

123
TABLE 18. Recovery of seeded enterovirus from soil by the Goyal
procedure versus ammonium sulfate flocculation.
Trial
Virus added
b
Goyal Procedure
% recovery
c
ASF
% recovery
1
Poliovirus-1
64
82
2
Poliovirus-1
87
81
3
Echovirus-5
52
46
4
Echovirus-5
48
91
5
Coxsackievirus B5
74
72
6
Coxsackievirus B5
63
103
Mean
d
recovery ± SD
65 ± 13
79 ± 18
a - One hundred ml of ^.01 M CaCl^ in 0.01 M imidazole, pH 6.5,
was seeded with 10 PFU enterovirus listed, and added to 50 g
soil. After centrifugation, 200 ml of 3% beef extract, pH
10.5, was added to soil pellet to elute adsorbed viruses.
Beef extract supernatant was collected by centrifugation and
split into equal halves (see text).
b - Goyal procedure - One half of beef extract eluate was adjusted
to pH 3.5, floe was centrifuged and resuspended in 5 ml of 5
mM glycine, pH 11. Concentrate was adjusted to pH 7.5 and
assayed (see text).
c - ASF - Ammonium sulfate flocculation. One half of beef extract
eluate was mixed with two volumes saturated ammonium sulfate,
floe was centrifuged and resuspended in 5 ml distilled water
(see text).
d - SD - standard deviation.

124
Again, we tried a different protein solution, 10% brain heart
infusion, instead of beef extract, as the primary eluting solution.
These results are shown in Table 19. Using ammonium sulfate
flocculation as the concentration step, the mean recovery of seeded
enteroviruses from soils was 49% if the eluting solution was BHI, as
compared to 79% recovery when 3% beef extract was used.
Discussion
During wastewater treatment, enteric viruses present in sewage
generally become associated with wastewater sludges. While many of
these viruses will be inactivated (24, 41, 47, 68, 166, 184) , some will
remain viable (89, 115, 122, 172, 186). After land disposal, sludge-
associated viruses, as well as viruses still in treated effluents, may
become associated with the soil, and under certain conditions, may
migrate through the soil matrix and contaminate groundwater supplies
(142, 174, 187). In order to assess the fate of these viruses, it is
important to learn more about the factors involved in these associations
as well as develop techniques which rapidly and reliably detect and
concentrate such viruses.
Virus Association to Clays
Clay minerals are one of the most important soil components
involved in the adsorption of viruses (22, 61, 141, 143). The mechanism
for virus adsorption to clays is not known. However, two general
theories of adsorption have been proposed. Both theories are based on
the observation that in the pH range of most natural water systems, both
the virus and the clay particles have a net negative charge, and

125
TABLE 19. Recovery of seeded enterovirus from soil by protein solutions
using ammonium sulfate flocculation procedure
concentration.
for
PROTEIN SOLUTION
b
3% BE
10% BHIC
Trial
Virus added
% recovery
% recovery
1
Poliovirus-1
82
66
2
Poliovirus-1
81
75
3
Echovirus-5
46
24
4
Echovirus-5
91
25
5
Coxsackievirus B5
72
50
6
Coxsackievirus B5
103
44
d
Mean recovery ± SD
79 ± 18
49 ± 15
a - One hundred ml7of 0.01 M CaCl^ in 0.01 M imidazole, pH 6.5, was
seeded with 10 PFU enterovirus listed and added to 50 g of soil.
After centrifugation, 200 ml of protein solution listed was added
to soil pellet to elute adsorbed virus and processed as described
below (see text).
b - 3% BE - 3% beef extract was used as primary eluting solution (see
text). Values from Table 18.
c - 10% BHI - 10% brain heart infusion was used as primary eluting
solution. Floe was formed by addition of one volume ammonium
sulfate, collected by centrifugation, and resuspended in 10 ml
distilled water (see text).
d - SD - standard deviation.

126
electrostatic repulsion blocks adsorption. The introduction of cations
or a decrease in pH can bring about adsorption by neutralizing the
negative charges by electropositive ions, or by lowering the
electronegativity of the particles by pH change.
Carlson et al. (22) noted that the molar concentration required for
maximum adsorption of T2 by clay was about 10 times greater for sodium
salt than for a calcium salt. These authors suggested that adsorption
of virus in the presence of these ions was dependent on reduction of the
thickness of the ion cloud surrounding the particles. This reduction is
sufficient to effect a proper charge distribution and allow the particle
to move within a minimum distance from each other to set up a
clay-cation-virus bridge according to the Schultz-Hardy rule of colloid
behavior (22).
A second theory of the mechanism of virus adsorption to clays has
been advanced by Schaub et al. (141, 143), based on theories of
coagulation. They hypothesized that virus adsorption is similar to the
mechanism of solids coagulation. Essentially, this theory states that
metal cations serve to reduce repulsive forces between the viruses and
clay particles, allowing these particles to come close enough together
to allow the shorter-range attractive forces, such as van der Waals
forces, to interact, thus resulting in virus adsorption.
Both of these theories indicate the importance of electrostatic
interactions in the promotion of virus adsorption to clays. Recent work
in our laboratory (40, 49, 115, 156) has indicated that the adsorption
and elution of viruses from negatively charged surfaces (membrane
filters) is influenced, in part, by hydrophobic interactions between the
viruses and filters. Therefore, we investigated the adsorption of

127
poliovirus-1 and bacteriophage T7 to clays to determine if hydrophobic
interactions were involved in this association.
Previous data (22, 111, 112) have indicated that proteinase
compounds reduced or blocked the adsorption of various viruses to clays.
Lipson and Stotsky suggested that this reduction in adsorption was due
to the protein competing with the virus for negatively charged sites on
the clay (111, 112). Little attention was paid to the disruption of
hydrophobic interactions that may have been caused by these protein
solutions.
By virtue of their amino acid composition, proteins will exhibit
some electrophoretic movement, and thus, their use in influencing
hydrophobic interactions between viruses and clays will also influence,
to a certain extent, electrostatic interactions. Therefore we chose to
evaluate the contribution of hydrophobic interactions between clay and
viruses by using a nonionic detergent, thereby minimizing any change in
electrostatic interactions. We found that at low pH, this detergent,
NP40, had no effect on the adsorption of poliovirus-1 or T7 to
bentonite. This confirmed previous results which indicated that viral
adsorption to negatively charged surfaces at low pH values appeared to
be mainly electrostatic in nature (156). At pH values at and above
neutrality, virus adsorption to bentonite was effectively reduced by the
presence of this nonionic detergent. This indicated that hydrophobic
interactions were indeed playing a role in the adsorption of both
viruses tested to bentonite. A minimum concentration of 0.1% detergent
was necessary to block adsorption to bentonite of 0.1%.
The effect of NP40 on virus adsorption to kaolin was not as
dramatic as that seen for virus adsorption to bentonite. Poliovirus-1

128
adsorption was not effected at all, while T7 adsorption was reduced only
50% as compared to a 78% reduction of adsorption to bentonite. This
indicated that the adsorption of T7 to kaolin appears to be more
hydrophobic in nature than poliovirus-1 association to kaolin. This
also indicates that the adsorption sites for these two viruses are
different. Other investigators have shown that adsorption sites are
different for different viruses (110, 145) and this appeared to be the
case for poliovirus-1 and T7 adsorption to kaolin. The use of ionic
detergents to block virus adsorption to kaolin indicated that
poliovirus-1 adsorption to kaolin appears to be to negatively charged
sites on the clay surface. A positively charged detergent, CTAB
complete blocked adsorption of poliovirus-1 to kaolin, while a
negatively charged detergent decreased but did not block adsorption to
the same extent as the cationic detergent.
Previous results have shown that once hydrophobic interactions have
been disrupted by the action of a detergent, certain salts can disrupt
electrostatic interactions (50, 156). This appeared to be the case of
T7 and poliovirus-1 adsorption to the clays. The addition of NaCl to
neutral detergent solutions resulted in a decrease in the adsorption of
both viruses to the clays. Alternatively, the charged group may be
incorporated as part of the detergent, and result in blocked adsorption
as well. However, this does not explain our results when the salt added
to the detergent solution was Na^SO^ or sodium citrate. These salts did
not decrease adsorption, but in fact promoted adsorption up to the
levels seen when viruses were suspended in buffer solution alone. An
explanation of these data can be found by examining the nature of these
salts.

129
Hatefi and Hanstein (73) were able to increase the solubility of
some proteins by using solutions of chaotropic ions. These chaotropic
ions are relatively large, singly charged ions such as trichloroacetate
or thiocyanate. Chaotropic ions can be viewed as decreasing the
structure of water and therefore making aqueous solutions more lipophilic
(73, 74). Solutions of chaotropic salts have been found to solubilize
membrane proteins and organic compounds such as riboflavin and adenine,
as well as disrupt antigen-antibody complexes (29, 40, 73). Recent
studies have indicated that chaotropic salts effectively eluted viruses
adsorbed to filters (49, 50, 155), wastewater sludges (48) and estuarine
sediments (177). Sodium chloride, a borderline chaotropic salt, was
found to increase the solubility of riboflavin. This indicated that in
addition to the disruption of electrostatic interactions, it is likely
that sodium chloride may have been acting to further disrupt hydrophobic
interactions.
In contrast, antichaotropic ions, such as citrate, magnesium or
sulfate, are generally small singly charged or multivalent ions. These
antichaotropic ions have been found to promote hydrophobic interactions,
presumably by increasing water structure (73, 74). As water structure
is increased, the ability of aqueous solutions to accommodate
hydrophobic groups is reduced. As such, hydrophobic interactions
between apolar groups in the solution are increased. These
antichaotropic salts have been shown to conteract the ability of
chaotropic salts to disrupt hydrophobic interactions (40, 49, 73, 74).
Additionally, antichaotropic salts can decrease the solubility of
organic compounds such as riboflavin and adenine (49, 73). We found
that Na^SO^ and sodium citrate both decreased the solubility of

130
riboflavin relative to the buffer solution by 51 and 71% respectively.
It appears that these antichaotropic salts were able to overcome the
disruption of hydrophobic interactions caused by the nonionic detergent,
and thus promote virus adsorption to the clays. The relatively higher
antichaotropic nature of citrate as compared to sulfate may explain why
adsorption of poliovirus-1 to kaolin was promoted to a greater extent by
the addition of citrate to the detergent solution.
Previous reports have indicated that the mechanism of virus
adsorption to clays is primarily electrostatic in nature (22, 110,
111, 112, 143, 145). Our results indicate that, at pH 9, virus
adsorption to clays is influenced, at least in part, by hydrophobic
interactions. Additional studies should be conducted to determine the
extent of these interactions.
Recovery of Enteroviruses Adsorbed to Sludges and Soils
Some enteric viruses have been shown to survive sewage treatment
processes and persist in wastewater sludges (115, 170, 186) and
effluents (68, 135, 185). These end products of sewage treatment are
likely to be disposed of on land. Therefore, the probability of
adsorption of these surviving viruses to soil particles is great.
Studies have indicated that, for the most part, viruses associated with
sludge and in wastewater effluents are effectively retained by
adsorption to the soil matrix (18, 99, 142), thus the possibility of
groundwater contamination due to breakthrough is reduced, but still
occurs. It should be remembered, however, that solid-associated viruses
have been shown to remain infective (18, 108, 122, 143). To determine
the fate of these solid-associated viruses, a variety of methods have
been developed to recover and concentrate viruses adsorbed to sludges

131
(6, 10, 47, 48, 62, 85, 186) and soils (13, 86, Goyal - personal
communication).
The Environmental Protection Agency (EPA) of the United States
government has established some standard procedures for the recovery of
viruses from environmental samples (9). The standard method for the
recovery of viruses from sludges or soils is the same and is based
on work by Berman and fellow EPA coworkers (6, 10). Viruses are eluted
from solids with a 10% buffered beef extract solution. After elution,
the beef extract is diluted to 3% and reconcentrated by organic
flocculation. We modified this procedure by omitting the dilution and
organic flocculation steps and replacing them with the ammonium sulfate
flocculation procedure developed during the course of this study (see
previous chapter). The recoveries of seeded enteroviruses adsorbed to
sludge using the ammonium sulfate flocculation procedure were
consistently higher than recoveries when the standard EPA procedure was
used. We also tested two other protein solutions for the primary
elution step, followed by salt flocculation. The recovery of
enteroviruses by elution with brain heart infusion were virtually the
same as those of the EPA procedure, while the use of tryptose phosphate
broth gave consistently lower recoveries.
While these results indicate that the 10% buffered beef extract
appears to be the best eluting solution, recoveries can be increased if
ammonium sulfate flocculation is used instead of organic flocculation as
the reconcentration step. In addition to higher recoveries, the
ammonium sulfate flocculation procedure has several advantages over the
EPA procedure. In both methods, dilution of the beef extract is
necessary. In the EPA procedure, this dilution is necessary before

132
flocculation can occur, while in our procedure, the dilution step, into
saturated ammonium sulfate, results in the formation of a floe. Another
advantage of our procedure is that no change in pH is required. The EPA
method requires that the diluted beef extract solution be adjusted to pH
3.5 and stirred at this pH level for 30 minutes for the floe to form.
This may result in the inactivation of acid-labile viruses. In addition,
the floe formed from organic flocculation is resuspended in a phosphate
solution at pH 9 which must be neutralized. In our ammonium sulfate
flocculation procedure, both the formation of the floe and the
resuspended concentrate are at neutrality without pH adjustment. A
final advantage of our procedure is due to the nature of the beef
extract itself. Several laboratories have indicated that different lots
and sources of beef extract vary in their ability to flocculation at low
pH (5, 84, 125, 132). Since the ammonium sulfate flocculation procedure
does not require a change in pH to bring about the formation of a floe,
this problem is eliminated.
We used our salt flocculation procedure to recover viruses adsorbed
to soils and compared it to the Goyal method, currently being tested for
use as a standard procedure by the ATSM (personal communication). The
method proposed by Goyal is very similar to the standard EPA procedure
(9), except that 3% beef extract, pH 10.5, is used as the eluting
solution. After elution, viruses are concentrated by organic
flocculation. We followed the Goyal procedure for the recovery of
viruses from soils up to the organic flocculation, which we replaced
with ammonium sulfate flocculation. Again, recoveries of enteroviruses
from soil were higher using our procedure. Results with another protein

133
solution as the eluate again indicated that beef extract was the eluting
solution of choice.
The advantages of our procedure over the Goyal procedure of the
recovery of enteroviruses from soils are similar to those listed above
for the recovery of viruses from sludge, i.e., little if any pH
flocculations are necessary, as well as no problems with floe variability
due to the source and lot of the beef extract used. One disadvantage
with our method in this case was in the volume of the beef extract eluate
to be centrifuged to collect floes. The ammonium sulfate flocculation
procedure requires an increase in the volume of beef extract solution to
form the floe by dilution into saturated ammonium sulfate. Since the
Goyal procedure uses a 3% solution of beef extract for elution, no
dilution is required before the organic flocculation step. However, the
volume of the final concentrate is the same (approximately 10 ml per 50
g soil) for both methods.
Our results indicate that ammonium sulfate flocculation as a
concentration step in procedures for the recovery of viruses from
sludges and soils is an attractive alternative to organic flocculation.

