Title: Rapid detection of enteroviruses in environmental samples
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Title: Rapid detection of enteroviruses in environmental samples
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Language: English
Creator: Preston, David Raymond, 1961-
Copyright Date: 1989
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RAPID DETECTION OF ENTEROVIRUSES IN ENVIRONMENTAL SAMPLES


BY

DAVID RAYMOND PRESTON





















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


1989

















ACKNOWLEDGEMENTS


The author wishes to thank the members of his

committee, Dr. Samuel R. Farrah, Dr. Phillip M. Achey, Dr.

Gabriel Bitton, Dr. Rasul Chaudhry, and Dr. Edward

Hoffmann for their patience and guidence during my studies

at the University of Florida. In particular, the author

wishes to thank Dr. Farrah for his companionship and

friendship during his years within as well as outside of

his laboratory. In addition, the author wishes to thank

the numerous undergraduate students, graduate students,

faculty, and staff of the University of Florida without

whose friendship, assistance, and guidance this work would

not have been completed.


















TABLE OF CONTENTS

page
ACKNOWLEDGEMENTS ................................. ii

ABSTRACT ................................. ....... v

CHAPTERS

1 INTRODUCTION........................... 1

Overview............................ 1
Viruses............................ 3
Concentration Methods............. 5
Detection Methods ................ 7
Identification....................... 9

2 MODIFICATION OF DIATOMACEOUS EARTH BY
IN SITU PRECIPITATION OF METAL
SALTS TO ENHANCE VIRUS
ADSORBING PROPERTIES................. 10

Introduction....................... 10
Materials and Methods............. 11
Results.......................... 14
Discussion........................ 22

3 MODIFICATION OF FIBERGLASS FILTERS
WITH CATIONIC POLYMERS TO ENHANCE
VIRUS ADSORBING PROPERTIES........... 24

Introduction....................... 24
Materials and Methods............. 25
Results ......................... 30
Discussion........................ 49








page


4 ENHANCED INFECTIVITY OF ENT'IPOVIRUSES
IN VITRO BY PRETREATING HOST CELL
MONOLAYERS WITH CATIONIC POLYMERS.... 52

Introduction........................... 52
Materials and Methods.............. 53
Results............ ............... 57
D scussion............... ...... ....... 74


5 PAID DETECTION OF ENTEROVIRUSES USING
IMMUNOLOGICAL METHODS................ 77

Introduction ...................... 77
Materials and Methods............. 79
Results ........................... 86
Discussion ........................ 94

6 RAPID DETECTION OF ENTEROVIRUSES
USING DOT BLOT HYBRIDIZATION
METHODS.............................. 97

Introduction .................... .. 97
Materials and Methods............. 98
Results........................... 105
Discussion ......................... 17

7 APPLICABILITY OF DNA HYBRIDIZATION
PROCEDURES FOR THE DETECTION
OF HUMAN IMMUNODEFICIENCY VI'US
IN WASTEWATER.......................... 120

Introduction....................... 120
Materials and Methods............. 121
Results............................ 125
Discusstion........................ 129

S SUMMARY AND CONCLUSIONS................ 132

IE':FERENCES ........................................ 137

BIOGRAPHICAL SKETCH................................ 148
















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



RAPID DETECTION OF ENTEROVIRUSES IN ENVIRONMENTAL SAMPLES

By

David Raymond Preston

December 1989


Chairman: Dr. Samuel R. Farrah
Major Department: Microbiology and Cell Science



Surfaces which are routinely used to concentrate or

remove viruses from water were modified by either: 1) in

situ or 2) heat precipitation of metal salts or, 3)

treatment with cationic polymers. These modifications

converted electronegative surfaces to electropositive

surfaces and greatly enhanced their virus-adsorbing

properties. Cationic polymer-modified filters were found

well suited for the routine monitoring of virus

contamination of surface and potable waters.

The speed and sensitivity of tissue culture-based

assays for enteroviruses were enhanced by pretreating host

cell monolayers with the cationic polymer polyethylinimine

(PEI). PEI was found to enhance poliovirus infectivity

five-fold by enhancing the adsorption of virus to host








cell. This technique showed a five- to ten-fold increase

in the numbers of indigenous viruses detected by

traditional tissue culture-based assays.

Due to the low sensitivity of immunological and

r-ucleic acid probing techniques to detect viruses in

environmental samples, PEI in conjunction with tissue

culture were used to amplify the low numbers of viruses

found in environmental samples to a levr] detectable by

immunoperoxidase staining of virus infected cells and dot

blot hybridization procedures. Routine analysis of

unamplified environmental samples using dot blot

hybridization procedures was found to be unreliable,

insensitive, are not suitable for routine deteci or, of low

numbers of viruses in natural samples.

Dot blot assays might be useful for detecting viruses

in large numbers, such as in untreated wastewater. Human

immunodeficiency virus (HIV) genome and provirus were

detected by dot blot hybridization techniques from Belle

Glade wastewater concentrates but not from wastewaters

from the University of Florida, Ocala, and Kanapaha

wastewater treatment plants in Florida.

The combined results of these studies indicate tfhKi

for the detection of cultureable enteroviruses,

traditional tissue culture-based assays provide the

sensitivity required for public heath requirements and

that the sensitivity can be enhanced using cationic

polymers such as PEI. Tissue culture-based assays are of

little value in the detection of Jnon-culterable,








fastidious or hazardous viruses, such as HIV. In these

instances, immunological or nucleic acid hybridization

procedures would be the method of choice.



















CHAPTER 1

INTRODUCTION


Overview


Pathogenic human enteric viruses that may be present

in polluted waters can belong to the families

picornaviridae, caliciviridae, reoviridae, adenoviridae,

herpesviridae, papovaviridae, and the enterovirus group

(65,87). The pathology of enterovirus infection includes

paralysis, meningitis, fever, respiratory disease, rash,

gastoenteritis, myocarditis, congenital heart anomalies,

pleurodynia, encephalitis, acute hemorrhagic

conjuctivitis, infectious hepatitis, infectious

mononucleosis, immunological deficiency syndrome, epidemic

vomiting, progressive multifocal leukoencephalopathy,

immunosupression, and death (65,87). Enteric viruses are

transmitted by the consumption or exposure to contaminated

water and contaminated shellfish, most notably raw oysters

and clams, that is, by the fecal-oral route (87).

Infections may be apparent or inapparent and the infected

individual may shed up to 106 infectious virus particles

per gram of fecal material (64), thus establishing an

epidemiological cycle reminiscent of the seasonal polio








outbreaks that have all but been eradicated in industrial

rations due to rigorous vaccination programs (87).

Contrary to popular perception, poliovirus has not

been eradicated (87). The neuroviru-l1 ert serotypes have

mostly been replaced with attenuated vaccine strains

capable of limited replication within a human host (87),

making poliovirus relatively easy to isolate from polluted

waters due to viruses being shed by vaccinated individuals

and herd immunity that results froin the use of a live,

oral poliovirus vaccine (39). A recent outbreak of

poliomyelitis in Finland (63) was traced to the policy of

using an inactivated poliovirus vaccine rather than a

live, attenuated vaccine strain of poliovirus, a practice

which has since been reversed. In developing nations,

poliomyelitis is still endemic, most likely due to poor

sanitary conditions, as well as a less than rigorous

vaccination program (54). The problem of human enteric

viruses in the United States water supply is therefore

still a major consideration as there are 100 different

types of viruses capable of being transmitted by the

fecal-oral route, 97 for which there are still no vaccine

available (65). Out of 170 epidemics of gastroenteritis in

the United States between 1975 and / 9"7- .7% were of

viral etiology (10,19)

In the United States, drinking water quality comes

under the Safe Drinking Water Act of 1974, whose

assurances come under the jurisdiction of the

Environmental. Protection Agency (EPA). There are currently









no set federal standards for the numbers of viruses

allowed in potable waters. Therefore, public water

supplies are not monitored for viruses except in

localities where wastewater is reused for human

consumption or used to replenish diminishing groundwater

supplies as determined by local governing bodies. The EPA

has substituted treatment guidelines rather than

monitoring programs to assure virus-free drinking water.

The decision to issue treatment rather than Maximum

Contaminant Levels (MCL's) of viruses in water was due to

the relatively high cost of assaying waters for enteric

viruses (6). Researchers are therefore focusing on

developing rapid and cost-effective methods for detecting

human enteric viruses in water.


Viruses


As shown in Table 1-1, a wide variety of viruses can

be found in polluted waters. Some of these viruses, such

as poliovirus, are easily culturable on a continuous

primate kidney cell line such as Buffalo Green Monkey

(BGM) cell monolayers. Other enteric viruses, such as

Norwalk virus, cannot be cultivated using existing cell

lines and require human volunteers as a source of viruses

(65). The most rapidly replicating, and therefore the most

studied enteric viruses, are those in the family

picornaviridae which include the polioviruses,

echoviruses, coxsakieviruses, and hepatitis type-A virus.








Picornaviridae are naked icosohedrons 20 nm in diameter

composed of twenty units of four coat proteins (VP1, 2, 3,

and 4) and one protein (VPS) covalently attached to the 5'

end of the 7.5 Kb single-stranded, positive-sense, poly-

adenylated, RNA genome (87). Not all picornaviruses are

cultureable using cell cultures, nor do they all replicate

at the same rate (65). For example, poliovirus will

replicate in approximately thirty hours (39), whereas

hepatitis type-A will replicate in four to six weeks (7).

Some echoviruses and coxsakieviruses are not culturable in

vitro (65). In general, picornaviruses tend to persist in

the environment (7,30). This is a property consistent with

the fecal-oral route of transmission (87). These viruses

must first endure the physical extremes of the human

gastro-intestinal tract, and then survive extended periods

of time in the aqueous phase within the environment in

order to infect another individual. These viruses also

have very low infectious doses, on the order of one to ten

virus particles as compared to the 10 to 106 human

pathogenic enteric bacteria required to initiate infection

(4). Therefore, even very low levels of enteric viruses,

on the order of 1 infectious unit per 40 liters of potable

waters represents a significant public health threat (1).