CHAPTER IV
CHARACTERIZATION OF VIRUSES
Review of the Literature
The adsorptive behavior of viruses to various solids is greatly
dependent upon the virus being studied (53). In addition, there appear
to be differences in adsorptive behavior not only between virus types,
but also between different strains of the same virus (55, 56). It has
been suggested that these differences in the adsorption of viruses to
various solids may be related to differences in virion surface charge
and hydrophobicity (11, 53, 156). A definative study on the
contributions of the electrostatic and hydrophobic nature of viruses on
adsorption is lacking.
Since viruses are largely protein and behave as colloidal
hydrophilic particles in aqueous solutions, procedures used to study
proteins have been shown to be useful in the concentration, purification
and characterization of virus particles (52, 64). Techniques used for
macromolecules, such as electrophoresis, affinity chromatography and
ion exchange chromatography, have been used to study the physical nature
of viruses (53, 64). Such information may be useful in predicting virus
adsorption to various solids.
Electrostatic Contributions
Most viruses have an outer protein coat composed of various amino
acids. Depending on the pH of the suspending solution, some side groups
of these amino acids may become ionized and give the virus capsid an
134

135
electrical charge. Since these ionizing groups can have variable
dissociation constants, most viruses will have net charges that vary
continuously with pH. At the isoelectric point, the net charge on the
virus is zero and it will not migrate in an electric field. Viruses
will be positively charged below their isoelectric point and negatively
charged above it. At the isoelectric point, there will be local areas
of positive and negative charges across the virus surface (121). As
such, isoelectric points, while useful, do not tell us about any
differences in the distribution of charged groups, nor do they yield any
information concerning the charge density of a virus. Gerba et al. (56)
were able to correlate differences in adsorptive behavior of viruses to
soils with the virus isoelectric points. These workers studied the
adsorption of several viruses to nine different soils, and used a
statistical analysis to determine if there were similarities in the
adsorptive behavior of these viruses. Viruses which adsorbed very
strongly to soils were considered Group II viruses, while those that
adsorbed to soils less efficiently were placed in Group I. An
examination of the viruses indicated that the isoelectric points of
viruses in Group II tended to be lower than those of Group I. Thus,
under defined conditions, the isoelectric point of a virus may be useful
in determining its adsorption to a charged surface.
Table 20 lists some isoelectric points for various viruses. As can
be seen from these data, isoelectric points vary not only with the type
and strain of viruses, but also with the method used to measure it.
Because microorganisms, such as viruses, have net surface charges,
they are able to interact with ion exchange resins. Basically, an ion
exchanger consists of an insoluble matrix to which charged groups have

TABLE 20. Virus Isoelectric Points.
a
Virus (strain)
PI
Method
Reovirus 3 (Dearing)
3.9
Column IEFb
Polio 1 (Bruenders)
7.4, 3.8
7
Polio 1 (Mahoney)
8.2
Column IEF
Polio 1 (Chat)
7.5, 4.5
Column IEF
Polio 1 (Brunhilde)
7.0, 4.5
Column IEF
Zone electrophoresis
Polio 1 (LSc)
6.6
Granulated gel
Echo 1 (V239)
5.3
Granulated gel
Echo 1 (V212)
6.4
Granulated gel
Echo 1 (4CH-1)
5.5
Granulated gel
Echo 1 (Farouk)
5.1
7
Coxsackie A21
6.1, 4.8
7
Vaccinia (Lister)
5.1
Horizontal IEF
Vaccinia (Lister)
3.8
Microelectrophoresis
Smallpox (Harvey)
5.9
Horizontal IEF
Smallpox (Harvey)
3.3
Microelectrophoresis
Influenza A (PRb)
5.3
Moving boundary
T2 bacteriophage
4.2
Moving boundary
T4 bacteriophage
4-5
Gel
MS2 bacteriophage
3.9
Moving boundary
a -
b
Adapted from 53.
IEF: isoelectric focusing.

137
been covalently bound. These charged groups are associated with mobile
counter-ions that can be reversibly exchanged with other ions of the
same charge (4). Thus, an anion exchanger would have covalently bound
positive charges and negatively charged exchangable counter-ions, while
a cation exchanger would have negatively charged ions covalently bound
to the matrix, and positively charged exchangable counter-ions.
Ion exchange resins have been used to purify and concentrate
viruses. Muller (126) packed columns with a cation exchange resin and
passed virus-containing tissue homogenates through them. He found that
these columns removed extraneous protein and nitrogenous material, while
viruses, such as Japanese encephalitis virus, equine encephalomyelitis
virus and rabies virus, all passed through the column without appreciable
loss in titer.
LoGrippo (114) found that a strong-based anion exchanger could be
used to adsorb both extraneous nitrogenous materials and viruses present
in tissue homogenates and fecal matter. He was able to selectively
elute the adsorbed virus (Lansing poliomyelitis strain) with a 10%
disodium acid phosphate solution and recover the purified virus without
considerable decrease in titer. Later, Muller and Rose (127) found that
influenza virus in a crude extract of chorioallantoic fluid was
selectively adsorbed to a cation exchange column. Elution of the
adsorbed influenza was accomplished with reduced volumes of 10% sodium
chloride, so that virus was concentrated 8- to 16-fold.
Ion exchange resins have also been used to separate bacteria based
on their adsorption/elution patterns. Daniels and Kempe (30) studied
the adsorption to and subsequent elution from anion exchange resins of
six bacterial species. They found that elution of some bacteria

138
adsorbed to anion exchange resins could be accomplished by a reduction
in pH, while different bacteria were eluted more efficiently by the
addition of salt. Some bacteria required both salt and pH reduction for
elution to occur. In another report, a mixture of Salmonella
typhimurium and E. coli was adsorbed to a DEAE cellulose column (72).
During elution with increasing salt concentration, S. typhimurium came
off the column at a low salt concentration (<200 mM NaCl), while E. coli
remained bound until salt concentrations were almost 400 mM, thus
affording good separation of these bacteria. These authors suggested
that ion exchange may be used to separate or classify bacteria based on
differences in the occurrence and distribution of ionizable groups.
These data indicated that ion exchange may be one means of
determining differences in surface charge among viruses and thus aid in
preparing adsorption behavior to solids.
Hydrophobic Contributions
Until the late 1960's, proteins were separated from each other by
methods which were based on differences in their size, charge,
solubility and other physical parameters (152, 153). The development of
affinity chromatography, which made use of recognition of specific
receptors, enzymes or antibodies covalently linked to an inert backbone,
greatly facilitated separation techniques. In 1972, Er-al and coworkers
(38) were attempting to purify and study the enzyme glycogen
phosphorylase b using affinity chromatography. They activated agarose
with cyanogen bromide to attach the activated glycogen to a "spacer arm"
attached to the agarose. They found that if the spacer arm was eight
carbon atoms long, the enzyme adsorbed to the glycogen-agarose column,
but if the spacer arm was four carbon atoms long, the enzyme did not

139
adsorb at all. Attempts to explain this difference led researchers to
synthesize a variety of sepharose affinity columns which differed only
in the number of carbons in the spacer arm (38, 152). Results indicated
that it was possible to resolve and purify proteins on the basis of a
new criterion, that is, the hydrophobic nature of their surface. Thus,
a new type of affinity chromatography, hydrophobic chromatography, was
born.
Early methods used to synthesize hydrophobic materials imparted a
charge on to the matrix. These gel materials therefore exhibited ion
exchange properties superimposed upon the hydrophobic character, and as
such, early reports should be viewed with caution (79, 81, 82). Hjerton
and coworkers (81) developed a coupling method which imparted no charge
to the matrix, thus eliminating the ion exchange character.
Hydrophobic gels, such as pentyl-, phenyl-, octyl-, and dodecyl-
sepharose, have been used extensively to determine and compare
hydrophobic natures of proteins and bacterial species. Adsorption to
hydrophobic gels will depend upon the ionic strength and nature of the
suspending salt solution, temperature, flow rate and the hydrophobicity
of the attached groups (3, 80, 128, 130, 153). It was shown that
deodecyl-sepharose adsorbed ovalbumin and human serum albumin equally
well, while pentyl-sepharose did not adsorb ovalbumin as well as human
serum albumin (130). In another report, Ogamo et al. (128) also found
that the substituent hydrophobic group played a role in adsorption. In
addition, these researchers showed that solutions of a
mucopolysaccharide, heparin, in chaotropic salts did not adsorb to
hydrophobic columns, while antichaotropic salts, such as ammonium
sulfate, increased the adsorption of heparin to these columns (128).

140
Smyth and coworkers (160) tested the ability of porcine
enteropathogenic strains of E. coli to adsorb to octyl- and phenyl-
sepharose. They found that strains of E. coli which had the K88 antigen
were strongly adsorbed to these hydrophobic gels, while K88 negative
strains did not adsorb. These researchers suggested that the
hydrophobic nature of this antigen may be a simple means of separation
of K88 positive from K88 negative strains. Similar experiments were
conducted using hemagglutinating strains of Yersina enterocolitica (39).
Strains which possessed hemagglutinating fimbriae were bound tightly to
octyl-sepharose, whereas those lacking fimbriae did not adsorb to the
hydrophobic gel. In addition to its use in distinguishing between
strains of bacteria based on hydrophobicity, octyl-sepharose has been
used to study changes in cell surface hydrophobicity brought about by
starvation of marine organisms (96), and caused by the intraperiplasmic
growth of Bdellovibrio (28).
While hydrophobic chromatography has seen many uses with proteins
and bacteria, only a few references are available for its use with
viruses. Hjerten et al. (81) used octyl-sepharose to purify satellite
tobacco necrosis virus. Adsorption to the octyl-sepharose was complete
with 4 M NaCl was used as the adsorbing suspension. Elution of the
virus occurred as a broad peak as salt concentration was dropped to
between 2 and 1 M NaCl. Prusiner et al. (137) used adsorption to
phenyl-sepharose to show that scrapie agent, a slow virus infecting sheep
and goats, contained a hydrophobic protein. This protein was bound very
tightly to the phenyl-sepharose and required a solution of 8.5 M
ethylene glycol, 4% Nonidet P-40 and 2% sarkosyl to bring about elution.

141
Another method for determining surface hydrophobicity which has not
seen much use is the salt aggregation test. This test is based on the
principle that the more hydrophobic the surfaces of a cell, the lower
the salt concentration required to aggregate the cells (109).
Antichaotropic salts, such as ammonium sulfate, work best. Aggregation
is measured visually on glass slides held against a dark background.
Faris et al. (39) used this method along with hydrophobic chromatography
with octyl-sepharose to show that there was a good correlation between
the production of a Yersina hemagglutinin, the presence of fimbriae and
high surface hydrophobicity. The same group of researchers used the
salt aggregation test to examine enterotoxigenic E, coli strains with
different surface protein antigens (109). There is no information
available concerning the use of salt aggregation tests to determine
hydrophobicity in viruses.
While several methods are available to study hydrophobic
characteristics, no single method adequately described hydrophobicity
because various non-hydrophobic effects often interfere with
measurements, and experimental conditions employed may influence the
observed hydrophobic interactions to some degree (139). Due to this
problem, Rosenberg and coworkers developed a method to determine cell
surface hydrophobicity by adherence to hydrocarbons (139). Bacteria
were washed and resuspended in a low salt buffer solution. Varying
amounts of a hydrocarbon, such as hexadecane or xylene were added.
After a period of mixing, species of bacteria known to have a high
degree of cell surface hydrophobicity were found to partition into the
hydrocarbon phase, while other species did not. Makey (116) used this
technique to show large increases in hydrophobicity occurred in frozen

142
and EDTA treated E. coli cells. This increase in hydrophobicity was
related to an increase in the sensitivity of these organisms to
hydrophobic antibiotics. More recently, Kjelleberg and Hermansson (96)
used a adherence to hydrocarbons in conjunction with hydrophobic
chromatography using octyl-sepharose to determine the starvation-induced
effects on bacterial surface characteristics. Again, no information on
hydrocarbon adherence as a means of determining hydrophobicity in
viruses is available.
Materials and Methods
A list of chemicals and their sources, and routine methods used in
animal and bacterial virus preparation and assay are presented in
appendix A. A complete list of media and solutions used in cell culture
work is presented in appendix B.
Hydrophobic and Ion Exchange Gels
Octyl-sepharose CL-4B (Pharmacia Fine Chemicals, Piscataway, NJ) is
a hydrophobic derivative of the cross-linked agarose gel, sepharose, and
contains the hydrophobic n-octyl group. DEAE-sepharose CL-6B (Pharmacia
Fine Chemicals, Piscataway, NJ), an anion exchanger, is a derivative of
the cross-linked agarose gel, sepharose, and contains the positively
charged group diethylaminoethyl (DEAE).
Batch Adsorption Experiments
The sepharose derivative was washed at least 20 times with the
adsorbing solution to be tested (see Tables). After this equilibration,
one ml of the gel was placed in a 50 ml tube. Next, 3 ml of the
adsorbing solution containing 10^ PFU of virus was added. The tubes
were placed on a rotary shaker and shaken at high speed for 2 hours to

143
allow for adsorption to occur, the tubes were centrifuged at 12000 x g
for 10 minutes at 22°C. A portion of the supernatant was removed and
transferred to a sterile 13 mm test tube. These tubes were centrifuged
for 5 minutes at high speed in a Whisperfuge (Fisher Scientific Co.,
Fair Lawn, NJ) to remove any remaining gel. The supernatant was then
assayed for the presence of virus. All experiments were run in
triplicate and values represent the means of six determinations.
Column Adsorption Experiments
The sepharose derivative was washed approximately 10 times with
adsorbing solution, and 3 ml was poured slowly into glass columns, 0.7 x
10 cm (Bio-Rad, Rockville Center, NY), to avoid formation of bubbles.
Packed columns were rinsed with 10 bed volumes of the adsorbing
solution. After this equilibration, 5 ml of adsorbing solution
containing 10^ PFU virus was passed through the column at a rate of 2.5
ml/hr. The column was rinsed with 2 bed volumes of adsorbing solution,
and the void volume and rise were assayed to confirm adsorption of
virus. Elution of adsorbed virus was accomplished by passage of 3 bed
volumes of solution listed (see Tables) at a rate of 6 ml/hr. All
columns were run at room temperature and in triplicate. Values
represent the means of the percent total virus eluted as each solution
was passed through
the column.
Hydrocarbon Adherence Experiments
Approximately 10^ PFU virus was added to a solution of IX PBS (see
appendix B). After addition of virus, 2.5 ml of this solution was
placed in sterile 13 mm test tubes and varying amounts of hexadecane

144
(Sigma Chemical Co., St. Louis, MO) were added. Tubes were mixed on a
vortex for 2 minutes and the hydrocarbon phase was allowed to settle
(ca. 15 minutes). Samples were removed from the aqueous phase and
assayed for the presence of virus. All experiments were run two to
three times, and values represent the means of four to six
determinations.
Results
Association of Viruses with DEAE-sepharose
Animal and bacterial virus adsorption to DEAE-sepharose was
examined at pH 7 and 5.5. The results of animal virus adsorption to the
anion exchanger are shown in Table 21. At pH 7, adsorption varied with
the virus tested. When virus was suspended in 1 mM imidazole, less than
70% of echovirus-5 and less than 20% of coxsackievirus B3 were adsorbed
to the DEAE-sepharose, while all other viruses adsorbed very
efficiently. When the buffer concentration was increased to 10 mM,
there was no detectable adsorption of coxsackievirus B3. Poliovirus-1
adsorption was decreased to less than 40% under these conditions, while
echovirus-5 adsorption was lowered to 51%. Echovirus-4 adsorption,
which was virtually complete in 1 mM imidazole, was less than 80% in 10
mM imidazole. When 0.1 M NaCl was added to the buffer, there was a
decrease in adsorption of all viruses tested, with coxsackievirus B3
showing the least amount of adsorption (3%) and coxsackievirus B5 showed
the largest amount of virus adsorbed (64%). When the salt concentration
was increased to 0.25 M or 0.5 M NaCl, there was a gradual decrease in
the amount of all viruses adsorbed to the DEAE-sepharose. However, at
concentrations of 0.5 M NaCl, the amount of coxsackievirus B3 and
echovirus-5 adsorbed was increased (53 and 57% respectively).

145
TABLE 21. Adsorption of animal viruses to DEAE-sepharose.3
b
Solution
% virus
adsorbed
PI
CB3
CB4
CB5
El
E4
E5
pH 7
1 mM imidazole
99
18
100
100
100
100
66
10 mM imidazole
38
0
100
100
100
79
51
0.1 M NaCl
46
3
54
64
52
53
40
0.25 M NaCl
46
30
47
59
47
53
37
0.5 M NaCl
35
53
41
46
14
37
57
pH 5.5
1 mM imidazole
97
19
99
100
100
99
53
10 mM imidazole
73
42
100
96
95
43
30
0.1 M NaCl
41
0
38
53
16
37
39
0.25 M NaCl
29
22
49
29
0
40
0
0.5 M NaCl
47
18
36
42
10
48
38
a - Pi: poliovirus-1, CB3: coxsackievirus B3, CB4: coxsackievirus B4,
CB5: coxsackievirus B5, El: echovirus-1, E4: echovirus-4, E5:
echovirus-5.
b - All NaCl solutions contained 10 mM imidazole buffer.

146
At pH 5.5, in the presence of 1 mM imidazole, the adsorption of the
viruses tested was again very efficient except for coxsackievirus B3
(19% adsorption) and echovirus-5 (53% adsorption). When the
concentration of the buffer solution was increased to 10 mM, greater
than 95% of coxsackievirus B4, coxsackievirus B5 and echovirus-1 were
adsorbed to DEAE-sepharose. Less than 70% of poliovirus-1 was adsorbed,
while less than 50% of coxsackievirus B3, echovirus-4 and echovirus-5
adsorbed under these conditions. When 0.1 M NaCl was added to the
adsorbing solution, less than 50% of all viruses tested were adsorbed,
except for coxsackievirus B5 (53%) . Increasing the concentration of
NaCl to 0.25 M or 0.5 M led to a decrease in the adsorption of all
viruses tested. However, at a salt concentration of 0.5 M, we again saw
an increase in the adsorption of some of the viruses (Table 21).
The results of bacteriophage adsorption to DEAE-sepharose are shown
in Table 22. At pH 7, when viruses were suspended in 1 or 10 mM
imidazole, adsorption of all phage tested was greater than 95%. The
addition of 0.1 M NaCl to the adsorbing solution resulted in a
tremendous decrease in the adsorption of 4>xl74 (23% adsorption) while
all other viruses were virtually uneffected. An increase in salt
concentration to 0.25 M NaCl decreased adsorption to less than 60% for
all viruses tested, except for T2, T4 and MS2, which remained almost
completely adsorbed at this salt concentration. When 05. M NaCl was
used as an adsorbing solution, adsorption of <{>xl74 was less than 30%,
while less than 10% of MS2 was adsorbed. Greater than 80% of T2
remained adsorbed under these conditions, while the percent adsorption
of the other phages ranged between 54 to 65%.

147
TABLE 22. Adsorption of Bacteriophage to DEAE-sepharose.
% virus adsorbed
Solution^ T2
T3
T4
T7
$X174
MS 2
f 2
pH
7 1 mM imidazole
100
100
100
100
97
100
100
10 mM imidazole
100
99
100
100
96
100
100
0.1 M NaCl
100
99
100
100
23
100
99
0.25 M NaCl
98
42
99
45
24
99
58
0.5 M NaCl
84
54
65
55
26
7
58
pH
5.5 1 mM imidazole
100
100
100
100
91
100
100
10 mM imidazole
100
100
100
100
16
99
100
0.1 M NaCl
99
100
99
97
9
100
99
0.25 M NaCl
98
20
99
19
23
99
60
0.5 M NaCl
52
28
39
28
14
47
36
a - All NaCl solutions contained 10 mM imidazole.

148
At pH 5.5, the adsorption patterns of bacteriophage were very
similar to those seen at pH 7. All the viruses tested adsorbed to the
DEAE-sepharose when the adsorbing solution contained either
concentration of buffer or 0.1 M NaCl, except for 4¡xl74, which showed a
large decrease in adsorption, beginning with 10 mM imidazole. When the
salt concentration was increased to 0.25 M NaCl, again the adsorption of
T2, T4 and MS2 was nearly complete, while less than 25% of T3, T7 and
Xl74 was adsorbed. At the highest salt concentration, less than 50% of
all bacteriophages, except T2, were adsorbed (Table 22).
Column elution experiments using DEAE-sepharose were also examined.
Viruses were adsorbed to the gel in 1 mM imidazole and eluted by
increasing concentration of salt solutions (data not shown). These
results were nearly identical to those in the batch adsorption
experiments, that is, solutions which promoted adsorption did not elute
the viruses adsorbed to the DEAE-sepharose. Conversely, solutions which
blocked adsorption effectively eluted the adsorbed viruses. In all
cases but one, greater than 70% of the viruses adsorbed to the
DEAE-sepharose columns were recovered. The exception was bacteriophage
MS2. Recovery of MS2 adsorbed to DEAE-sepharose columns was never
greater than 25% (data not shown). This indicated that some
inactivation may have been taking place.
Association of Viruses with Octyl-sepharose
Viruses were suspended in 4 M NaCl, pH 7, and adsorbed to octyl-
sepharose in 10 cm columns. Elution of adsorbed viruses was
accomplished by decreasing salt concentrations. The results for the
elution of animal viruses adsorbed to octyl-sepharose are shown in Table
23. All viruses were completely adsorbed in 4 M NaCl. As 2 M NaCl was

TABLE 23. Elution of animal viruses adsorbed to octyl-sepharose3
% virus eluted (cummulative)
NaCl concentration
4 M
2 M
1 M 0.1 M 0
Tween 80
Virus
mean
sd
mean
sd
mean
sd
mean
sd
mean
sd
mean
sd
Poliovirus-1
0
0
1
0
31
15
66
17
69
15
83
11
Coxsackievirus B3
0
0
5
4
10
3
60
17
63
17
72
17
Coxsackievirus B4
0
0
7
7
42
18
83
17
88
17
100
15
Coxsackievirus B5
0
0
8
2
57
15
72
19
74
19
78
19
Echovirus-1
0
0
28
13
66
12
79
21
79
21
80
21
Echovirus-4
0
0
14
14
44
12
72
24
74
25
82
31
Echovirus-5
0
0
0
0
0
0
1
1
4
2
65
14
Echovirus-7
0
0
47
9
64
8
80
13
80
13
88
12
a - All solutions contained 0.05 M imidazole and were adjusted to pH 7.
b - SD standard deviation
149

150
passed through the column, greater than 45% of echovirus-7 was eluted.
Almost 30% of echovirus-1 was eluted by this solution, while less than
15% of any of the other viruses tested was removed. When the salt
concentration was dropped to 1 M NaCl, the total amount of adsorbed
virus eluted at this point was greater than 55% for coxsackievirus B5,
echovirus-1 and echovirus-7. Intermediate levels (between 30-45% total
virus eluted) of elution of adsorbed virus were found for poliovirus-1,
coxsackievirus B4 and echovirus-4. Less than 10% of coxsackievirus B3
or echovirus-5 were eluted. As the concentration of salt was decreased
so than only the buffer solution, 50 mM imidazole, was passed through
the columns, the amount of total virus eluted was over 60% for all the
animal viruses tested except for echovirus-5, which required a solution
of detergent to elute the adsorbed virus.
These same experiments were run using bacteriophages, and these
results are shown in Table 24. Again, the adsorbing solution, 4 M NaCl
had no effect of the elution of viruses adsorbed to octyl-sepharose.
When 2 M NaCl was passed through the column, almost 35% of the Xl74 was
eluted while only a little of the adsorbed T3 or T7 was eluted. This
solution had no effect on MS2 or f2 adsorbed to octyl-sepharose. When
1 M NaCl was passed through the column, the total amount of xl74 eluted
at this point was 87%. The amount of T3 and T7 was 29 and 28%
respectively. Still, little or no MS2 or f2 was eluted. As the
concentration of salt was decreased to until just the buffer solution
was passed through, the total amount of T3 eluted was 45%,and for T7 was
53%. Little difference in the total amount of virus eluted was noted
for other bacteriophages tested. The passage of Tween 80 through the
column increased the amount of total viruses eluted to greater than 65%