The detection of these low levels of viruses in water has

led to the development of several methods for the

concentration of viruses from environmental samples

including water (29,85), soil (24), sludges (32),

sediments (7), and shellfish (37) to examine the flow of





5



viruses through the environment and their risk to the

public's health.


Table 1-1: Human pathogenic viruses which may be present
in polluted water.a



Number of
Virus Group Serotypes




Poliovirus 3
Echovirus 34
Coxsackievirus A 24
Coxsackievirus B 6
Enterovirus types 68 to 71 4
Hepatitis A 1
Norwalk virus 2
Rotavirus 4
Reovirus 3
Parvovirus 3
Adenovirus >30
Cytomegalovirus 1
Papovavirus 2



a From Rao, V.C. and J.L. Melnick. 1986.
Environmental Virology. American Society for Microbiology,
Washington, D.C. (65).


Concentration Methods


Presently, the method of choice for concentrating

viruses from the aqueous environment involves the

adsorption of viruses to a filter, eluting adsorbed

viruses with a complex proteinacious solution, such as

beef extract, and a secondary concentration step such as


flocculation (36).








Filters which have been routinely used in virus

concentration procedures include Filterite

electronegativee epoxyfiberglass) (30,38) and Virasorb 1-

MDS electropositivee charge-modified resins)

(41,73,77,79). Electronegative filters require that the

water sample be pretreated by adjusting the pH to 3.5 and

the addition of 0.05 M MgC12 or 0.0005 M AlCl3 to enhance

virus adsorption to acceptable levels (38). Virasorb 1-MDS

filters, however, do not require that the water be

retreated as long as the pH is less than 7 (73,77).

The major disadvantage of electropositive filters is

the cost. Many environmental laboratories would not be

able to afford the three to four-fold increase in

expenditures to switch from electronegative to

electropositive filters. Electropositive filters also tend

to clog more readily than electronegative filters, making

them much less efficient for the detection of viruses in

waters with high particulate and organic content (78).

These filters may also require that the pH be adjusted to

less than 7 (73,77).

Following adsorption, viruses are eluted from the

filters with 3% beef extract at pH 9 (36). More defined

eluents have been used for theoretical studies of virus

adsorption and recovery (28,33,71,74), but have not

received significant attention for the practical aspects

of environmental virology. In order to reduce the volume

of the beef extract eluent, which may be one liter or

more, a secondary concentration procedure is employed to









reduce the volume of simple to be assayed. Such procedures

include precipitating the beef extract eluent with acid

(50) or ammonium sulfate (72). The flock is separated by

centrifugation and resuspended in physiological buffer

prior to assay for infectious virus particles (50).


Detection Methods


The "gold standard" for the detection of viruses in

environmental concentrates has been the development of the

cytopathic effect (CPE) or plaque-forming-units (PFU) on a

monolayer of a competent continuous-ce1. line, sucl as the

Buffalo Green Monkey kidney cell-line (BGM). Other cell-

lines of importance include MA-104 for the detection of

reoviruses, especially rotaviruses, and Chinese hamster

ovary cells (CHO). For routine detection of human

enteroviruses, BGM cell monolayers are still the cell line

of choice as they are less fastidious and more sensitive

than other cell-lines (17). It is important to note that

no single cell line is available to detect all human

pathogenic viruses which may be present in polluted waters

(17).

Novel detection systems which have been recently

investigated include solid-phase enzyme-linked

immunoassays (ELISA) (22), iwmmunofluorescent-staining of

virus infected cells (42), immunoperoxidase-staining of

virus infected cells (62), and radioimmuno-detection of

virus infected cells (52). ELISA methods have not received








significant attention for the direct detection of viruses

in environmental samples as the sensitivity is much too

low, on the order of 106 PFU of poliovirus, whereas a

se-rsitivity of one PFU would be required. Therefore,

ini unofluorescent, immunoperoxidase, and radjioimmuno

assays directed against virus infected cells were

introduced as they allow the host cells to amplify the

amount of viral antigen present within the cell. Of the

three immunological techniques available, the

immunoperoxidase techniiqce is probably the most applicable

to the wastewater and environmental laboratory as no

special equipment (such as i fluorescent microscope) or

expensive reagents (such as radioisotopes) are required

for the assay.

Nucleic acid probes have recently been evaluated for

the detection of viruses in water (66) and in clinical

samples (68) in an effort to reduce the dependence on

expensive tissue-culture based assays. Presently, cDNA

probes of poliovirus type-1 (P1) (66,68) and Simian

Adeiovirus 40 (SV40) (21) and RNA probes of Hepatitis

type-A virus (HAV) (70) are being used with some

difficulty for the direct detection of viruses in

environmental concentrates. Reports of sor;sitivities rangZy

from less than 1 PFU to 303 PFU of PI, rotaviruses, a&nd

HAV.










Identification


Following isolation of viruses from environmental

samples, it is desirable to identify the virus type to

determine if the isolate is a threat to the public health.

Normally, viruses are identified by a neutralization test

whereby the virus isolate is mixed with a specific

neutralizing antibody preparation, incubated for one hour

at 370C, and assayed for infectivity in cell culture (65).

In addition, Melnick et al. (55) have used pooled antisera

to identify 42 of the most commonly isolated human enteric

viruses.

















CHAPTER 2

MODIFICATION OF DIATOMACEOUS EARTH BY IN SITU PRECIPITATION
OF METAL SALTS TO ENHANCE VIRUS ADSORBING PROPERTIES


Introduction


ProcedCures for the concentration of viruses from

; c ter most commonly uti lize i a electronegative or

electropositive filter material to adsorb viable viruses

from water (36,60). Viruses are then recovered with a

complex eluent such as beef extract ,and assayed on

competent cell monolayers such as Buffalo grrein monkey

(BGM) kidney cells (65). In addition to filter materials,

insoluble salts of aluminum, calcium, and magnesium have

been used to adsorb viruses from water (29,30,82,83).

Ferric chloride has also been used to enhance the

formation of virus-adsorbing flocs of beef extract eluents

as an efficient secondary concentration procedure by

recovering flocs by centrifugation (61). Early attempts to

use flocs in a continuous-flow system had proven

inpractable as the filters cloggaV- at an unacceptable rate

(31). Recently, Farrah and Preston (31) were able to

precipitate ferric and aluminum hydroxi]des in situ onto

and within cellulose filters to greatly enhance their








virus adsorbing properties at pHi values indicative of

environmental water sample..;. In addition to celluose

filters, microporous filters have been modified by in situ

precipitation of metal salts (81).

In this study, diatomaceous earth was modified by in

situ precipitation of aluminum, calcium, ferric, and

magnesium hydroxides and heat precipitates to enhance its

virus adsorbing properties. Diatomaceous earth was chosen

as the solid-phase support for in situ precipitation as

several existing public health applications exist for

diatomaceous earth filtration to include swimming pool

filters and filters used to treat surfact- waters destined

for human consumption.


Materials and Methods


Viruses and Viral Assays

Enteric Viruses. Poliovirus type-1 (P1) was

quantitated on Buffalo Green Monkey kidney (BGM) cell

inonola years a c plaque-forming units (PFU) using a

methylcellulose or agar overlay as previously described

(71). All viruses were harvested from cell lysates by

freezingj and thawing three times, centrifuging at 14,000 x

g for ten minutes, and then treating the supernatant twice

with trichloro-trifluoro ethane (freon).


Indigenous Bacteriophage. Indigenous bacteriophage

from trickling filter filterate (TFF) from the University

of Florida Wastewater Treartment Plant were quantitated as








plaque-forming units (PFU) using Escherichia coli C3000 as

the host using a soft agar overlay technique previously

described (76).

Coliphage MS2. Coliphage MS2 was propagated using E.

coli C3000 as the host strain and quantitated as PFU using

a soft agar overlay technique previously described (76).


Chemicals

The following chemicals with their sources were used

for these studies: aluminum chloride, arinonium hydroxide,

calcium chloride, ferric chloride, freon, imidazole,

magnesium chloride, and sodium chloride were from Fisher

Scientific Co. (Fair Lawn, NJ); diatomaceous earth (DE)

and glycine were from Sigma Chemical Co. (St. Louis, MO);

beef extract was from Scott Laboratories (Fiskesville,

RI).


Filters

The following filters with their sources were used

for these studies: Filterite epoxyfiberglass filters (0.45

um pore size) from pleated filter cartridges (Doufine

Filterite, Timonium, MD.) and G25 fiberglass prefilters

(Fisher Scientific Co., Fair Lawn, NJ).


Modification of Diatomaceous Earth

Twenty-five Jrar.s of DE were placed in a 250 ml

polycarbonate centrifuge tube to which 100 ml of either 1)

deionized water, 2) 1 M AIC13, 3) 1 M CaCI2, 4) 1 M MgCl2,

5) 1 M FeCl3, 6) 1 M FeC13 and 1 M AIC13, 7) 1 M FeC13 and








1 M CaCl2, or 8) 1 M FeC13 and 1 M MgCl2 were added. The

bottles were then shaken at 100 rpm on a rotary platform

shaker for thirty minutes, centrifuged at 14,000 x g for

ten minutes, and the supernatant discarded. The pellets

were then either 1) heated in a crucible over a bunsen

burner for at least fifteen minutes until no odor of HC1

was detected (heat precipitated) or 2) dried at 370C,

treated with 50 ml of 3 M NH40H in a 250 ml polycarbonate

centrifuge tube with constant agitation for thirty

minutes, and then centrifuged at 14,000 x g for ten

minutes. The pellets were air dried at 350C (in situ

precipitation). The final products were stored in glass

bottles.