TABLE 24. Elution of bacteriophage adsorbed to octyl-sepharose3
% virus eluted (cummulative)
NaCl concentration
Virus
4
mean
M H
,b
sd
2
mean
M
sd
1
mean
M
sd
0.1
mean
M
sd
0
mean
sd
Tween
mean
80
sd
T3
0
0
8
6
29
13
38
17
45
19
66
17
T7
0
0
2
2
28
15
44
17
52
17
68
18
*X174
0
0
34
16
87
10
92
11
93
12
93
12
MS 2
0
0
0
0
0
0
0
0
2
2
54
11
f 2
0
0
0
0
1
1
4
2
5
3
13
4
a - All
solutions contained
0.05 M
imidazole and
were adjusted
to pH
7.
b - SD standard deviation

152
for T3 and T7. The MS2 was not eluted to an appreciable extent until
this solution was passed through, and resulted in 54% elution of the MS2
adsorbed to octyl-sepharose. Bacteriophage f2 was not effectively
recovered under these eluting conditions.
Virus Association to Hydrocarbons
The removal of selected animal viruses into a hexadecane phase is
shown in Figure 18. Coxsackievirus B5 did not partition into the
hexadecane phase regardless of the amount of hexadecane added. When the
amount of hexadecane was 0.5 ml or less, greater than 80% of echovirus-1
and coxsackievirus B4 were still found in the aqueous layer. Addition
of 0.75 ml of hexadecane decreased the amount of coxsackievirus B4 found
in the aqueous phase to 66%, while having little effect on echovirus-1.
The addition of 1 ml hexadecane removed most of the coxsackievirus B4
from the aqueous phase and decrease the amount of echovirus-1 to 47%.
Coxsackievirus B3 in the aqueous phase was rapidly removed as the
amount of hexadecane added was increased. When the amount of hexadecane
added was 0.5 ml or greater no coxsackievirus B3 was detected in the
aqueous phase. The partitioning of echovirus-5 and poliovirus-1 into
the hexadecane phase was quite similar, although echovirus-5 appeared to
be removed from the aqueous phase slightly more at lower hexadecane
concentrations.
The removal of bacteriophage from an aqueous phase by hexadecane is
shown in Figure 19. Bacteriophage <{>xl74 was found to remain in the
aqueous phase for the most part. Even when 1 ml of hexadecane was
added, almost 70% remained associated with the aqueous phase. For all
other bacteriophages tested, as the amount of hexadecane increased, the
amount of virus found in the aqueous phase decreased. This decrease

FIGURE 18. Removal of animal viruses from an aqueous phase by the addition of varying amounts
of hexadecane.

% VFUS N AQUEOUS PHASE
AMOUNT HEXADECANE ADDED CmD
154

FIGURE 19. Removal of bacterial viruses from an aqueous phase by the addition of varying
amounts of hexadecane.

% VRUS N AQUEOUS PHASE
IOCH
T4
AMOUNT HEXADECANE ADDED CmO

157
was very similar for T2 and T4. Bacteriophages T3 and T7 behaved
similarly also, with less than 50% removed when the amount of hexadecane
was 0.5 ml or less, and greater than 80% when the amount of hexadecane
added was 0.75 ml or greater. When 0.5 ml hexadecane was added, the
amount of MS2 found in the aqueous phase was 32%. As with other phages,
at higher hexadecane concentrations, less than 20% of MS2 was found in
the aqueous phase.
Discussion
When studying the factors that may influence virus adsorption to
solids, it is not always possible nor practical to evaluate these
parameters using a wide variety of viruses. Therefore, these studies
are often conducted using just one or two viruses. Poliovirus-1 has
often been used to serve as a model for enterovirus behavior in the
development of procedures to detect and/or concentrate viruses from
samples of tapwater (44, 70, 155, 163, 164), estuarine water (16, 45,
133) and sediments (14, 177), wastewater effluents (23, 83, 107) and
sludges (6, 62, 131), and soils (13, 86). However, recent studies have
indicated that the associations of poliovirus-1 with solids are not
necessarily similar to those observed for other enteroviruses. Studies
have shown that there are major differences between virus groups and
their associations with solids such as membrane filters (50, 154) ,
estuarine sediments (90, 100), clays (112, 145) and soils (55, 56).
The behavior of different viruses in their associations with
various solids is believed to be the result of differences in virion
surface charge or the hydrophobicity of the virus surface (11, 53, 156).
This, in turn, is related to the protein capsid which surrounds most
viruses. We have used ion-exchange chromatography, hydrophobic

158
interaction chromatography and hydrocarbon adherence to examine
differences in the electrostatic and hydrophobic nature of a variety of
animal and bacterial viruses.
Electrostatic contributions
The principle of ion-exchange is simple. An insoluble matrix has a
charged group covalently bound to it. Oppositely charged counter-ions
are attracted to the covalently bound ions. Exchange occurs when a
charged substance with a higher affinity for the bound ion replaces the
mobile counter-ion (4). We studied the adsorption of viruses to
DEAE-sepharose, an anion exchanger. We chose to use an anion exchanger
since under conditions found in most natural samples, viruses are often
above their isoelectric point and are therefore negatively charged (53,
174, 183).
Our results with adsorption to DEAE-sepharose indicated that there
were some distinct differences in the adsorption of various viruses.
For example, coxsackievirus B3 and echovirus-5 adsorbed very poorly to
the ion exchanger regardless of the pH or salt concentration. On the
other hand, all of the bacteriophages tested adsorbed well to the
DEAE-sepharose, with T2, T4 and MS2 adsorbing almost completely even in
relatively high salt solutions. A comparison of the adsorption patterns
of animal and bacterial viruses to DEAE-sepharose indicated that there
is not much similarity between the two. This raises the question of
whether or not bacteriophages, as a group, are suitable as models for or
indicators of enterovirus behavior.
We divided the viruses we tested into four groups, based on their
adsorption to DEAE-sepharose (Table 25). Group A consists of viruses
that were poorly adsorbed under the conditions of our experiments, and

159
includes coxsackievirus B3 and Echovirus-5. Poliovirus-1 is the only
member of Group B, which is characterized by weak adsorption to the
DEAE-sepharose. Group C is made up of viruses which adsorbed well in
the presence of low salt solutions at both pH values tested, and a
majority of the viruses we tested fall into this category. Group D
consists of viruses which remained bound to the DEAE-sepharose at both
pH 5.5 and 7 at higher salt concentrations. Members of this group
include bacteriophages T2, T4 and MS2. Under the conditions of the
batch adsorption tests, T2 remained bound to the DEAE-sepharose at the
highest salt concentration tested at pH 7. However, experiments were
run in which T2 was adsorbed to a packed column of DEAE-sepharose (data
not shown). Over 70% of this virus was eluted by a 1 M NaCl solution at
pH 7, indicating that T2 was not inactivated by adsorption to the ion
exchanger.
Isoelectric point data are not available for all the viruses we
tested, but generally, those viruses with lower isoelectric points were
found to adsorb more strongly to the DEAE-sepharose than those viruses
with higher isoelectric points. This is not surprising, as one would
expect the viruses with lower isoelectric points to be more strongly
negatively charged, and thus bind more tightly to the anion exchanger.
Gerba and coworkers examined the adsorption of different viruses to
soils and found that they could group these viruses based on the
strength of the adsorption to soils (55, 56). This grouping is
summarized in Table 25. Viruses in Group II, which adsorbed strongly to
the soils tested, tended to have lower isoelectric points. A comparison
of our grouping of viruses based on adsorption to DEAE-sepharose to the
grouping of viruses based on adsorption to soils yields some interesting

TABLE 25. Comparison of viruses adsorbed to soil and DEAE-sepharose.
Virus adsorption to soil3
Group I
Group II
Group III
weakly adsorbed
strongly
adsorbed
very weakly adsorbed
Coxsackievirus B4
Echovirus-1
4>Xl74
MS2
Poliovirus-1
Echovirus-7
Coxsackievirus B3
T4
T2
f 2
Virus adsorption to DEAE-
sepharose
Group A
Group B
Group C
Group D
very weakly adsorbed
weakly adsorbed
strongly adsorbed
very strongly adsorbed
Coxsackievirus B3
Echovirus-5
Poliovirus-1
Coxsackievirus
Coxsackievirus
Echovirus-1
Echovirus-4
4>Xl74
T3
T7
f 2
B4
B5
T2
T4
MS 2
a - adapted from 56.
160

161
points. Poliovirus-1 and coxsackievirus B3 were found to adsorb
strongly to soils, yet we found that these viruses did not adsorb well
to DEAE-sepharose. Conversely, Gerba et al. (56) found that
coxsackievirus B4, echovirus-1, Xl74 and MS2 did not adsorb well to
soils, yet our data indicates that these viruses adsorbed well to
DEAE-sepharose. These discrepancies can be reconciled by remembering
that the complex nature of soils will effect the adsorption patterns of
viruses greatly. Obviously, electrostatic attraction will not be the
only parameter that determines adsorption. It is therefore equally
obvious that these differences in adsorption are due to some other
force, perhaps hydrophobic interactions.
Hydrophobic contributions
We used two methods to determine the hydrophobic character of
viruses: adsorption to and subsequent elution from octyl-sepharose and
adherence to hydrocarbons.
We experience difficulty with some viruses when we attempted to
determine their hydrophobic nature by binding to octyl-sepharose. When
elution of bacteriophages T2 and T4 adsorbed to octyl-sepharose was
attempted, we recovered greater than 1000% the total input virus (data
not shown). This indicated that aggregates of viruses were somehow
being formed and subsequently broken up during the elution process.
Subsequent examination of the experimental conditions indicated that
suspension of T2 or T4 in 4 M NaCl (the adsorbing solution used for
octyl-sepharose) resulted in 90% decrease in PFU's. Electron microscopy
did not indicate the presence of viral aggregates, yet dilution of
viruses from 4 M NaCl solutions into protein solutions resulted in a 50
to 75% increase in PFU's detected. This indicated that the decrease in

162
PFU when T2 and T4 were placed in high salt solutions was not due to
inactivation. We believe two possible explanations exist. It has been
suggested that aggregation of microorganisms in salt solutions can serve
as a measurement of surface hydrophobicity (109). Since T2 and T4 were
found to adhere to hydrocarbons, it is possible that this type of
aggregation may have been occurring, and the preparation of the viruses
for electron microscopy may have disrupted these aggregates (19, 119).
Aggregation could be confirmed by differential centrifugation in sucrose
gradients (51). An alternative explanation lies in the structure of
these two phages. Both T2 and T4 are tailed phages, and studies have
shown that tail fiber configurations may greatly affect attachment
mechanisms (27, 53). It is possible that the high salt concentration
needed for adsorption to octyl-sepharose may have somehow altered the
tail configuration so that attachment to the host bacterium was reduced,
resulting in a decrease in PFU's detected. Another virus which
exhibited strange behavior during hydrophobic interaction chromatography
was f2, of which less than 15% was recovered from octyl-sepharose
columns. Batch adsorption experiments indicated that f2 adsorption was
greatly reduced as the salt concentration was dropped below 1 M NaCl
(data not shown). This indicated that once f2 was adsorbed to
octyl-sepharose, some sort of inactivation was occurring. Other work
in this laboratory has indicated that f2 is readily inactivated under a
variety of conditions (unpublished data).
Hydrophobic interactions chromatography results indicated that some
viruses were not very tightly bound to the octyl-sepharose, suggesting
that these viruses (echoviruses-1, -4, and -7, coxsackievirus B4, and
bacteriophage xl74) are not very hydrophobic in nature. On the other

163
hand, viruses such as echovirus-5 and bacteriophage MS2, adsorbed very
strongly to the octyl-sepharose, and needed a detergent solution to
cause elution.
Hydrocarbon adherence tests were conducted on some of the viruses.
For the most part, results of this test concurred well with
octyl-sepharose data. Viruses which adsorbed strongly to
octyl-sepharose were found to be removed from the aqueous phase by low
levels of hexacane. Conversely, those viruses which were not tightly
bound to the octyl-sepharose were not readily removed from the aqueous
phase by the addition of hexadecane, again indicating that the
hydrophobic nature of these viruses was less.
We ranked the viruses according to the strength of hydrophobic
interactions based on hydrocarbon partitioning, and did the same ranking
for hydrophobic interaction chromatography. We assigned arbitrary
values for the least to most hydrophobic, combined these values and came
up with a relative ranking of viruses based on the relative
hydrophobicity of their surfaces. This is shown in Figure 20.
Bacteriophage MS2 and echovirus-5 were found to be most hydrophobic in
nature of the viruses tested. The fact that MS2 is so hydrophobic in
nature is interesting. Zerda (188) examined the amino acid sequences
for the coat protein of MS2 using a computer program, found that most of
the amino acids are hydrophobic. Our results would tend to confirm this
finding.
Our ranking based on hydrophobic interactions can also be used to
help explain discrepancies indicated earlier concerning the adsorption
of viruses to soils and DEAE-sepharose. For example, coxsackievirus B3
was found to adsorb strongly to soils, yet it adsorbed very poorly to

FIGURE 20. Ranking of viruses based on relative hydrophobicity.

Most
Hydrophobic
MS 2 T2 CB4
> CB3 > T3 > T7 > PI > > >
E5 T4 E4
Least
Hydrophobic
f 2 >> El > CB5 > E7 > 4>x174
165

166
DEAE-sepharose. Results from hydrophobic tests indicate that
coxsackievirus B3 appears to have a high degree of surface
hydrophobicity. Since soils are quite complex, it would appear that the
strong adsorption of coxsackievirus B3 is the result of a combination of
electrostatic and hydrophobic interactions. An examination of the
relative hydrophobicity of viruses shows that xl74 is the least
hydrophobic of the viruses tested. This could be why xl74 was found to
adsorb so poorly to soils by Gerba et al. (56).
Because virus adsorption to DEAE- or octyl-sepharose varies with
different viruses, it is theoretically possible to separate viruses
based on these differences. For example, a mixture of poliovirus-1,
coxsackievirus B3 and echovirus-1 could be separated using adsorption to
DEAE-sepharose in 1 mM imidazole at pH 7. Coxsackievirus B3 should pass
through the column virtually unadsorbed. Poliovirus-1 adsorbed to the
DEAE-sepharose could be eluted by a solution of 10 mM imidazole, while
echovirus-1 would remain adsorbed. Echovirus-1 could then be eluted by
a NaCl solution.
Similarly, viruses could be separated based on adsorption to and
subsequent elution from octyl-sepharose. We successfully separated a
mixture of poliovirus-1 and bacteriophage MS2 using this resin (data not
shown). A mixture of viruses was adsorbed to octyl-sepharose, and over
70% of the adsorbed poliovirus-1 was recovered in a buffer eluate, while
MS2 remained adsorbed and was recovered by elution with Tween 80.
These data show that variability in adsorption and elution patterns
observed for different viruses (50, 55, 56, 154) can be related to the
relative electrostatic or hydrophobic nature of the viruses.

CHAPTER V
SUMMARY
In this study, we have modified a procedure for the concentration
of enteroviruses from tapwater. These modifications are: i) the use of
acetic acid in place of hydrochloric acid to condition the tapwater
prior to virus adsorption onto a Filterite filter; ii) the use of a
chaotropic salt, sodium trichloroacetate, for elution of the adsorbed
virus; and iii) a second-stage concentration procedure using a second
membrane filter (Seitz S) that differs from the first filter.
We characterized four positively charged and two negatively charged
filters by contact angle and capillary rise measurements. Based on
these results, we were able to rank these filters according to their
relative hydrophobic and hydrophilic nature. We found this correlated
well to the ability of different solutions to elute viruses adsorbed to
the filters. We utilized these differences to develop a two-step
procedure for the concentration of bacteriophages from various natural
waters using dissimilar filters in each step.
The ability of ammonium sulfate to flocculate proteins was
incorporated into several concentration procedures. Ammonium sulfate
flocculation of beef extract solutions gave consistently higher
recoveries of viruses than organic flocculation of the same virus-
containing solutions. Ammonium sulfate flocculation was used
successfully as a second stage concentration procedure in the recovery
of viruses from effluents, wastewater sludges and soils.

168
The association of viruses to clays at pH 9 was examined. Results
indicated that hydrophobic interactions between the virus and clay
particles were important in the association of poliovirus-1 and
bacteriophage T7 to both kaolin and bentonite.
Finally, viruses were evaluated to determine their relative
hydrophobic and electrostatic character. Adsorption to and elution from
octyl-sepharose, as well as hydrocarbon adherence were used to
characterize the relative hydrophobicity of the viruses. Adsorption to
DEAE-sepharose was used to determine the relative strength of
electrostatic interactions. We found that viruses differed greatly in
their relative hydrophobic and electrostatic nature. These differences
can be used to explain variability in virus adsorption to and elution
from various solids.

APPENDIX A
ROUTINE METHODS USED IN ANIMAL
AND BACTERIAL VIRUS PROPAGATION AND ASSAY
Cell Culture Preparation
Buffalo green monkey kidney (BGM) cells (Flow Laboratories,
McLean, VA) were grown in Eagle's Minimal Essential Media (MEM)
supplemented with 10% fetal calf serum (FCS) (Flow Laboratories) (see
appendix B) in a 32 oz. bottle until a confluent monolayer was obtained.
This monolayer was washed twice with 10 ml of Gey's A (see appendix B)
to remove both salts and traces of serum. The monolayer was then
covered with 10 ml of a trypsin-versene solution (see appendix B) for 60
seconds. After removal of 9 ml, the remaining trypsin was kept on the
monolayer until cells had come off the glass (approximately 3 minutes).
Nine ml of MEM plus 10% FCS was added and pipetted up and down twice to
dislodge any cells remaining adhered to the glass and to break up clumps
of cells. Next, 150 ml of MEM/MT plus 10% FCS (see appendix B) was
added to the cell mixture. This mixture was dispensed into 6-well
microtiter plates (Flow Laboratories) (3 ml per well) and incubated at
37°C for 48 hrs, until a confluent monolayer was obtained, and were then
used for animal virus assay.
Virus Stock Preparation
Animal viruses used in this study were Poliovirus-1 (LSc strain,
ATCC VR 59), Coxsackievirus B3 (Nancy), Coxsackievirus B4 (natural
isolate), Coxsackievirus B5 (ATCC VR 689), Echovirus-1 (Farouk),

170
Echovirus-4 (natural isolate), Echovirus-5 (ATCC 35), and Echovirus-7
(Wallace). To prepare all enteroviruses used in this study, a 32 oz.
bottle of BGM cells in monolayer was rinsed with IX Phosphate Buffered
Saline (PBS) plus antibiotics (see appendix B) and 0.25 ml of virus was
added and bottle was left undisturbed for 30-40 minutes to allow for
viral adsorption. After adsorption period, 50 ml of MEM plus 10% FCS
was added and cells were incubated at 37°C until cytopathic effect (CPE)
was visible (approximately 2 days). The bottle containing virus was
frozen and thawed 3 times. The virus-cell mixture was transferred to a
blender (Waring) and mixed at high speed with an equal volume of
trichlorotrifluorethane (freon). This was placed in a sepatory funnel
and the bottom phase, containing extracted cellular debris and serum
proteins, was disgarded. The top phase, which contained viruses, was
subjected to extraction by freon at least two more times to remove all
traces of serum and other proteins. After the final extraction, the
phase containing virus was transferred to test tubes and centrifuged at
15,000 x g for 10 minutes to remove cellular debris. The supernatant
was transferred to sterile 25 ml tubes and centrifuged at 50,000 x g for
one hour. The pellet, containing the concentrated virus, was
resuspended using PBS with antibiotics (see appendix B) in 1/10 the
original volume. The concentrated virus was titered and stored at -20°C
until needed.
Animal Virus Assays
Viruses were assayed on BGM cells with a routine plaque procedure.
One-tenth ml of virus-containing solution was adsorbed to a monolayer of
BGM cells and allowed to attach. After 30 minutes, a 1.5% methyl
cellulose solution (see appendix B) was used as an overlay, and cells

171
were incubated for 48 hours at 37°C. Following the incubation period,
the methyl cellulose solution was removed and monolayers were rinsed
gently with tap water, followed by staining with a 1% crystal violet
solution. The dye was rinsed off with monolayer with tap water, and the
plaques were counter after stained monolayers were allowed to dry
(usually overnight).
Bacteria
Bacteria used in this study, obtained from the American Type
Culture Collection (Rockville, MD), were Escherichia coli B (ATCC
11303), E. coli K-13 (ATCC 15766), E. coli C (ATCC 13706) and E. coli
C-3000 (ATCC 15597). Each culture was grown on Standard Plate Count
Agar slants (BBL, Fisher Scientific Co., Fair Lawn, NJ) at 30°C,
then streaked out on MacConkey Agar (BBL) to check for purity. When
needed, a typical colony was picked from the MacConkey plate and
transferred to a 3% solution of trypticase soy broth (TSB) (BBL) and
incubated at 37°C overnight.
Bacteriophage and Preparation of High-Titer Phage Stocks
Bacteriophages used in this study were: MS2 (Host: E. coli
C-3000), f 2 (Host: E. coli K-13), xl74 (Host: E. coli C) , T2, T3, T4,
and T7 (Host for T phage: E. coli B). To prepare high titer stock, 10
plates showing confluent lysis were prepared with phage and appropriate
host. A total volume of 30 ml of either 3% TSB or M9 media (1) was
poured on the plates. The top agar was carefully scraped off of each
plate and collected in a sterile beaker with stirring bar. Three ml of
chloroform was added and the beaker was mixed on a magnetic stirring
plate for 10 minutes. This mixture was placed in glass centrifuge tube

172
and spun at 2000 RMP for 10 minutes to remove chloroform and agar
debris. The supernatant was collected and transferred to sterile
centrifuge tubes and spun at 15,000 x g for 10 minutes to remove
cellular debris. Phage was titered and stored at 4°C until needed.
Bacteriophage Assay
Bacterial viruses were assayed using a standard soft-agar overlay
procedure (1). Briefly, one-tenth ml of host bacteria and one-tenth ml
of virus containing solution were mixed and incubated at room
temperature for 10 minutes. Four ml of warm (50°C) top agar overly
(0.6% agar in 3% TSB) were added, and the solution was poured over a
Standard Plate Count Agar plate supplemented with 0.1% beef extract.
The plates
were incubated at 37°C for 12 hours and checked for plaque production.
Chemicals
The chemicals used in this study and their sources were as follows:
potassium hydrogen phthalate, sodium chloride, calcium chloride,
magnesium chloride, sodium sulfate, sodium citrate, potassium phosphate
monbasic, potassium phosphate dibasic, chloroform, imidazole,
hydrochloric acid, sodium hydroxide, orthotolidine, carbon
tetrachloride, and t-butanol from Fisher Scientific Co., Fair Lawn, NJ;
sodium trichloroacetate, lysine, aluminum chloride,
cetyltrimethylammonium bromide, Tween 80, Triton QS-9, Nonidet P-40,
riboflavin, citric acid, ammonium sulfate and hexadecane from Sigma
Chemical Co., St. Louis, MO; brain heart infusion, tryptose phosphate
broth and casiton from Difco Laboratories, Detroit, MI; and beef extract
from Scott Laboratories, Miami, FL.

APPENDIX B
COMPOSITON OF MEDIA AND SOLUTIONS
USED IN CELL CULTURE WORK
1.Gey's salt solutions are common ingredients for cell culture media
and solutions:
Gey's A (10X): 70 grams NaCl
3.7 grams KC1
3.01 grams Na HPO • 12H 0
0.237 grams Kf^PCT
100 ml 0.1% phenol red
10 grams glucose
900 ml distilled water
5 ml chloroform
This stock solution of Gey's A is stored at room
temperature unautoclaved, and is diluted 1:10 and
autoclaved when needed.
Gey's B:
Gey's C:
0.42 grams MgCl^
0.14 grams MgSO^
0.34 grams CaCl
100 ml distilled
• 6H 0
• 7^0
water
2.25 grams NaHCO^
100 ml distilled water
Gey's B and C are autoclaved without further dilution.
2. Hepes buffer (1 M) stock solution:
47.7 grams Hepes
190 ml Gey’s A (IX)
10 ml Gey's B
16 ml 2 M NaOH
Adjust to pH 7.31, dispense and autoclave.
3. Streptomycin-penicillin (1000X) stock solution:
Solution I: 1.0 gram streptomycin
9 ml Gey's A (IX)
0
Solution II: 10 units of penicillin
4 ml of solution I
Solution II contains 125 mg of streptomycin and 2.5 X 10
units of penicillin. This solution is filter sterilized
and frozen until needed.

174
Gentamycin (1000X) stock solution:
0.5 grams of gentamycin sulfate
10 ml distilled water
This solution contains 50 mg of gentamycin and is filter
sterilized and frozen until needed.
Eagle's Minimal Essential Media (MEM) plus 10% fetal calf serum
(FCS):
Regular MEM:
Microtiter MEM:
(MEM/MT)
400 ml sterile distilled water
50 ml 10X MEM
25 ml Gey's C
15 ml Hepes buffer
5 ml 1-glutamine
0.5 ml streptomycin-penicillin stock
50 ml FCS
400 ml sterile distilled water
50 ml 10X MEM
15 ml Gey's C
25 ml Hepes buffer
5 ml 1-glutamine
0.5 ml streptomycin-penicillin stock
0.5 ml gentamycin stock
50 ml FCS
MEM and MEM/MT are kept refrigerated until needed.
Solutions required for trypsination of cells:
Solution I (pre-trypsin wash):
100 ml Gey's A (IX)
4 ml Gey's C
This solution removes all traces of serum as well as
magnesium and calcium ions.
Solution II (versene stock) :
2 grams ethylenediaminetetraacetic acid
(EDTA)
10 ml 2 M NaOH
20 ml Gey's A (10X)
170 ml distilled water
Adjust to pH 7.2, dispense and autoclave.

175
Solution III (standard trypsin-versene solution):
100 ml Gey's A (IX)
5 ml Gey's C
4 ml 2.5% trypsin
4 ml stock versene
This solution is made just prior to use, and is only good
for one day.
7. Methyl cellulose overlay:
Solution I: 300 ml distilled water
6 grams methyl cellulose (1500 centipoise)
Autoclave and then allow to cool to room temperature,
shaking vigorously every hour to avoid layering.
Refrigerate.
Solution II (2X MEM/MT):
350 ml sterile distilled water
120 ml 10X MEM
60 ml Gey's C
60 ml FCS
50 ml Hepes buffer
12 ml 1-glutamine
1.2 ml streptomycin-penicillin stock
1.2 ml gentamycin stock
Combine equal amounts of solutions I and II to make methyl
cellulose overlay. Refrigerate.
8. Phosphate-buffered saline (PBS):
8 grams NaCl
0.2 grams KC1
1.15 grams Na HPO^
0.2 grams KH^pO^
1000 ml distilled water
Adjust to pH 7.2 and autoclave. Add antibiotics as
needed.