Virus Adsorption and Elution Studies

Buffer. One hundred milliliters of buffer, 20 mM

glycine and 20 mM immidazole at pH 8 and seeded to

approximately 105 PFU per ml with MS2 and P1, were passed

through 25 mm diameter filter holders with a 0.45 um pore

size Filterite filter as the base containing approximately

0.5 gm of DE modified as described previously. Next, 10 ml

of 3% beef extract with 1 M NaCl at pH 9 were passed

through the filters to elute adsorbed viruses. The number

of viruses in the beef extract eluate and in the filter

effluent were compared with the number in the initial

sample to determine the percent of virus adsorbed and

recovered.








Trickling Filter Filterate. The procedure described

above for buffer was used except that TFF from the

University of Florida Wastewater Treatment Plant at pH 8

was used in place of buffer.


Determination of Zeta Potential of Modified Diatomaceous
Earth

One gram of diatomaceous earth modified as previously

described was suspended in 100 ml of 3 mM phosphate buffer

at pH 3, 5, 7, or 8.5 for 24 hours and the pH adjusted if

required. Zeta potentials were determined directly with a

meter (model 501; Lazer Zee; Penken, Inc., Bedford Hills,

N.Y.)


Results


As shown in Table 2-1, DE samples modified by in situ

precipitation of metal salts were evaluated for their

ability to adsorb and concentrate bacteriophage MS2 and P1

from buffer and at pH 8. It was found that DE modified

with AlC13, MgC12, FeCl3, or FeC13 with AlC13, CaC12, or

MgC12 were best suited for the adsorption of phage MS2 and

P1 from buffer at pH 8. For the adsorption and recovery of

P1 from buffer, DE modified by in situ precipitation of

FeC13 with AIC13 was found to be best suited for this

task. When these same surfaces were evaluated for their

ability to adsorb and concentrate indigenous coliphage and

seeded P1 from TFF at pH 8 (Table 2-2), a similar trend

was noted, where DE modified with FeC13 with AlC13 was








best suited for the adsorption and recovery of these

viruses from water.

As shown in Tables 2-3 and 2-4, DE modified by heat

precipitation were evaluated for their ability to adsorb

phage MS2 and P1 from buffer and indigenous phage and

seeded P1 from TFF at pH 8. Similar trends were noted for

the adsorption of these viruses to DE modified by in situ

or heat precipitation of metal salts. One striking

difference was that surfaces modified by in situ

precipitation were well suited for the eventual recovery

of adsorbed viruses, whereas DE modified by heat

precipitation tended to give much lower recovery of

adsorbed viruses with this eluent. Adsorbed viruses may

have been inactivated by these surfaces. One very notable

example is the adsorption and recovery of P1 from TFF

using DE modified by in situ and heat precipitation of

FeC13 with AlC13. In situ precipitated FeC13 with AiC13

showed 100% adsorption and 91% recovery of P1 whereas DE

modified by heat precipitated FeC13 with AiC13 showed 96%

adsorption and only 40% recovery.

Since it has been shown that electropositive filters

are better suited for the adsorption and subsequent

recovery of viruses from environmental water samples at

ambient pH (73), Zeta potentials of DE modified by in situ

precipitation of metal salts were determined (Table 2-5).

In general, it was noted that all surfaces modified by in

situ precipitation of metal salts showed a more

electropositive Zeta potential as compared to the strongly





16



electronegative unmodified DE. It is interesting to note

that the two types of DE most suited to the adsorption and

recovery of viruses from water, DE modified by the in situ

precipitation of A1C13 or FeCl3 with A1C13, showed a net

change in surface charge at pH 3. DE modified with A1C13

showed a point of zero charge at pH 5.








Table 2-1: Adsorption and recovery of Poliovirus-1 and
bacteriophage MS2 from buffer, pH 8, using
diatomaceous earth modified by In Situ
precipitation of metal salts.a


MS2 P1


DEb % Ads. % Rec. % Ads. % Rec.


UT 1+2 1+1 79+2 47+7

NH4OH 4+4 2+1 63+11 28+11

IM A1C13 100+0 92+10 99+2 34+14

1M CaC12 4+6 2+1 68+4 15+3

1M MgCl2 86+6 94+10 100+0 51+8

1M FeCl3 98+4 80+14 99+2 65+13

1M FeCl3 +
IM AIC13 92+10 67+13 100+0 118+6

1M FeC13 +
IM CaC12 100+0 92+3 100+1 49+19

1M FeC13 +
1M MgC12 100+0 95+5 100+1 43+19

a One-hundred milliliters of buffer, 20 mM glycine +
20mM immidazole, adjusted to pH 8 and seeded to ca. 105
pfu of MS2 and P1 were passed through 25mm diameter filter
holders with a 25mm 0.45um Filterite filter as the base
and filled with ca. 0.5 gm of DE modified by in situ
precipitation of the indicated solution. Next, 10 mi of 3%
beef extract with 1M NaCI at pH 9 were passed through the
filters to elute adsorbed viruses. The number of viruses
in the beef extract eluate and in the filter effluent were
compared with the number in the initial sample to
determine the percent of virus adsorbed and recovered.
Values indicate the mean and standard deviation of
triplicate determinations.

b Diatomaceous earth was modified by in situ
precipitation as described in the material and methods. UT
indicates untreated DE and NH4OH indicates DE treated
with deionized water in place of a metal solution.









Table 2-2: Adsorption and recovery of Poliovirus-1 and
indigenous bacteriophage from trickling filter
filterate, pH 8, using diatomaceous earth
modified by In Situ precipitation of metal
salts.a


Indigenous Phage P1


DEb % Ads. % Rec. % Ads. % Rec.


UT 0+0 0+0 45+11 13+5

NH4OH 0+0 8+3 36+14 21+9

1M A1C13 99+0 25+4 98+3 87+10

1M CaC12 3+5 6+2 30+27 19+3

1M MgC12 46+23 50+8 83+14 61+13

IM FeC13 92+6 34+9 93+5 60+11

1M FeCl3 +
1M A1C13 97+5 41+21 100+0 91+25

iM FeCl3 +
1M CaCl1 71+10 17+5 82+11 61+6

1M FeC13 +
1M MgCl2 58+22 40+23 78+3 50+9


a One-hundred milliliters of TFF adjusted to pH 8 and
seeded to ca. 10 pfu of P1 were passed through 25mm
diameter filter holders with a 25mm 0.45um Filterite
filter as the base and filled with ca. 0.5 gm of DE
modified by in situ precipitation of the indicated
solution. Next, 10 ml of 3% beef extract with IM NaC1 at
pH 9 were passed through the filters to elute adsorbed
viruses. The number of viruses in the beef extract eluate
and in the filter effluent were compared with the number
in the initial sample to determine the percent of virus
adsorbed and recovered. Values indicate the mean and
standard deviation of triplicate determinations.

b Diatomaceous earth was modified by in situ
precipitation as described in the material and methods. UT
indicates untreated DE and NH4OII indicates DE treated
with deionized water in place of a metal solution.









Table 2-3: Adsorption and recovery of Poliovirus-l and
bacteriophage MS2 from buffer, pH 8, using
diatomaceous earth modified by heat
precipitation of metal salts.a


MS2 P1


DEb % Ads. % Rec. % Ads. % Rec.


UT 1+2 1+1 79+2 47+7

Heat 13+10 2+1 90+8 45+12

1M AlCl3 88+10 41+43 98+2 83+14

1M CaC1, 45+27 1+1 95+6 19+3

1M MgC12 99+1 90+9 100+0 22+4

1M FeC13 2+4 2+0 77+6 25+3

1M FeC13 +
1M AlC13 99+1 84+13 96+7 49+15

IM FeC13 +
IM CaCl2 14+10 1+1 82+21 12+3

1M FeC13 +
1M MgCl2 88+13 60+22 91+13 13+7


a One-hundred milliliters of buffer, 20 mM glycine +
20mM immidazole, adjusted to pH 8 and seeded to ca. 105
pfu of MS2 and P1 were passed through 25mm diameter filter
holders with a 25mm 0.45um Filterite filter as the base
and filled with ca. 0.5 gm of DE modified by heat
precipitation of the indicated solution. Next, 10 ml of 3%
beef extract with 1M NaC1 at pH 9 were passed through the
filters to elute adsorbed viruses. The number of viruses
in the beef extract eluate and in the filter effluent were
compared with the number in the initial sample to
determine the percent of virus adsorbed and recovered.
Values indicate the mean and standard deviation of
triplicate determinations.

bDiatomaceous earth was modified by heat
precipitation as described in the material and methods. UT
indicates untreated DE and heat indicates DE treated with
deionized water in place of a metal solution.









Table 2-4: Adsorption and recovery of Poliovirus-1 and
indigenous bacteriophage from trickling filter
filterate, pH 8, using diatomaceous earth
modified by heat precipitation of metal salts.a


Indigenous Phage P1


DEb % Ads. % Rec. % Ads. % Rec.


UT 0+0 0+0 45+11 13+5

Heat 23+5 4+2 54+8 25+6

IM AIC13 72+9 5+2 96+3 51+14

IM CaC12 24+6 6+1 66+7 38+5

1M MgC12 75+10 36+13 84+2 49+14

IM FeC13 8+14 23+ 4 81+4 51+1

lM FeC13 +
1M AlC13 76+11 17+9 96+4 40+6

1M FeCl, +
IM CaC1 5+6 12+2 81+8 86+1

1M FeCl1 +
IM MgC12 91+6 55+6 97+2 59+18


a One-hundred milliliters of TFF adjusted to pH 8 and
seeded to ca. 10 pfu of P1 were passed through 25mm
diameter filter holders with a 25mm 0.45um Filterite
filter as the base and filled with ca. 0.5 gm of DE
modified by heat precipitation of the indicated solution.
Next, 10 ml of 3% beef extract with 1M NaCl at pH 9 were
passed through the filters to elute adsorbed viruses. The
number of viruses in the beef extract eluate and in the
filter effluent were compared with the number in the
initial sample to determine the percent of virus adsorbed
and recovered. Values indicate the mean and standard
deviation of triplicate determinations.

b Diatomaceous earth was modified by heat
precipitation as described in the material and methods. UT
indicates untreated DE and heat indicates DE treated with
deionized water in place of a metal solution.