BIBLIOGRAPHY
1. Adams, N.H. 1959. Bacteriophages, Interscience Publishers, Inc.,
New York, NY.
2. American Public Health Association. 1975. Standard Methods for
the Examination of Water and Wastewater, 14th ed., American Public
Health Association, Inc., New York, NY.
3. Anon. 1976. Octyl-sepharose CL-4B, Phenyl-Sepharose CL-4B for
hydrophobic interaction chromatography, Pharmacia Fine Chemicals,
Uppsala, Sweden.
4. Anon. 1983. Ion Exchange Chromatography, Principles and Methods,
Pharmacia Fine Chemicals, Uppsala, Sweden.
5. Armón, R., Y. Kott and I. Neeman. 1984. Ghost cells as a sorption
matrix for virus concentration from water. Appl. Environ.
Microbiol. 47:1337-1340.
6y Berg, G., D. Berman and R.S. Safferman. 1982. A method for
concentrating viruses recovered from sewage sludges. Can. J.
Microbiol. 28:553-556.
7/ Berg, G., D.R. Dahling, G.A Brown and D. Berman. 1978. Validity
of fecal coliforms, total coliforms and fecal streptococci as
indicators of viruses in chlorinated primary sewage effluents.
Appl. Environ. Microbiol. 36:880-889.
8. Berg, G. and T.G. Metcalf. 1978. Indicators of viruses in water,
p. 267-296. In G. Berg (ed.), Indicators of Viruses in Water and
Food, Ann Arbor Science, Ann Arbor, MI.
9. Berg, G., R.S. Safferman, D.R. Dahling, D. Berman and C.J. Hurst.
1984. USEPA Manual of Methods for Virology. EPA-600/4-84-013.
10. Berman, D., G. Berg and R.S. Safferman. 1981. A method for
recovering viruses from sludges. J. Virol. Methods 3:283-291.
J
11. Bitton, G. 1975. Adsorption of viruses onto surfaces in soil and
water. Water Res. 9:473-484.
12. Bitton, G. 1980. Introduction to Environmental Virology, John
Wiley and Sons, Inc., New York, NY.
13. Bitton, G., M.J. Charles and S.R. Farrah. 1979. Virus detection
in soils: A comparison of four recovery methods. Can. J.
Microbiol. 25:874-880.
1 7£

177
14. Bitton, G., Y.-J. Chou and S.R. Farrah. 1982. Techniques for
virus detection in aquatic sediments. J. Virol. Meth. 4:1-8.
15. Bitton, G., S.R. Farrah, O.C. Pancorbo and J.M. Davidson. 1981.
Fate of viruses following land application of sewage sludge. I.
Survival and transport patterns in core studies under natural
conditions, p. 133-136. In M. Goddard and M. Butler (eds.),
Viruses and Wastewater Treatment, Pergamon Press, New York, NY.
16. Bitton, G., B.N. Feldberg and S.R. Farrah. 1979. Concentration of
enteroviruses from seawater and tapwater by organic flocculation
using non-fat dray milk and casein. Water, Air and Soil Pollut.
12:189-195.
17. Bitton, G., N. Masterson and G.E. Gifford. 1976. Effect of a
secondary treated effluent on the movement of viruses through a
cypress dome soil. J. Environ. Qual. 5:370-375.
18. Bitton, G., O.C. Pancorbo and S.R. Farrah. 1984. Virus transport
and survival after land application of sewage sludge. Appl.
Environ. Microbiol. 47:905-909.
19. Brenner, S. and R.W. Horne, 1959. A negative staining method for
high resolution electron microscopy of viruses. Biochim. Biophys.
Acta 34:103-110.
20. Burge, W.D. and N.K. Enkiri. 1978. Adsorption kinetics of
bacteriophage <|>xl74 on soil. J. Environ. Qual. 7:536-541.
21. Burge, W.D. and N.K. Enkiri. 1978. Virus adsorption by five
soils. J. Environ. Qual. 7:73-76.
22. Carlson, G.F., Jr., F.E. Woodard, D.F. Wentworth and O.J. Sproul.
1968. Virus inactivation on clay particles in natural waters. J.
Water Pollut. Control Fed. 40:R89-R106.
23. Chang, L.T., S.R. Farrah and G. Bitton. 1981. Positively charged
filters for virus recovery from wastewater treatment plant
effluents. Appl. Environ. Microbiol. 42:921-924.
24. Clark, N.A., R.E. Stevenson, S.L. Chang and P.W. Kabler. 1961.
Removal of enteric viruses from sewage by activated sludge
treatment. Am. J. Public Health 51:1118-1129.
25. Cliver, D.O. 1967. Enterovirus detection by membrane
chromatography, p. 139-149. In G. Berg (ed.), Transmission of
Viruses by the Water Route, John Wiley and Sons, New York, NY.
26. Cliver, D.O. 1968. Virus interaction with membrane filters.
Biotechnol. Bioeng. 10:877-889.

178
27. Cookson, J.T. 1967. Adsorption of viruses on activated carbon:
Adsorption of E. coli bacteriophage T4 on activated carbon as a
diffusion-limited process. Env. Sci. and Technol. 1:157-160.
28. Cover, W.H. and S.C. Rittenberg. 1984. Change in the surface
hydrophobicity of substrate cells during Bdelloplast formation by
Bdellovibrio bacteriovirus 109J. J. Bacteriol. 157:391-397.
29. Dandiker, W.B., R. Alonso, V.A. deSaussure, F.Kierszenbaum, S.A.
Levinson and H.C. Schapiro. 1967. The effect of chaotropic ions
on the dissociation of antigen-antibodies complexes. Biochemistry
6:1460-1467.
30. Daniels, S.L. and L.L. Kempe. 1966. The separation of bacteria by
adsorption onto ion exchange resin. Chem. Eng. Prog., Symp. Ser.
62:142-148.
31. Deetz, T.R., E.M. Smith, S.M. Goyal, C.P. Gerba, J.J. Vollet, L.
Tsain, H.L. DuPont and B.H. Keswick. 1984. Occurrence of rota-
and enteroviruses in drinking and environmental water in a
developing nation. Water Res. 18:576-571.
3^ Dingier, L.C. 1985. Factors affecting the association of phage
with sludge floes, Master's thesis, University of Florida,
Gainesville, FL.
33. Dobbs, R.A., R.H. Wise and R.B. Dean. 1972. The use of
ultraviolet adsorbsance for monitorying the total organic carbon
content of water and wastewater. Water Res. 6:1173-1180.
34. Drewry, W.A. and R. Eliassen. 1968. Virus movement in
groundwater. J. Water Pollut. Control Fed. 40:R257-R271.
35. Drury, D.F. and D.C. Wheeler. 1982. Applications of a Serratia
marcescens bacteriophage as a new microbial tracer of aqueous
environments. J. Appl. Bacteriol. 53:137-142.
36. Dubouise, S.M., B.P. Sagik, B.E.D. Moore and J.F. Malina, Jr.
1974. Virus migration through soils, p. 233-240. In J.F. Malina,
Jr. and B.P. Sagik (eds.), Virus Survival in Water and Wastewater
Systems, Center for Research in Water Resources, Austin, TX.
37. Ellford, W.J. 1931. A new series of graded collodion membranes
suitable for general bacteriological use, especially in filterable
virus studies. J. Pathol. Bacteriol. 34:505-535.
38. Er-el, Z., Y. Zaidenzaig and S. Shaltiel. 1972. Hydrocarbon
coated sepharoses: Use in purification of glycogen phosphorylase.
Biochem. Biophys. Res. Commun. 49:383-391.
Faris, A., M. Lindahl, A. Ljungh, D.C. Old,and T. Wadstrom. 1983.
Autoaggregating Yersinia enterocolitica express surface fimbriae
with high surface hydrophobicity. J. Appl. Bacteriol. 55:97-100.
39.

179
40. Farrah, S.R. 1982. Chemical factors influencing adsorption of
bacteriophage MS2 to membrane filters. Appl. Environ. Microbiol.
43:659-663.
41. Farrah, S.R. 1982. Isolation of virus associated with sludge
particles, p. 161-170. In C.P. Gerga and S.M. Goyal (eds.),
Methods in Enviromental Virology, Marcel Dekker, New York, NY.
42. Farrah, S.R. and G. Bitton. 1979. Low molecular weight
substitutes for beef extract as eluants for poliovirus adsorbed to
membrane filters. Can. J. Microbiol. 25:1045-1051.
43. Farrah, S.R. and G. Bitton. 1982. Methods (other than microporous
filters) for concentration of viruses from water, p. 117-150. In
C.P. Gerba and S.M. Goyal (eds.), Methods in Environmental
Virology. Marcel Dekker, Inc., New York, NY.
44. Farrah, S.R., C.P. Gerba, C. Wallis and J.L. Melnick. 1976.
Concentration of viruses from large volumes of tap water using
pleated membrane filters. Appl. Environ. Microbiol. 31:221-226.
45. Farrah, S.R., S.M. Goyal, C.P. Gerba, C. Wallis and J.L. Melnick.
1977. Concentration of enteroviruses from estuarine water. Appl.
Environ. Microbiol. 33:-1192-1196.
46. Farrah, S.R., S.M. Goyal, C.P. Gerba, C. Wallis and P.T. B.
Shaffer. 1976. Characteristics of humic acid and organic
compounds concentrated from tapwater using the Aquella Virus
Concentrator. Water Res. 10:897-901.
47. Farrah, S.R. and S.A. Schaub. 1983. Viruses in wastewater
sludges, p. 147-161. In G. Berg (ed.), Viral Pollution of the
Environment, CRC Press, Boca Raton, FL.
48. Farrah, S.R., P.R. Scheuerman and G. Bitton. 1981. Urea-lysine
method for recovery of enteroviruses from sludge. Appl. Environ.
Microbiol. 41:455-458.
49. Farrah, S.R., D.O. Shah and L.O. Ingram. 1981. Effect of
chaotropic and antichaotropic agents on elution of poliovirus
adsorbed on membrane filters. Proc. Natl. Acad. Sci. U.S.A.
62:1129-1136.
50. Farrah, S.R. and P.A. Shields. 1982. Factors influencing the
association of viruses with membrane filters, p. 101-106. In M.
Butler, A.R. Medien and R. Morris (eds.), Viruses and Disinfection
of Water and Wastewater, Univ. of Surrey Press, England.
51. Floyd, R. and D.G. Sharp. 1978. Viral aggregation: Quantitation
and kinetics of the aggregation of poliovirus and reovirus. Appl.
Environ. Microbiol. 35:1079-1083.

180
52. Gerba, C.P. 1983. Methods for recovering viruses from the water
environment, p. 19-35. In G. Berg (ed.), Viral Pollution of the
Environment, CRC Press, Boca Raton, FL.
y
53. Gerba, C.P. 1985. Applied and theroetical aspects of virus
adsorption to surfaces. Adv. Appl. Microbiol. 30:133-168.
54. Gerba, C.P., S.R. Farrah, S.M. Goyal, C. Wallis and J.L. Melnick.
1978. Concentration of enteroviruses from large volumes of tap
water, treated sewage and seawater. Appl. Environ. Microbiol.
35:540-548.
55. Gerba, C.P. and S.M. Goyal. 1978. Adsorption of selected
enteroviruses to soils, p. 225-232. In H.L. McKim (ed.), State of
Knowledge in Land Treatment of Wastewater, vol 2, US Army CREEL,
Hanover, NH.
56. Gerba, C.P., S.M. Goyal, I. Chec and G.F. Bogdan. 1981.
Quantitative assessment of the adsorptive behavior of viruses to
soils. Environ. Sci. Technol. 15:940-944.
57. Gerba, C.P., S.M. Goyal, R.L. LaBelle, I. Cech and G.F. Bodgan.
1979. Failure of indicator bacteria to reflect the occurence of
enteroviruses in marine water. Am. J. Publ. Health 69:116-1119.
58. Gerba, C.P. and J.C. Lance. 1978. Poliovirus removal from primary
and secondary sewage effluent by soil filtration. Appl. Environ.
Microbiol. 36:247-251.
59. Gerba, C.P., J.B. Rose and S.R. Singh. 1985. Waterborne
gastroenteritis and viral hepatitis. CRC Critical Reviews in
Environ. Controls 15:213-236.
v/
60. Gerba, C.P., C. Wallis and J.L. Melnick. 1975. Viruses in water:
the problem, some solutions. Environ. Sci. and Technol.
9:1122-1126.
61. Gerba, C.P., C. Wallis and J.L. Melnick. 1975. Fate of wastewater
bacteria and viruses in soil. J. Irr. Drain. Div., ASCE
101:157-174.
62. Glass, J.S., R.J. Van Slius and W.A. Yanko. 1978. Practical
method for detecting poliovirus in anaerobic digester sludge.
Appl. Environ. Microbiol. 35:983-985.
/
63. Goyal, S.M. 1983. Indicators of viruses, p. 211-230. In G. Berg
(ed.), Viral Pollution of the Environment, CRC Press, Inc., Boca
Raton, FL.
64. Goyal, S.M. and C.P. Gerba. 1982. Concentration of viruses from
water by membrane filters, p. 59-116. In C.P. Gerba and S.M. Goyal
(eds.), Methods in Environmental Virology, Marcel Dekker, New York,
NY.