Table 2-5: Zeta potentials of diatomaceous earth modified
by In Situ precipitation of metal salts.a


Zeta Potential (mV) at Indicated pH


DEb 3 5 7 8.5


UT -66 -65 -63 -71

NH4OH -34 -53 -57 -66

1M A1C13 20 0 -13 -22

1M CaCl2 -45 -21 -41 -55

1M MgC12 -26 -13 -14 -21

IM FeC13 -3 -34 -45 -48

1M FeC13 +
1M A1C13 4 -22 -35 -37

IM FeCl3 +
1M CaC12 -12 -29 -24 -12

1M FeC13 +
IM MgC12 -12 -21 -22 -24


a Zeta Potentials (mV) of DE modified by in situ
precipitation of metal salts were determined directly
using a Lazer Zee Meter model 501 (Penkem, Inc., Bedfor
Hills, NY) in the following manner. Diatomaceous earth was
suspended in 3mM sodium phosphate buffer at a rate of 1 gm
per liter and allowed to settle. The supernatant was
poured off and replaced with an equal volume of 3mM
phosphate buffer at the indicated pH. The pH was checked
and adjusted with 3mM phosphate buffer (phosphoric acid or
sodium phosphate tribasic) if required.

bDiatomaceous earth was modified by in situ
precipitation as described in the material and methods. UT
indicates untreated DE and NH4OH indicates DE treated
with deionized water in place of a metal solution.









Discussion


Although previous studies have shown that a wide

variety of filters can be modified by in situ

precipitation of metal hydroxides to enhance their virus-

adsorbing properties (31,81), this is the first study to

report the development of such a surface applicable to

water treatment facilities rather than water monitoring

procedures. In addition, this is the first study to report

the formation of metal oxides onto surfaces to enhance

their virus adsorbing properties.

Presently, diatomaceous earth filters are used in

swimming pools to remove particulates and microorganisms

which affect clarity and health standards. Diatomaceous

earth filters are also used to treat surface waters

destined for human consumption and to remove pathogenic

bacteria as well as cysts of human parasites such as

Giardia and Cryptosporidium (18,67). These diatomaceous

earth filters have not been rigorously investigated for

their ability to remove human pathogenic viruses from

water (80).

The results of this study indicate that diatomaceous

earth can be readily and inexpensively modified by the

formation of either hydroxides or oxides to enhance their

virus-adsorbing properties. In addition, it was found that

metal hydroxides would be best suited for the

concentration of viable viruses from the environment,

whereas metal oxides would be better suited for the





23



removal of viruses from water destined for human

consumption as viruses were not easily recovered from

these surfaces.



















CHAPTER 3

MODIFICATION OF FIBERGLASS FILTERS WITH CATIONIC
POLYMERS TO ENHANCE VIRUS ADSORBING PROPERTIES


Introduction


Popular procedures for the detection of viruses in

waters have taken advantage of the phenomenon of virus

adsorption to and elution from microporous filters to

concentrate viruses from a wide variety of water sources

(36,60). Filters used in these procedures have been

characterized as electronegative or electropositive based

on their electrophoretic mobilities (78,79) and

characterized as to their relative hydrophobicities (73).

In order for viruses to adsorb to electronegative filters,

the water must be pretreated by lowering the pH or by the

addition of salts to enhance virus adsorption to

acceptable levels so that the virus and filter have

opposite charges (8,36). Electropositive filters, however,

can adsorb viruses over a broader pH range without the

addition of salts and so do not require pretreatment of

the water prior to virus adsorption (16,73,78). Recent

advances in the modification of surfaces to enhance their

virus adsorbing properties (31,81,92) have shown that








simple modifying procedures are available. This study we

report a simple and inexpensive procedure similar to that

of Brown et al. (14) to modify fiberglass filters to

enhance their virus adsorbing properties under ambient

water conditions. This modification procedure entails

soaking a fiberglass filter in an aqueous solution of

cationic polymer and then allowing the filters to air-dry

at room temperature. The cationic polymers

polyethylenimine and Nalco cationic polymer 7111 can be

used together to produce a filter which can recover

enteroviruses from environmental waters as effectively as

presently available electropositive filters. The

modification converts the electronegative fiberglass

surface to an electropositive surface as determined by

Zeta potential measurements of disrupted filter materials.


Materials and Methods


Viruses and Viral Assays

Bacteriophages MS2, T2, and OX174 were determined as

plaque-forming-units (PFU) using Escherichia coli C3000,

E. coli B, and E. coli ATCC (13607), respectively, as

hosts according to previously described procedures (76).

Indigenous bacteriophage from a trickling filter filtrate

(TFF) were assayed as PFU using E. coli C3000 as host

cells by previously described procedures (76). Poliovirus

type-i (P1), coxsackievirus type-B5 (CB5), echovirus type-

1 (El), and echovirus type-5 (E5) were determined as PFU









using BGM cell monolayers and a methylcellulose overlay as

previously described (71).


Chemicals and Filters

The chemicals used in this study were as follows.

Glycine, hydrochloric acid, and sodium hydroxide from

Fisher Scientific (Fair Lawn, NJ); immidazole and

polyethylenimine (PEI) from Sigma Chemicals (St. Louis,

MO), Dellchem cationic polymer BASF CF 600 (gift of Mike

New, Kanapaha wastewater treatment plant, Gainesville,

Florida); LCI cationic polymer (gift of Mike New, Kanapaha

wastewater treatment plant, Gainesville, Florida); Nalco

cationic polymer, 90% charge, high molecular weight (gift

of Kenneth E. DeGarmo, Leahchem Industries, Inc.,

Titusville, FL). Filters used in this study were as

follows. Filterite Duofine 0.2-1.0 um from pleated filter

cartridges (Filterite, Timonium, MD); G25 fiberglass

prefilters (MSI, Westbourough, MS); and Virasorb 1-MDS

filters (AMF Cuno, Meriden, CN). All filters were 25 mm in

diameter and were kept in appropriate filter holders.


Modification of Epoxyfiberglass Filters

Filterite filters, 25mm diameter, were modified with

PEI and Nalco cationic polymer in the following manner.

PEI was made 0.5% (w/v) in deionized water and Nalco

polymer was made 0.05% (w/v) in deionized water. Filters

were soaked for two hours at room temperature and air

dried overnight on adsorbent paper towels. One or four








layers for the filter types Nalco and PEI were used in

adsorption experiments. N/P filters had two layers of

Nalco treated filters on top of two layers of PEI treated

filters whereas N+P filters represent four layers of

filters treated with 0.5% PEI and 0.05% Nalco polymers

mixed together. N->P filters were first soaked for two

hours in 0.05% Nalco polymer, blotted an adsorbent paper

towels to remove excess liquid, soaked in 0.5% PEI for one

hour, air dried overnight, and four layers were used in

adsorption experiments. P->N filters were treated with PEI

followed by Nalco polymer in the same manner as N->P

filters.


Determination of Zeta Potentials of Filter Materials

Filterite filter material (0.2 um pore size) was

disrupted in deionized water by blending at high speeds

and air-dried at room temperature. Next, disrupted filter

material was soaked for two hours at room temperature in

0.5% PEI, 0.05% Nalco polymer, or deionized water and

subsequently centrifuged at 14,000 x g for ten minutes.

The pellets were collected, air dried at room temperature,

and resuspended in 3mM phosphate buffer at pH 3, 5, 7, and

10. Zeta potentials of disrupted filter material were

determined directly using a Lazer Zee Meter Model 501

(Penkem, Inc., Bedford Hills, NY).









Virus Adsorption-Elution Studies

Buffer. One-hundred milliliters of buffer (20mM

glycine and 20mM immidazole) adjusted to pH 5, 7, or 9

with IN HCL or IN NaOH as required, and seeded to

approximately 105 PFU/ml with P1 were passed through four

layers of the indicated filters in the following order, pH

5, 7, and then 9. Next, 10ml of 3% Beef Extract (Scott

Laboratories, Fiskeville, RI) with 1M NaC1, pH 9 were

passed through the filters to elute adsorbed viruses. The

number of viruses in the beef extract eluate and in the

filter effluents were compared with the number of viruses

in the initial sample to determine the percent of virus

adsorbed and eluted. Values indicate the mean and standard

deviation of quadruplicate determinations.

Trickling Filter Filtrate, Unchlorinated Secondary
Effluent, and Raw Wastewater. TFF from the

University of Florida Wastewater Treatment Plant

(Gainesville, FL) prefiltered with an AP20 filter to

remove suspended solids were seeded to approximately 105

PFU/ml of P1 and adjusted to pH 5, 7, or 9. One-hundred

milliliters of this water were passed through the

indicated filters containing two layers of Nalco-treated

filters (0.05%) and two layers of filters treated with

PEI. The number of viruses in the filtrates were compared

with those in the initial samples to determine the

percentage of viruses adsorbed. Indigenous bacteriophage

from unchlorinated secondary effluent or raw wastewater

were determined by first prefiltering the water sample









with a G25 fiberglass filter to remove suspended solids.

One hundred milliliter aliquots of these waters were then

passed through one filter layer at approximately 1 ml/sec.

Recovery and determinations of adsorption and recovery of

viruses were performed as described previously.