181
65. Goyal, S.M., B.H. Keswick and C.P. Gerba. 1984. Viruses in
groundwater beneath sewage irrigated cropland. Water Res.
18:299-302.
66. Goyal, S.M., S.A. Schaub, F.M. Wellings, D. Berman, J.S. Glass,
C.J. Hurst, D.A. Brashear, C.A. Sorber, B.E. Moore, G. Bitton, P.H.
Gibbs and S.R. Farrah. 1984. Round robin investigation of methods
for recovering human enteric viruses from sludge. Appl. Environ.
Microbiol. 48:531-538.
Goyal, S.M., K.S. Zerda and C.P. Gerba. 1980. Concentration of
coliphages from large volumes of water and wastewater. Appl.
Environ. Microbiol. 39:85-91.
Grabow, W.O.K. 1968. The virology of wastewater treatment. Water
Res. 2:675-701.
69. Guttman-Bass, N. and J. Catalano-Sherman. 1985. Effects of humic
materials on virus recovery from water. Appl. Environ. Microbiol.
49:1260-1264.
70. Guttman-Bass, N., T. Hostovsky, M. Lugten and R. Armón. 1985. A
comparison of current methods of poliovirus concentration from
tapwater. Water Res. 19:85-88.
71. Guttman-Bass, N. and A. Nasser. 1984. Simultaneous concentration
of four enteroviruses from tap, waste, and natural waters. Appl.
Environ. Microbiol. 47:1311-1315.
72. Hall, A.N., S.D. Hogg and G.O. Phillips. 1976. Gradient elution
of Salmonella typhimuruim and Escherichia coli strains from a
DEAE-cellulose column. J. Appl. Bacteriol. 41:189-192.
73. Hatefi, Y. and W.G. Hanstein. 1969. Solubilization of particulate
proteins and nonelectrolytes by chaotropic agents. Proc. Natl.
Acad. Sci. U.S.A. 62:1129-1136.
74. Hatefi, Y. and W.G. Hanstein. 1974. Destabilization of membranes
with chaotropic ions. Methods Ezymol. 31:770-790.
75. Havelaar, A.H. and W.M. Hogeboom. 1983. Factors affecting the
enumeration of coliphages in sewage and sewage-polluted waters.
Antonie von Leeuwenhoek 49:389-397.
76. Hejkal, T.W., B. Keswick, R.L. LaBelle, C.P. Gerba, Y. Sanchez, G.
Dreesman and B. Hafkin. 1982. Viruses in a community water supply
associated with an outbreak of gastroenteritis and infectious
hepatitis. J. Amer. Water Works Assoc. 74:318-327.
77. Hiemenz, P.C. 1977. Principles of Colloid and Surface Chemistry,
p. 224. Marcel Dekker, Inc. New York, NY.
78. Hilton, M.C. and G. Stotzky. 1973. Use of coliphages as
indicators of water pollution. Can. J. Microbiol. 19:747-751.

182
79. Hjerten, S. 1976. Hydrophobic interaction chromatography of
proteins on neutral adsorbents, p. 233-243. In N. Catsimpoolas
(ed.), Methods of Protein Separation, Plenum Press, New York, NY.
80. Hjerten, S. 1978. Fractionation of membrane proteins by
hydrophobic interaction chromatography and by chromatography on
agarose equilibrated with a water-alcohol mixture of low or high
pH. J. Chromatogr. 159:85-91.
81. Hjerten, S., J. Rosengren and S. Pahlman. 1974. Hydrophobic
interaction chromatography: The synthesis and use of some alkyl
and aryl derivatives of agarose. J. Chromatogr. 101:281-288.
82. Hofstee, B.H.J. 1976. Hydrophobic adsorption chromatography of
proteins, p. 245-278. In N. Catsimpoolas (ed.), Methods of Protein
Separation, Plenum Press, New York, NY.
83. Homma, A., M.D. Sobsey, C. Wallis and J.L. Melnick. 1973. Virus
concentration from sewage. Water Res. 7:945-950.
84. Hurst, C.J., D.R. Dahling, R.S. Safferman and T. Goyke. 1984.
Comparison of commercial beef extracts and similar materials for
recovering viruses from environmental samples. Can. J. Microbiol.
30:1253-1263.
85. Hurst, C.J., S.R. Farrah, C.P. Gerba and J.L. Melnick. 1978.
Development of quantitative methods for the detection of
enteroviruses in sewage during activation and following land
disposal. Appl. Environ. Microbiol. 36:81-89.
86. Hurst, C.J. and C.P. Gerba. 1979. Development of quantitative
method for the detection of enteroviruses in soil. Appl. Environ.
Microbiol. 37:626-632.
87. Hurst, C.J. and T. Goyke. 1983. Reduction of interfering
cytotoxicity associated with wastewater sludge concentrates assayed
for indigenous enteric viruses. Appl. Environ. Microbiol.
46:133-139.
88. IAWPRC Study Group on Water Virology. 1983. The health
significance of viruses in water. Water Res. 17:121-132.
/
89. Irving, L.G. and F.A. Smith. 1981. One year survey of
enteroviruses, adenoviruses and reoviruses isolated from effluent
at an activated-sludge purification plant. Appl. Environ.
Microbiol. 41:51-59.
Johnson, R.A., R.D. Ellender and S.-C. Tsai. 1984. Elution of
enteric viruses from Mississippi estuarine sediments with lecithin-
supplemented eluents. Appl. Environ. Microbiol. 48:581-585.
Joyce, G. and H.H. Weiser. 1967. Survival of enteroviruses and
bacteriophages in farm pond waters. J. Am. Water Works Assoc.
59:491-499.
90.
J
91.

183
92. Katzenelson, E., B. Fattal and T. Hostovesky. 1976. Organic
flocculation: an efficient second-step concentration method for
the detection of viruses in tap water. Appl. Environ. Microbiol.
32:638-639.
93. Kauzmann, W. 1959. Some factors in the interpretation of protein
denaturation. Adv. Protein Chem. 14:1-63.
:
94. Kessick, M.A. and R.A. Wagner. 1978. Electrophoretic mobilities
of virus adsorbing filter materials. Water Res. 12:263-268.
95. Keswick, B.H., C.P. Gerba, H.L. DuPont, and J.B. Rose. 1984.
Detection of enteric viruses in treated drinking water. Appl.
Environ. Microbiol. 47:1290-1294.
96. Kjelleberg, S. and M. Hermansson. 1984. Starvation-induced
effects on bacterial surface characteristics. Appl. Environ.
Microbiol. 48:497-503.
97. Konawalchuk, J. and J.I. Speirs. 1971. An evaluation of three
agents of eluting adsorbed enterovirus from Millipore membrane
filters. Can. J. Microbiol. 17:1351-1355.
98. Kott, Y., N. Rose, S. Sperber and N. Betzer. 1974. Bacteriophages
as viral pollution indicators. Water Res. 8:165-171.
99. Koya, K.V.A. and M. Chaudhuri. 1977. Virus retention by soil.
Prog. Water Technol. 9:43-52.
100. LaBelle, R.L. and C.P. Gerba. 1979. Influence of pH, salinity and
organic matter on the adsorption of enteric viruses to estuarine
sediment. Appl. Environ. Microbiol. 38:93-101.
101. Lance, J.C. and C.P. Gerba. 1980. Poliovirus movement during high
rate land filtration of sewage water. J. Environ. Qual. 9:31-34.
102. Lance, J.C. and C.P. Gerba. 1984. Effect of ionic composition of
suspending solution on virus adsorption by a soil column. Appl.
Environ. Microbiol. 47:484-488.
103. Lance, J.C. and C.P. Gerba. 1984. Virus movement in soil during
saturated and unsaturated flow. Appl. Environ. Microbiol.
47:335-337.
104. Lance, J.C., C.P. Gerba and J.L. Melnick. 1976. Virus movement in
soil columns flooded with secondary sewage effluent. Appl.
Environ. Microbiol. 32:520-526.
105. Lance, J.C., C.P. Gerba and D.-S. Wang. 1982. Comparative
movement of different enteroviruses in soil coumns. J. Environ.
Qual. 11:347-351.

184
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
Landry, E.F., J.M. Vaughn, M.Z. Thomas and C.A. Bechwith. 1979.
Adsorption of enteroviruses to soil cores and their subsequent
elution by artificial rainwater. Appl. Environ. Microbiol.
38:680-687.
Landry. E.F., J.M. Vaughn, M.Z. Thomas and T.J. Vicale. 1978.
Efficiency of beef extract for the recovery of poliovirus from
wastewater effluents. Appl. Environ. Microbiol. 36:544-548.
Lefler, E. and Y. Kott. 1974. Virus retention and survival in
sand, p. 84-91. In J.F. Malina, Jr. and B.P. Sagik (eds.), Virus
Survival in Water and Wastewater Systems, Center for Research in
Water Resources, Austin, TX.
Lindahl, M., A. Faris, T. Wadstrom and S. Hjerten. 1981. A new
test based on 'salting out' to measure relative surface
hydrophobicity of bacterial cells. Biochim. Biophys. Acta
677:471-476.
Lipson, S.M. and G. Stotzky. 1983. Adsorption of reovirus to clay
minerals: effects of cation-exchange capacity, cation saturation
and surface area. Appl. Environ. Microbiol. 46:673-682.
Lipson, S.M. and G. Stotzky. 1984. Effect of proteins on reovirus
adsorption to clay minerals. Appl. Environ. Microbiol. 48:525-530.
Lipson, S.M. and G. Stotzky. 1985. Specificity of virus
adsorption to clay minerals. Can. J. Microbiol. 31:50-53.
Logan, K.B., G.E. Rees, N.D. Seeley and S.B. Primrose. 1980.
Rapid concentration of bacteriophages from large volumes of
freshwater: evaluation of positively charged, microporous filters.
J. Virol. Methods. 1:87-97.
LoGrippo, G.A. 1950. Partial purification of viruses with an
anion exchange resin. Proc. Soc. Exp. Biol. Med. 47:208-211.
Lund, E. and V. Ronne. 1973. On the isolation of virus from
sewage plant sludges. Water Res. 7:863-871.
Mackey, B.M. 1983. Changes in antibiotic sensitivity and cell
surface hydrophobicity in Escherichia coli injured by heating,
freezing, drying or gamma radiation. FEMS Microbiol. Lett.
20:395-399.
Malina, J.F., Jr., K.R. Ranganathan, B.E.D. Moore and B.P. Sagik.
1974. Poliovirus inactivation by activated sludge, p. 95-106. In
J.F. Malina, Jr. and B.P. Sagik (eds.), Virus Survival in Water and
Wastewater Systems, Center for Reserach in Water Resources, Austin,
TX.
Mandel, B. 1971. Characterization of type 1 poliovirus by
electrophoretic analysis. Virology 44:554-568.

185
119. Mercer, E.H. 1959. An electron microscopic study of thin sections
of bacteria and bacteriophage grown on agar plates. Biochim.
Biophys. Acta 34:84-89.
120. Metcalf, T.D., C. Wallis, and J.L. Melnick. 1974. Environmental
factors influencing isolation of enteroviruses from polluted
surface waters. Appl. Microbiol. 27:920-926.
121. Mix, T.W. 1974. The physical chemistry of membrane-virus
interactions. Dev. Ind. Microbiol. 15:136-142.
122. Moore, B.D.E., B.P. Sagik and J.F. Malina, Jr. 1975. Viral
association with suspended solids. Water Res. 9:197-203.
123. Moore, R.S., D.H. Taylor, M.M. Reddy and L.S. Sturman. 1981.
Adsorption of reovirus by minerals and soils. Appl. Environ.
Microbiol. 44:852-859.
124. Moore, R.S., D.H. Taylor, L.S. Sturman, M.M. Reddy and G.W. Fuhs.
1981. Polivirus adsorption by 34 minerals and soils. Appl.
Environ. Microbiol. 42:963-975.
125. Morris, R. and W.M. Waite. 1980. Evaluation of procedures for
recovery of viruses from water. I. Concentration systems. Water
Res. 14:791-793.
126. Muller, R.H. 1950. Application of ion exchange resins to the
purification of certain viruses. Proc. Soc. Exp. Biol. Med.
73:239-241.
127. Muller, R.H. and H.M. Rose. 1952. Concentration of influenza
virus (strain PR8) by cation exchange resin. Proc. Soc. Exp. Biol.
Med. 80:27-29.
128. Ogamo, A., K. Matsuzaki, H. Uchiyama and K. Nagasawa. 1981.
Hydrophobic interaction chromatography of mycopolysaccharides:
Examination of fundamental conditions for fractionation of heparin
on hydrophobic gels. J. Chromatogr. 213:439-451.
129. Osipow, L.I. 1977. Surface Chemistry, p. 232-234. Robert E.
Krieger Publishing Co., Huntington, New York, NY.
130. Pahlman, S., J. Rosengren and S. Hjerten. 1977. Hydrophobic
interaction chromatography on uncharged sepharose derivatives:
Effects of neutral salts on the adsorption of proteins. J.
Chromatogr. 131:99-108.
131. Pancorbo, O.C., P.R. Scheuerman, S.R. Farrah and G. Bitton. 1981.
Effect of sludge type on poliovirus association with and recovery
from sludge floes. Can. J. Microbiol. 27:279-286.
132. Payment, P., S. Fortin, and M. Trudel. 1984. Ferric chloride
flocculation for nonflocculationg beef extract preparations. Appl.
Environ. Microbiol. 47:591-592.