Tap Water. Tap water was dechlorinated with sodium

thiosulfate and confirmed with o-tolidine, seeded with

approximately 105 PFU/ml of P1, El, E5, or CB5 and

adjusted to pH 3.5 or left at ambient pH (pH 8.0). One-

hundred milliliters of the tap water were passed through

the indicated filters and the adsorbed viruses were eluted

with 10ml of 10% Beef Extract, pH 9. The number of

viruses in the beef extract eluates and filtrates were

compared with the number of viruses in the initial samples

to determine the percent of viruses adsorbed and eluted.

Values indicate the mean and standard deviation of

quadruplicate determinations.


Stability of PEI-Treated Filters

PEI-treated and untreated Filterite filters were

placed in paper envelopes at 250C and in sealed plastic

storage bags at 25, 4, and -200C. At the indicated time

intervals, 100ml of G25 prefiltered TFF, pH 7

(approximately 200 PFU/ml), were passed through the

filters and the filter effluents and initial sample

assayed for indigenous bacteriophage.










Results


As shown in Table 3-1, the removal of bacteriophage

MS2 from buffer at pH 7 was enhanced from 55% to 100% by

treating Filterite fiberglass filters with aqueous PEI at

a concentration of 0.01% PEI or greater. For later

experiments, 0.1 or 0.5% PEI was used to modify Filterite

filters.

As shown in Table 3-2, the pH influenced virus

removal from buffer by three layers of untreated and PEI-

treated Filterite filters. When all bacteriophages and

viruses are considered, untreated filters removed a

greater percentage of viruses at pH 3.5 (97+7) than did

PEI-treated filters (71+31). However, this condition was

reversed at pH 7 and 9. There was little difference in the

percent removal of viruses by treated and untreated

filters at pH 5. It is evident that treating Filterite

filters with PEI greatly enhanced the removal of both

bacteriophages and animal viruses at pH 7 and 9. One very

notable exception is the removal of P1 at pH 7, where PEI-

treated and untreated filters removed less than 10% of P1

from buffer.

In addition to polyethylenimine, several other

cationic polymers were investigated for their ability to

enhance virus removal by Filterite fiberglass filters by

the simple modification procedure described in the

Materials and Methods and in Table 3-3. For the removal








of bacteriophage MS2, the cationic polymers produced by

Nalco, Dellchem, and LCI were found to be as efficient as

PEI, with 100% of MS2 being removed from 100 ml of buffer

at pH 5, 7, and 9. No single polymer-treated filter was

able to remove P1 at all the pH values tested. However,

filters treated with Nalco polymer were able to remove

greater than 99% of P1 from buffer at pH 5 and 7 whereas

PEI treated filters were able to remove 97% of P1 at pH 9.

These polymer-treated filters were further investigated

for their ability to recover adsorbed bacteriophage MS2

and P1 (Table 3-4). The recovery of bacteriophage MS2 from

PEI and Nalco polymer-treated filters with 3% beef extract

was 0% and 5%. However, 22% and 18% of bacteriophage MS2

were recovered from filters treated with Dellchem and LCI

polymers, respectively. A totally different pattern was

found for P1 recovery where PEI-treated filters gave 85%

recovery and Nalco polymer-treated filters gave 42%

recovery of P1. We were not able to recover P1 from

Dellchem and LCI polymer treated filters under the

conditions tested.

Untreated, PEI-treated, and Nalco polymer-treated

Filterite filters were then evaluated for their ability to

recover P1, CB5, El, and E5 from dechlorinated tap water

at ambient pH (pH 8.1), (Table 3-5). For the four viruses

tested, filters treated with cationic polymers adsorbed a

greater percentage of viruses (96+7) and allowed a greater








percentage of virus recovery (99+12) than did untreated

filters (20+12% adsorbed and 18+25% recovered).

Zeta potentials of Filterite filter material modified

with cationic polymers following disruption in a blender

are shown in Figure 3-1. At the pH values investigated,

untreated filter material showed a negative charge, PEI-

treated filter material showed a positive charge, and

Nalco-treated filter material showed an intermediate

charge.

The ability of PEI-treated Filterite filters to

recover indigenous bacteriophage from 100 ml of

prefiltered secondary unchlorinated effluent and raw

wastewater at ambient pH is presented in Table 3-6. A

single layer of PEI treated filter material was able to

remove 100 and 98% of indigenous bacteriophage from 100 ml

of secondary unchlorinated effluent and raw wastewater,

respectively. The recovery of adsorbed bacteriophage with

3% beef extract was 18% recovery from secondary

unchlorinated effluent and 39% from raw sewage.

Table 3-7 shows the breakthrough volume of a single

layer of PEI treated Filterite filter for the adsorption

of indigenous phage from unchlorinated secondary effluent

at ambient pH. The virus breakthrough volume of a single

25mm filter under these conditions was found to be between

200 and 300 ml.

Fiberglass filters treated with the cationic polymers

PEI and Nalco 7111 alone and in combination were

evaluated for their ability to adsorb seeded P1 from









buffer at pH 5, 7, and 9 (Figure 3-2). The most efficient

filter type resulted when PEI-treated filters and Nalco-

treated filters were stacked upon each other (N/P) rather

than combining the two polymers to treat a single filter.

It was noted that when PEI and Nalco polymer were mixed

together at their effective concentrations, a precipitate

resulted. Filterite filters modified with this precipitate

in solution showed greatly reduced flow rates and did not

adsorb P1 well at pH 7 (P+N), a situation which was not

improved by treating a filter with one polymer and then

another (N->P and P->N).

Later studies therefore concentrated on the use of

several layers of filters, where each filter was treated

with either PEI or Nalco polymers rather than combining

PEI and Nalco polymers onto a single filter layer. The

concentration of PEI used to treat Filterite filters was

maximized for the adsorption of P1 from trickling filter

filtrate (TFF) at pH 5, 7, and 9 (Figure 3-3). Solutions

of Nalco polymer are highly viscous and filters modified

with high concentrations of this polymer showed reduced

flow rates. Therefore N/P filters modified with 0.05%

(w/v) of Nalco polymer were used as this concentration of

Nalco polymer showed little reduction in flow rate but

maintained high virus adsorbing capacity (Figure 3-3). In

later studies 0.5% (w/v) PET and 0.05% (w/v) Nalco

polymers were used. As shown in Table 3-8, we were able to

recover adsorbed P1 quantitatively from N/P filters using









1M NaCi, pH 9; 1M NaCI + 0.1% Tween 80, pH 9; or 10% beef

extract, pH 9.

The relative efficiencies of N/P filters, untreated

fiberglass filters, and Virasorb 1-MDS filters to adsorb

and recover P1, El, E5, and CB5 from dechlorinated tap

water was also investigated (Table 3-9).

In general, N/P filters were able to adsorb and

recover a greater percentage of all the viruses tested

from dechlorinated tap water at pH 8 than Virasorb-1MDS

filters at pH 8 or Filterite filters at pH 3.5. Autoclaved

N/P filters showed a slight decrease in flow rate and

virus adsorbing capability. The stability of PEI-treated

filters could be greatly extended by sealing filters in

air-tight plastic bags as opposed to open air storage in

paper envelopes (Figure 3-4).















Table 3-1: Removal of bacteriophage MS2 by Filterite
filters treated with polyethylenimine.a


Percent aqueous
polyethylenimine
used to treat filters b


10-3
10-2
10-1
0.5
1.0


Percentage
Bacteriophage MS 2
Removed


55
49
55
55
100
100
100
100


a One hundred milliliters of buffer, pH 7.0, seeded
with approximately 10 pfu/ml were passed through one
filter layer (0.2 um pore size) in a 25mm holder. The
results are expressed as the percentage of virus in the
buffer prior to filtering. Values indicate the mean of
duplicate determinations. The standard error was less than
20% of the mean for all values.

b Filterite filters were treated with the indicated
percentage of polyethylenimine in deionized water for two
hours at room temperature and allowed to dry at room
temperature on adsorbent paper towels


- -------------------
















Table 3-2: Influence of pH on virus removal by untreated
and polyethylenimine treated Filterite filters.a


Percentage Removed


Polyethylenimine
pH Treated Filters


Virus


Untreated
Filters


Bacteriophage MS2




Bacteriophage T2




Bacteriophage OX174


Poliovirus 1




Coxsackievirus B5


3.5
5
7
9

3.5
5
7


87
100
100
100

100
100
100
100

76
29
100
100


3.5
5
7


3.5
5
7
9

3.5
5
7
9


100
97
28
41


100
100
7
0

100
35
0
0


72
100
100
100


__ ~I


__ ~ _~I__I___~














Table 3-2--continued


Percentage Removed


Polyethylenimine Untreated
Virus pH Treated Filters Filters


Bacteriophages
and Viruses 3.5 71+31 97+7
5 80+31 76+29
7 80+45 17+13
9 100+11 25+26

Bacteriophages b 3.5 88+12 94+8
5 76+41 82+22
7 100+0 25+5
9 100+0 42+18

Viruses b 3.5 46+37 100+0
5 86+20 68+46
7 50+70 4+5
9 99+ 1 0


a The procedure described in Table 3-1 was used
except three layers of filters (0.25 um pore size) were
used. Filterite filters were treated as described in Table
1 using 0.5% polyethylenimine. The results are expressed
as the percentage of virus in the buffer prior to
filtering and the values indicate the mean of duplicate
determinations. The standard error was was than 20% of the
mean for all values.

b Values presented indicate the mean and standard
error of the values presented for the groupings indicated.















Table 3-3: Effects of pH on the removal of viruses from
buffer by Filterite filters modified with
cationic polymers. a



Percentage Removed


pH Polymer Bacteriophage MS2 Poliovirus-1


5 None 22+6 100+ 0
PEI 100+0 58+15
Nalco 100+0 100+ 0
Dellchem 100+0 84+ 6
LCI 100+0 96+ 6

7 None 28+1 21+ 2
PEI 100+0 3+ 6
Nalco 100+0 100+ 1
Dellchem 100+0 74+16
LCI 100+0 89+ 5

9 None 41+11 12+16
PEI 100+0 97+ 3
Nalco 100+0 0+ 0
Dellchem 100+0 0+ 0
LCI 100+0 0+ 0



a The procedure described in Table 3-1 was used
except that 100ml volumes of seeded buffer were passed
through three layers of filters (0.25 um pore size) in
the following order, pH 5, 7, and 9. Filters were treated
with 0.1% of the indicated polymer as described in
Materials and Methods. The results are expressed as the
percentage of virus in the buffer prior to filtering.
Values indicate the mean and standard deviation of of four
determinations.

















Table 3-4: Adsorption and recovery of viruses to
Filterite filters modified with cationic
polymers. a




Bacteriophage MS2 Poliovirus 1
Polymer used
to modify
filters %Adsorbed %Recovered %Adsorbed %Recovered


None 0 0 0 0

PEI 100 0 100 85

Nalco 100 5 68 42

Dellchem 98 22 34 0

LCI 100 18 39 5



a The procedure described in Table 3-1 was used
except three layers of filters (0.25 um pore size) were
used and the buffer pH was 9.0. Adsorbed viruses were
recovered by passing 10 ml of 3% beef extract through the
filter following the seeded buffer. The results are
expressed as the percentage of virus in the buffer prior
to filtering. Values indicate the mean of duplicate
determinations. The standard error was less than 20% of
the mean for all values.

b Filterite filters were treated with 0.1% of the
indicated polymer in deionized water for two hours at 25C.
Filters were then dried on adsorbent paper towels at 25C.
















Table 3-5: Adsorption and recovery of enteroviruses from
dechlorinated tap water using Filterite
filters modified with cationic polymers,a


Virus


CB5


b-
Polymerb
%Ads %Rec %Ads


%Rec %Ads


%Rec %Ads %Rec


None 37+13 55+11 8+13 3+2 20+13 5+1 15+11 7+2

PEI 79+7 75+7 99+1 104+7 99+1 99+2 99+1 111+5

Nalco 100+1 100+0 98+3 112+17 95+1 94+5 98+1 98+7



a One hundred milliliters of dechlorinated tp water
at ambient pH (8.1) seeded with approximately 10 pfu/ml
of the indicated virus were passed through 3 filter layers
(0.2 um pore size) in a 25mm filter holder. Adsorbed
viruses were recovered using 10 ml of 3% Beef Extract
(Difco Certified) + 1 M NaC1, pH 9.0. The results are
expressed as the percentage of virus in the tap water
prior to filtering. Values indicate the mean and standard
deviations of triplicate determinations.

b Filterite filters were treated as described in
Table 3-1 using 0.5% PEI or 0.05% Nalco polymer.


















Table 3-6: Recovery of indigenous bacteriophage from
unchlorinated secondary effluent and raw
sewage by PEI treated Filterite filters. a


Water Type


% Adsorbed


% Recovered


Secondary unchlor-
inated effluent 6.4 100 18

Raw sewage 7.0 98 39



a One hundred milliliters of the indicated water type
prefiltered with a G25 fiberglass filter were passed
through one layer of PEI treated Filterite filter (0.25 um
pore size). Adsorbed virus were recovered using 10ml of 3%
beef extract, pH 9.0. The results are expressed as the
percentage of viruses in the water prior to filtering.
Values indicate the mean of duplicate determinations. The
standard error was less than 20% of the mean for all
values.



















Table 3-7: Virus breakthrough volume of PEI treated
Filterite filters for indigenous phage from
unchlorinated secondary effluent. a


Volume (ml)


100
200
300
400
500


% Adsorbed


100
99
44
27
7


a Five one-hundred milliliter aliquots of G25
prefiltered secondary unchlorinated effluent, pH 6.5, were
passed through a single layer of PEI treated Filterite
filter material (0.25 um pore size). These aliquots were
collected and assayed separately. The results are
expressed as the percentage of virus in the water prior to
filtering. Values indicate the mean of duplicate
determinations. The standard error was less than 20% of
the mean for all values.
















































O -01 pH

m20

m30

I, m40
N
=50

i60

m70

mS0a

i901


















Figure 3-1: Zeta potentials of Filterite filter material
treated with cationic polymers.











Table 3-8: Recovery of poliovirus type 1 from PEI-Nalco
treated filters



Eluent, pH % Recovered


Bufferb, 5 1+1

Buffer, 7 2+2

Buffer, 9 26+8

1M NaC1, 7 88+13

1M NaCI, 9 118+11

0.1% Tween 80, 7 3+2

0.1% Tween 80, 9 63+15

1M NaCI + 0.1% Tween 80, 7 74+9

1M NaCI + 0.1% Tween 80, 9 107+9

3% Beef Extract, 7 70+23

10% Beef Extract, 9 108+7



a One-hundred milliliters of trickling filter
filtrate seeded with approximately 105 pfu/ml of P1 were
passed through two layers of PEI-treated (0.5%) and two
layers of Nalco-treated (0.05%) Filterite filters (0.2um
pore size, 25mm diameter) and the effluent assayed for
virus (96+3% adsorbed). Next, 10ml of the indicated eluent
were passed through the filters and the effluent assayed
for virus. The results are expressed as the percentage of
virus in the water prior to filtering. Values indicate the
mean and standard deviation of four determinations.

b Glycine (20mM) and immidazole (20mM) were used as
buffer.















Table 3-9: Adsorption and recovery of enteroviruses from
dechlorinated tap water using PEI-Nalco
treated filters, Virasorb-1MDS, and untreated
Filterite filters




P1 El E5 CB5


Filter pH %Ads %Rec %Ads %Rec %Ads %Rec %Ads %Rec


8 98+1 113+16


100+0 121+20


100+0 88+27100+0 113+28


N/P Auto-
claved 8 88+10 103+8


8 51+23 38+5


3.5 51+18 54+8


70+21 82+16


95+2 87+9


75+5 89+19 55+11 40+3


ND ND


a One-hundred mlliliters of dechlorinated tap water
pH 8 seeded to ca 10 pfu/ml of the indicated virus were
passed through the indicated filter (25mm diameter) and
the effluents assayed. Next 10ml of 10% beef extract
(Scott Laboratories), pH 9 were passed through the filters
and the eluates assayed. For untreated Filterite filters,
the water was adjusted to pH 3.5 prior to filtering. The
results are expressed as the percentage of virus in the
buffer prior to filtering. Values indicate the mean and
standard deviation of triplicate determinations.

b ND indicates not determined.


N/P


1MDS


Filter-
ite


NDb


--------------------------- ----


------------~-----






















a
60

S40

201

L II l 1
pH 5 7 9 579 5 7 9 57 57 9 579 5 7
%PEI 0 0.1 0.2 0.5 0.6 0.7 1.0
%NALCO 0 005 0.05 0.05 0.05 0.05 005





Figure 3-2: Adsorption and recovery of Poliovirus type-1
from buffer at pH 5, 7, and 9 using Filterite
filters modified with cationic polymers.


One-hundred milliliters of buffer (20mM glycine and
20mM immidazole) adjusted to pH 5,7, or 9 with 1N HC1 o
1N NaOH as required, and seeded to approximately 10
PFU/ml with P1 were passed through four layers of the
indicated filters in the following order, pH 5, 7, and
then 9. Next, 10 ml of 3% beef extract (Scott
Laboratories, Fiskeville, RI) + 1 M NaC1, pH 9 were passed
through the filters to elute adsorbed viruses. The number
of viruses in the beef extract eluate and in the filter
effluents were compared with the number of viruses in the
initial sample to determine the percent of viruses
adsorbed and total virus eluted. Values indicate the mean
and standard deviation of quadruplicate determinations.





























0


S60










UT NALCO PEl PN P+ N N-p p N
40


0 20



PH 5 1 9 R 5 7 9R 5 79 R 5 7 9 R 57 9 R 579 R 579 R
UT NALCO PEI P/N P+ N N-p P- N








Figure 3-3: Adsorption of poliovirus type-i from
trickling filter filtrate at pH 5, 7, and 9
using filterite filters modified with
cationic polymers.

One-hundred milliliters of trickling filter effluent
(TFE) prefiltered with an AP20 filter to remove suspended
solids were seeded to approximately 105 PFU/ml of P1 and
adjusted to pH 5, 7, or 9 and were passed through the
indicated filters containing two layers of Nalco-treated
filters (0.05%) and two layers of filters treated with PEI
at differing concentrations. The number of viruses in the
filtrates were compared with those in the initial samples
to determine the percentage of viruses adsorbed. Values
indicate the mean and standard deviation of quadruplicate
determinations.























* paper 25C 0 plaste 4C
o plaoti 2SC A plastic -20C


4
41
49
0


Figure 3-4: Stability of PEI-treated filters.

PEI-treated and untreated Filterite filters were
placed in paper envelopes at 250C and sealed in plastic
storage bags at 25, 4, and -200C. At the indicated time
intervals, 100ml of AP20 prefiltered TFE, pH 7
(approximately 20PFU/ml of indigenous bacteriophage), were
passed through the filters and the filter effluents and
initial sample assayed for indigenous bacteriophage.
Values represent the mean and standard error of duplicate
determinations of a paired difference analysis.










Discussion


The ability of electronegative and electropositive

filters to adsorb viruses from waters has been well

documented and these filters have been used to detect

viruses in environmental water samples (8,36). In general,

electronegative filters do not adsorb viruses well under

ambient water conditions whereas electropositive filters

are more efficient for this task. The relative isoelectric

points of virus and filter are responsible for this

phenomenon. Electropositive filters such as Seitz

(asbestos-containing filters), Zeta-plus (diatomaceous

earth and anion-exchange resin-containing filters), and

Virasorb 1-MDS filters (charge modified resin-containing

filters) have been used to concentrate viruses from

surface and wastewaters (73,78,79). These electropositive

filters, however, have some noted disadvantages. Seitz and

Zeta-plus filters have slow flow rates which prohibit the

analysis of large volumes of water whereas Virasorb 1-MDS

filters are expensive relative to other filter types.