186
133. Payment, P., C.P. Gerba, C. Wallis and J.L. Melnick. 1976.
Methods for concentrating virus from large volumes of estuarine
water on pleated membranes. Water Res. 10:893-896.
134. Payment, P. and M. Trudel. 1980. A simple low cost apparatus for
conditioning large volumes of water for virological analysis. Can.
J. Microbiol. 26:548-550.
135. Payment, P., M. Trudel and R. Plante. 1985. Elimination of
viruses and indicator bacteria at each step of treatment during
preparation of drinking water at seven treatment platns. Appl.
Environ. Microbiol. 49:1418-1428.
136. Primrose, S.B., N.D. Seely and K. Logan. 1981. The recovery of
viruses from water: methods and applications, p. 211-231. In M.
Goddard and M. Butler (eds.), Viruses and Wastewater Treatment,
Pergamon Press, Oxford, UK.
137. Prusiner, S.B., M.P. McKinley, D.F. Groth, K.A. Bowman, N.I. Mock,
S.P. Cochran and F.R. Masiarz. 1981. Scrapie agent contains a
hydrophobic protein. Proc. Natl. Acad. Sci. U.S.A. 78:6675-6679.
138. Rao, N.U. and N.A. Labzoffsky. 1969. A simple method for the
detection of low concentrations of viruses in large volumes of
water by the membrane filter technique. Can. J. Microbiol.
15:399-403.
139. Rosenberg, M., D. Gutnick and E. Rosenberg. 1980. Adherence of
bacteria to hydrocarbons: A simple method for measuring
cell-surface hydrophobicity. FEMS Microbiol. Lett. 9:29-33.
140. Safferman, R.S. and M.E. Morris. 1976. Assessment of virus
removal of a multistage activated sludge process. Water Res.
10:413-420.
141. Schaub, S.A. and B.P. Sagik. 1975. Association of enteroviruses
with natural and artificially introduced colloidal solids in water
and efectivity of solids-associated virions. Appl. Microbiol.
30:212-222.
142. Schaub, S.A. and C.A. Sorber. 1977. Virus and bacterial removal
from wastewater by rapid infiltration through soil. Appl. Environ.
Microbiol. 33:609-619.
143. Schaub, S.A., C.A. Sorber and G.W. Taylor. 1974. The association
of enteric viruses with natural turbidity in the aquatic
environment, p. 71-83. In J.F. Malina, Jr. and B.P. Sagik (eds.),
Virus Survival in Water and Wastewater Systems, Center for Research
in Water Resources, Austin, TX.
144. Scheuerman, P.R., G. Bitton, A.R. Overman and G.E. Gifford. 1979.
Transport of viruses through organic soils and sediments. J.
Environ. Eng. Div., ASCE, 105:629-640.

187
145. Schiffenbauer, M. and G. Stotzky. 1982. Adsorption of coliphages
T1 and T7 to clay minerals. Appl. Environ. Microbiol. 43:590-596.
146. Schrier, E.E. and E.B. Schrier. 1967. The salting-out behavior of
amides and its relation to the denaturation of proteins by salts.
J. Phys. Chem. 71:1851-1860.
147. Scutt, J.E. 1971. Virus retention by membrane filters. Water
Res. 5:183-185.
148. Seeley, N.D., G. Hallard and S.B. Primrose. 1979. A portable
device for concentrating bacteriophage from large volumes of
freshwater. J. Appl. Bacteriol. 43:103-116.
149. Seely, N.D. and S.B. Primrose. 1979. Concentration of
bacteriophage from natural waters. J. Appl. Bacteriol. 46:103-116.
150. Seely, N.D. and S.B. Primrose. 1982. The isolation of
bacteriophages from the environment. J. Appl. Bacteriol. 53:1-17.
151. Selna, M.W. and R.P. Miele. 1977. Virus sampling in wastewater-
field experiences. J. Environ. Eng. Div., ASCE. 103:693-705.
152. Shaltiel, S. 1974. Hydrophobic chromatography. Methods Enzymol.
34:126-140.
153. Shaltiel, S. 1984. Hodrophobic chromatography. Methods Enzymol.
104:69-96.
154. Shields, P.A. 1982. Contribution of Electrostatic and Hydrophibic
Interactions in Virus-filter Associations, Masters Thesis,
University of Florida, Gainesville, FL.
155. Shields, P.A., S. Berenfeld and S.R. Farrah. 1985. Modified
membrane-filter procedure for concentration of enteroviruses from
tapwater. Appl. Environ. Microbiol. 49:453-455.
156. Shields, P.A. and S.R. Farrah. 1983. Influence of salts on
electrostatic interactions between poliovirus and membrane filters.
Appl. Environ. Microbiol. 45:526-531.
157. Singh, S.N. and C.P. Gerba. 1983. Concentration of coliphage from
water and sewage with charged-modified filter aid. Appl. Environ.
Microbiol. 45:232-237.
158. Slade, J.S. and B.J. Ford. 1983. Discharge to the environment of
viruses in wastewater, sludges and aerosols, p. 3-18. In G. Berg
(ed.), Viral Pollution of the Environment, CRC Press, Boca Raton,
FL.
159. Smith, E.M. and C.P. Gerba. 1982. Development of a method for
detection of human rotavirus in water and sewage. Appl. Environ.
Microbiol. 43:1440-1450.

188
160. Smyth, C.J., P. Jonsson, E. Olsson, O. Soderlind, J. Rosengren, S.
Hjerten and T. Wadstrom. 1978. Differences in hydrophobic surface
characteristics of procine enteropathogenic Escherichia coli with
or without K88 antigen as revealed by hydrophobic interaction
chromatography. Infect. Immun. 22:462-472.
161. Sobsey, M.D., C.H. Dean, M.E. Knuckles and R.A. Wagner. 1980.
Interactions and survival of enteric viruses in soil materials.
Appl. Environ. Microbiol. 40:92-101.
162. Sobsey, M.D., C.P. Gerba, C. Wallis and J.L. Melnick. 1977.
Concentration of enteroviruses from large volumes of turbid estuary
water. Can. J. Microbiol. 23:770-778.
163. Sobsey, M.D. and J.S. Glass. 1980. Poliovirus concentration from
tap water with electropositive adsorbent filters. Appl. Environ.
Microbiol. 40:201-210.
164. Sobsey, M.D. and B.L. Jones. 1979. Concentration of poliovirus
from tap water using positively charged microporous filters. Appl.
Environ. Microbiol. 37:588-595.
165. Sobsey, M.D., C. Wallis, M. Hendersen and J.L. Melnick. 1973.
Concentration of enteroviruses from large volumes of water. Appl.
Microbiol. 26:529-534.
166. Sorber, C.A. 1983. Removal of viruses from wastewter and
effluents by treatment processes, p. 39-52. In G. Berg (ed.) Viral
Pollution of the Environment, CRC Press, Boca Raton, FL.
Ú
167. Stetler, R.E. 1984. Coliphages as indicators of enteroviruses.
Appl. Environ. Microbiol. 48:668-670.
168. Stotzky, G. and L.T. Rem. 1966. Influence of clay minerals on
microorganisms. I. Montmorillonite and kaolinite on bacteria.
Can. J. Microbiol. 12:547-563.
169. Stotzky, G., M. Schiffenbauer, S.M. Lipson and B.H. Yu. 1981.
Surface interactions between viruses and clay minerals and
microbes: Mechanisms and implications, p. 199-204. In M. Goddard
and M. Bulter (eds.), Viruses and Wastewater Treatment, Pergamon
Press, New York, NY.
170. Subrahmanyan, T.P. 1977. Persistence of enteroviruses in sewage
sludge. Bull. W.H.O. 55:431-434.
171. Taylor, D.H., R.S. Moore and L.S. Sturman. 1981. Influence of pH
and electrolyte composition on adsorption of poliovirus by soils
and minerals. Appl. Environ. Microbiol. 42:976-984.
172. Tierney, J.T., R. Sullivan and E.P. Larkin. 1977. Persistence of
poliovirus in soil and on vegetables grown in soil previously
flooded with inoculated sewage, sludge or effluent. Appl. Environ.
Microbiol. 33:109-113.

189
173.Valentine, R.C. and A.C. Allison. 1959. Virus particle
adsorption. I. theory of adsorption and experiments on the
attachement of nonbiological surfaces. Biochem. Biophys. Acta.
34:10-23
174. Vaughn, J.M. and E.F. Landry. 1983. Viruses in soils and
groundwaters, p. 163-210. In G. Berg (ed.), Viral Pollution of the
Environment, CRC Press, Boca Raton, FL.
175. Vaughn, J.H. and T.G. Metcalf. 1975. Coliphages as indicators of
enteric viruses in shellfish and shellfish raising estuarine
waters. Water Res. 9:613-618.
176. Vilker, V.L. 1978. An adsorption for prediction of virus
breakthrough from fixed beds, p. 381-421. In H.L. McKim (ed.),
State of Knowledge in Land Treatment of Wastewater, vol. 2, US Army
CRREL, Hanover, NH.
177. Wait, D.A. and M.D. Sobsey. 1983. Method for recovery of enteric
viruses from estuarine sediments with chaotropic agents. Appl.
Environ. Microbiol. 46:379-385.
178. Wallis, C., M. Henderson and J.L. Melnick. 1972. Enterovirus
concentration on cellulose membranes. Appl. Microbiol.
23:476-480.
179. Wallis, C., A. Homma and J.O. Melnick. 1972. Apparatus for
concentrating vurises from large volumes of water. J. Am. Water
Works Assoc., 64:189-196.
180. Wallis, C. and J.L. Melnick, 1967. Concentration of viruses from
sewage by adsorption on millipore membranes. Bull. W.H.O.
36:219-225.
181. Wallis, C. and J.L. Melnick. 1967. Concentration of enteroviruses
on membrane filters. J. Virol. 1:472-477.
182. Wallis, C. and J.L. Melnick. 1967. Concentration of viruses on
aluminum and calcium salts. Am. J. Epidemionl. 85:459-468.
183. Wallis, C., J.L. Melnick and C.P. Gerba. 1979. Concentration of
viruses from water by membrane chromatography. Annu. Rev.
Microbiol. 33:413-437.
184. Ward, R.L. 1983. Destruction of viruses in sludges by treatment
processes, p. 95-114. In G. Berg (ed.), Viral Pollution of the
Environment, CRC Press, Boca Raton, FL.
185. Wellings, F.M., A.L. Lewis and C.W. Mountain. 1974. Virus
survival following wastewater spary irrigation of sandy soils, p.
253-260. In J.F. Malina, Jr. and B.P. Sagik (eds.), Virus Survival
in Water and Wastewater Systems, Center for Reserach in Water
Resources, Austin, TX.

190
186. Wellings, F.M., A.L. Lewis and C.W. Mountain. 1976. Demonstration
of solids-associated viruses in wastewater and sludge. Appl.
Environ. Microbiol. 32:354-358.
187. Wellings, F.M. A.L. Lewis, C.W. Mountain and L.V. Pierce. 1975.
Demonstration of virus in groundwater after effluent discharge into
soil. Appl. Microbiol. 29:751-757.
188. Zerda, K.S. 1982. Virus Adsorption to Charged Solids, Ph.D.
Dissertation, Baylor College of Medicine, Houston, TX.
189. Zerda, K.S. and C.P. Gerga. 1984. Agarose isoelectrofocusing of
intact virions. J. Virol. Methods 9:1-6.
190. Zerda, K.S., C.P. Gerba, K.C. Hou and S.M. Goyal. 1985.
Adsorption of viruses to charge-modified silica. Appl. Environ.
Microbiol. 49:91-95.

BIOGRAPHICAL SKETCH
Patricia Ann Shields was born in Washington, D.C., in 1958, and was
raised in Adelphi, Maryland. Upon graduation from Regina High School in
1976, she entered Catholic University of America, where she received a
Bachelor of Science degree in the biological sciences in 1980. She then
moved to Gainesville, Florida, to continue her studies at the University
of Florida. In 1982, she was awarded the degree of Master of Science in
microbiology, and has continued her studies toward the Doctor of
Philosophy degree at the University of Florida.
1 Q1

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Samuel R. Farrah, Chairman
Associate Professor of
Microbiology and Cell Science
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
i
< > '/
Gabriel Bitton
Professor of Environmental
Engineering Sciences
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Lonnie 0. Ingram '
Professor of
Microbiology and Cell Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
!
- ' V â– - . -
Dinesh 0. Shah
Professor of
Chemical Engineering
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
)
O
/ --
Stephen G. Zam
Associate Professor of
Microbiology and Cell Science
This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
May 1986
Dean, Graduate School

UNIVERSITY OF FLORIDA
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