Although PEI-treated glass surfaces have been used to

immobilize yeast cells (23), this is the first study to

utilize this novel approach to modify microporous filters

for the concentration of viruses from water. The results

of this study indicate that the virus adsorbing properties

of electronegative Filterite fiberglass filters can be

greatly enhanced by treating the filters with an aqueous








solution of a cationic polymer such as PEI. This

modification results in a filter which adsorbs viruses

well under ambient conditions while retaining the high

flow rates possible with untreated Filterite filters.

Although modified filters adsorb bacteriophages (MS2, T2,

and OX174) and enteroviruses (P1, El, E5, and CB5) well

from buffer and tap water at pH values indicative of

ambient water conditions (pH 5, 7, and 9), no single

polymer was able to adsorb P1 at all tested pH values. The

recovery of bacteriophage MS2 and indigenous bacteriophage

with 3% beef extract following adsorption to PEI-treated

Filterite filters was also not efficient. The type of

cationic polymer used to modify Filterite filters was

found to be important for both the adsorption and recovery

of viruses from buffer. For example, LCI and Dellchem

polymers were found to be better suited for the recovery

of bacteriophage MS2, whereas PEI and Nalco polymers were

found to be best suited for P1 recovery under the same

conditions. All polymers tested, however, were found to be

equally effective for the removal of bacteriophage MS2

from buffer at pH 5, 7, and 9 whereas the adsorption and

recovery of P1 were found to be dependent on the type of

polymer used to treat the filters as well as the pH of the

buffer.

The use of beef extract rather than defined media as

an eluent is presently the method of choice as several

second-step concentrating procedures are available which

rely on the flocuation of beef extract with acid, (36,50)









or ammonium sulfate (72). The low levels of P1 recovered

using Tween 80 as an eluent reflects weak hydrophobic

interaction between the viruses and the N/P filters

whereas the ability of 1M NaCl, pH 9, to recover P1

quantitatively indicates that electrostatic interactions

play an important role in the adsorption of P1 to N/P

filters (28,33,71). This is supported by zeta-potential

measurements of PEI- and Nalco-treated filters and this

indicates that these cationic polymers convert

electronegative Filterite filters to electropositive

filters,

Although our studies indicate that PEI-treated

filters can be stabilized by storage in air-tight plastic

bags, the simplicity of the filter-modifying procedure

does not make filter preservation a major consideration.

The results of this study have shown that fiberglass

filters can be modified with cationic polymers simply and

inexpensively to enhance their virus adsorbing properties

to include ambient water conditions. We feel that the use

of filters modified with cationic polymers is most

promising for the detection of indigenous bacteriophage

and enteroviruses in waters.


















CHAPTER 4

ENHANCED INFECTIVITY OF ENTEROVIRUSES IN VITRO
BY PRETREATING HOST CELL MONOLAYERS
WITH CATIONIC POLYMERS


Introduction


Cationic polymers such as DEAE-dextran (DEAE-D)

(27,84,86,90,91), protamine (84,90), and platinum

polyamines (15) have been used to enhance picornavirus

(15,84,86), herpesvirus (84), adenovirus (84), and

rabiesvirus (49,90,91) infectivity in vitro using

mammalian cell cultures. Conversely, anionic polymers such

as carrageenan, (40), protamine sulfate (84,86) and

dextran sulfate (40,49,57,91) have been shown to reduce

virus infectivity in vitro. Dextran sulfate has also been

shown to prolong scrapie incubation period in vivo (27).

The phenomenon of cationic polymer enhanced infectivity of

enteroviruses has been applied to the study of

environmental virology to enhance the probability of

detecting the very low numbers of viable viruses found in

environmental concentrates (40). Enteroviral infectivity

has also been enhanced in vitro by adding 5-Iodo-2'-

Deoxyuridine to the culture medium (3). This study reports

the enhancement of virus infectivity of both laboratory








strains of picornaviruses and viruses concentrated from

raw wastewater by increasing the number of viruses

detected as well as the rate at which they are detected by

retreating cell monolayers with the cationic polymer

polyethylenimine (PEI). PEI was found to be a more

effective enhancer of virus infectivity than DEAE-D.

Mechanistically, we propose that PEI enhances virus

infectivity by enhancing the attachment of virus to host

cell.


Materials and Methods


Virus and Viral Assays

Poliovirus type-1 (P1), coxsackievirus type-B5 (CB5),

and echovirus types-1 and 5 (El and E5) were quantitated

on Buffalo Green Monkey kidney (BGM) cell monolayers as

plaque forming units (PFU) using a methylcellulose or agar

overlay as previously described (71) or as the cytopathic

effect (CPE) using a liquid overlay as previously

described (42). All viruses were harvested from cell

lysates by freezing and thawing three times, centrifuging

at 14,000 x g for ten minutes, and then treating the

supernatant twice with trichloro-trifluoro ethane (freon).


Chemicals and Filters

The following chemicals and their sources were used

in this study: glycine, hydrochloric acid, sodium

hydroxide, and trichloro-trifluoro ethane were from Fisher

Scientific Co. (Fair Lawn, N.J.); crystal violet, DEAE-








dextran, immidazole and polyethylenimine (PEI) were from

Sigma Chemical Co. (St. Louis, Mo.); Dellchem cationic

polymer (BASF CF 600) and LCI cationic polymer were gifts

from Mike New (Kanapaha Wastewater Treatment Plant,

Gainesville, Fla.); Nalco cationic polymer (90% charge,

high molecular weight) was a gift from Kenneth E. DeFarmo

(Leachem Industries, Inc., Titusville, Fla.). The

following filters were used in this study: epoxyfiberglass

filters (pore size, 0.2 to 1.0 ur; from pleated filter

cartridges; Duofine Filterite, Timonium, Md.).


Tissue Culture Media and Plasticware

The following tissue culture media and buffers were

used in this study: fetal bovine serum (FBS), minimum

essential medium Eagle (MEM) and trypsin solution from

M.A. Bioproducts (Walkersville, MD); L-glutamine from Flow

Laboratories, Inc. (McLean, VA); hepes buffer from

Research Organics, Inc. (Cleveland, OH), methyl cellulose

(MC) 1500 centipoise from Fisher Scientific (Fair Lawn,

NJ); granulated agar from BBL Microbiology Systems

(Cockeysville, MD); penicillin-G and streptomycin from

Sigma Chemical Co. (St. Louis, MO). The following tissue

culture plastic ware were used in this study: Linbro six-

well 9.6cm2 microtiter tissue culture plates from Flow

Laboratories, Inc. (McClean, VA); and Lux 25cm2 tissue

culture flasks from Miles Scientific (Naperville, IL).









Preparation of PEI

A one percent (w/v) aqueous solution of PEI was

filtered with a 0.25 um Filterite filter and one ml

aliquots were frozen at -20C. When needed, the PEI stock

solution was diluted in PBS supplemented with 2% FBS (PBS-

2%FBS).


Enhanced Infectivity Studies

Methylcellulose overlay. After the spent media from

BGM cell monolayers was poured off, cell monolayers were

pretreated with either PBS-2% FBS or PEI in PBS-2%FBS

(0.1 ml per well for six-well microtiter tissue culture

plates, 9.6 cm2) and incubated for two minutes at room

temperature. Next, 0.1 ml of virus in PBS-2%FBS were added

to the monolayers which were further incubated at room

temperature. The infected cell monolayers were then

overlayed with 3 ml of methylcellulose supplemented with

MEM (MEM-MC). For kinetic studies, infected cell

monolayers were washed three times with 1 ml of PBS prior

to MEM-MC overlay. Infected cell monolayers were then

incubated at 370C until plaques developed (48 to 72

hours). Plaque visualization was enhanced by staining cell

monolayers with a 1% crystal violet solution.

Liquid overlays. For the determination of CPE, cell

monolayers were overlayed with MEM rather than MEM-MC and

observed microscopically every 24 hours.









Enhanced Infectivity of Viruses From Raw Wastewater

Concentration of viruses. Enteroviruses from one

liter of raw wastewater from the Kanapaha Wastewater

Treatment Plant, Gainesville, Florida were concentrated

100 fold by organic flocculation. Briefly, one liter of

raw wastewater was adjusted to pH 3.5 and centrifuged at

3,000 x g for 10 minutes. Next, the pellet was suspended

in 10 ml of 3% beef extract at pH 9 and centrifuged at

12,000 x g for 10 minutes. The supernatant was then

adjusted to neutral pH with 0.01 N HC1. Enteroviruses from

40 liters of raw wastewater from the Belle Glade

Wastewater Treatment Plant, Belle Glade, Florida were

concentrated 400 fold by adsorption-elution from a

Filterite cartridge filter. Briefly, 40 liters of raw

wastewater were adjusted to pH 3.5 and passed through a

Filterite pleated filter cartridge (10 inches in length

and 0.45 um pore size) under positive pressure. Adsorbed

viruses were recovered by passing 1 liter of 3% beef

extract, pH 9, through the filter. Viruses were further

concentrated by the organic flocculation procedure of

Katzenelson et al. (50).


Detection of Indigenous Viruses

Most-probable numbers (MPN). MPN's of enteroviruses

were determined using 25 cm2 flasks containing cell

monolayers which were pretreated with 0.3 ml of MEM-2%FBS

or 0.3 ml of 5.0 x 10-3 % PEI in MEM-2%FBS, using ten-fold

dilutions of the virus concentrate. Viruses were allowed









to adsorb to the monolayers for 30 minutes after which

they were overlayed with 5 ml of MEM-2%FBS. Flasks were

observed for CPE every 24 hours and the spent media

replaced with 5 ml of fresh MEM-2% FBS every three days.

MPN's were determined using previously published tables

(1).

Plaque-forming units. PFU's were determined under a

1.2% agar overlay as previously described (5) using BGM

cell monolayers in 25 cm2 tissue culture flasks. Cell

monolayers were either pretreated with 0.3 ml of MEM-2%FBS

or 0.3 ml of 5.0x10-3% PEI in MEM-2%FBS and allowed to

incubate 30 minutes at room temperature after inoculation

and incubated at 370C for 5 days. Plaques were counted

every 24 hours and plaques were picked and passed in BGM

cells for confirmation.


Results


As shown in Table 4-1, several cationic polymers were

tested for their ability to enhance P1 infectivity in

vitro. P1 infectivity measured as PFU was increased 5.0

fold by treating cells with 1.0 x 10-2% (w/v)

polyethylenimine (PEI) as opposed to 2.2 fold by Dellchem

cationic polymer, 0.9 fold by Nalco cationic polymer, and

1.7 fold by LCI cationic polymer. The concentration of PEI

used to treat BGM cell monolayers that produced the

maximum increase in PFU of P1 was found to be 5.0 x 10-3%

(w/v) (Figure 4-1); several enteroviruses were








investigated for enhanced infectivity using this

concentration of PEI (Table 4-2). P1 infectivity was

enhanced 5.5 fold, El 1.2 fold, E5 5.2 fold, and CB5 4.9

fold in vitro. In comparison, P1 infectivity was increased

2.0 fold when cell monolayers were pretreated with 5.0 x

10-2% (w/v) of the cationic polymer DEAE-D (Table 4-3).

PEI-treated cells were then evaluated for their

ability to enhance the detection of naturally occurring

viruses isolated from raw wastewater as quantitated by

MPN's (Table 4-4) or by PFU under an agar overlay

(Table 4-5). When indigenous viruses were quantitated via

MPN's, unwashed PEI-treated cells showed a 10.8 fold

increase in titer over that of untreated cells after 48

hours of incubation; however, only a 1.9 fold increase in

titer was noted after 5 days of incubation with an overall

average of a 3.7 fold increase in titer over the 5 days.

When natural viruses were quantitated as PFU's under an

agar overlay, PEI-treated cells showed a 3.3 fold increase

in titer over untreated cells.

The relative rates of virus attachment to PEI-treated

and untreated cell monolayers were investigated by washing

cell monolayers three times with 1 ml of PBS two and

thirty minutes after infection of P1 and El (Table 4-6).

In general, P1 and El infectivity was much more resistant

to washing of the cell monolayer when the cell monolayers

were pretreated with PEI. When compared to untreated cell

monolayers, P1 infectivity was enhanced 42.7 fold and El

infectivity was enhanced 4.7 fold when cell monolayers









were washed two minutes after infection. When cell

monolayers were washed thirty minutes after infection, P1

infectivity was enhanced 9.2 fold and El infectivity was

enhanced 2.3 fold.

As shown in Figure 4-2 and Figure 4-3, P1 and El CPE

was enhanced in the presence of PEI. In general, P1 and

El CPE at a specific dilution was noted 24 hours earlier

when cell monolayers were pretreated with PEI.

To determine if PEI enhanced the rate of virus

replication or the number of virus produced per infected

cell, single-step growth curves of P1 on BGM cell

monolayers were constructed in the presence and absence

of PEI (Figure 4-4). No differences were noted between

these two curves.

To determine if PEI exerted an effect after viral

replication had been initiated, PEI was added to infected

cell monolayers following washes to remove unadsorbed

viruses from the culture fluid (Table 4-7). When PEI was

added before infection with P1 and allowed to incubate for

30 minutes there was a 7.6 fold increase in P1 titer.

However if PEI was added after 30 minutes of P1 infection,

there was a 2.8 fold increase in P1 titer. Much lower

increase in titers were noted when PEI was added two

minutes (1.0 fold increase in titer) or ten minutes (1.9

fold increase in titer) after infection.

The possibility that PEI could dissaggregate

multiviral particles and therefore give an increase in








titer was investigated by treating P1 cell lysates

sequentially with procedures which would promote the

formation of monodispersed viruses and then determining

the P1 titer in the presence and absence of PEI (Table 4-

8). When cell lysates were frozen and thawed three times,

P1 infectivity was increased 2.7 fold when assayed on

untreated and PEI-treated BGM cell monolayers, low speed

centrifugation gave 2.1 fold increase in titer, one freon

treatment gave 2.5 fold increase in titer, a second freon

treatment gave 4.0 fold increase in titer, and filtration

through a 0.2 ur pore size membrane filter gave a 3.3 fold

increase in titer.

To determine if PEI could have an effect on P1

replication in an uncompetent cell line, P1 was used to

infect BGM (competent) and HL3 (uncompetent) cell lines

(Table 4-9). Cell monolayers were observed for

cytopathology, stained by the immunoperoxidase method of

Payment and Trudel (62) to detect viral antigens within

the cells, and for infectious P1 in the culture

supernatant. The results of these studies indicate that

PEI does not enable P1 to infect or replicate within an

uncompetent cell line such as the mouse-macrophage cell

line HL3.















Table 4-1: Enhanced plaquing efficentcy of poliovirus-1 by
pretreating BGM cell monolayers with cationic
polymers.a


Concentration Fold Increase
Polymer of Polymer (%) in Plaques


Polyethylenimine
(PEI) 1.0 x 10-2 5.0+1.3
-3
1.0 x 10-3 2.3+1.0
1.0 x 10-4 1.6+0.3

Dellchem 1.0 x 10-2 2.2+1.4
1.0 x 10-3 1.3+0.6
1.0 x 104 1.5+0.7
-4

Nalco 1.0 x 10-2 Toxic
1.0 x 10-3 0.5+0.3
-4
1.0 x 104 0.9+0.2

ICI 1.0 x 10-2 1.7+0.3
1.0 x 10-3 1.1+1.4
1.0 x 10-4 1.2+0.05



a BGM cell monolayers grown in microtiter plates (9.6
cm2 approximate surface area) were treated in the
following manner to increase P1 infectivity. The old media
was discarded and replaced with 0.1 ml of the indicated
cationic polymer in PBS-2%FBS or 0.1 ml of PBS-2%FBS.
Within two minutes, 0.lml of P1 diluted in PBS-2%FBS were
added to the monolayer and subsequently incubated at room
temperature for thirty minutes prior to a methylcellulose
overlay. Microtiter plates were incubated at 37 C for 2 to
3 days prior to staining with a crystal violet solution to
visualize plaques. Values represent the fold increase in
poliovirus-1 titer over BGM cell monolayers not treated
with cationic polymers and represent the mean and standard
deviation of triplicate determinations.




























Table 4-2: Enhanced plaquing of enteroviruses by
pretreating BGM cell monolayers with PEI.a


Virus


Poliovirus-1

Echovirus-1

Echovirus-5

Coxsackievirus-B5


Fold Increase
In Plaques


5.5+0.9

1.2+0.3

5.2+1.0

4.9+0.7


a The procedure described in Table 4-1 was used
except only the cationic polymer PEI diluted to 5.0 x 10-
% (w/v) in PBS-2%FBS was used. Values represent the mean
and standard deviation of twelve determinations.


- --------










Table 4-3: Enhanced plaquing of poliovirus-1 on BGM cell
monolayers using the cationic polymer DEAE-
dextran.a




Washed or Concentration of Fold Increase
Unwashed DEAE-Dextran (%) in Plaques


Unwashed


1.0

0.5

0.1

0.05

0.01

0.005

0.001


1.0

0.5

0.1

0.05


Washed


Toxic

0.7+3


1.6+0.2

2.0+0.2

1.1+0.2

1.0+0.1

1.1+0.1


1.3+0.2

1.0+0.2

1.6+0.2

2.0+0.3

2.0+0.3

1.2+0.1

1.0+0.2


0.01


0.005

0.001


a The procedure described in Table 4-1 was used
except the cationic polymer DEAE-dextran diluted in PBS-
2%FBS was used. The values indicate the mean and standard
deviation of triplicate determinations.

b Cell monolayers were either unwashed or washed
three times with 1 ml of PBS after 30 minutes of
incubation at room temperature after the addition of the
cationic polymer and P1.





64













Table 4-4: Enhanced infectivity of enteroviruses from raw
wastewater using PEI-treated BGM cell
monolayers.a


Washed or
Unwashed


Concentration
of PEI (%)


MPN/ liter of sewage on day

1 2 3 4 5


Unwashed


Washed


0 80 180 180 460


5.0 x 10-3

0

5.0 x 10-3


0 860

0 180

0 180


a The procedure described


methods was used for these experiments.


860 860 860

180 180 180

460 460 460


in the materials and


- I --




















Table 4-5: Enhanced infectivity of indigenous viruses
from raw wastewater under an agar overlay.a




PFU/ liter of raw sewage


Sample Untreated Cells PEI-Treated Cells PEI/UTb



Kanapaha
#1 30 120 4.0

Kanapaha
#2 20 70 3.5

Belle
Glade 30 70 2.3



a The procedure described in the materials and
methods was used for these experiments using 5.0 x 10- %
PEI.

b PEI/UT represents the ratio of viurs titers using
PEI-treated cells and untreated cells.













Table 4-6: Effects of PEI and washing of infected cell
monolayers on enterovirus titers.a



Titer (PFU/ml)


Time of Untreated PEI-Treated PEI4
Virus Incubation Cells Cells UT
Before Washing



P1 2 Minutes 7.5x105 3.2x107 42.7

30 Minutes 1.2x106 l.1x107 9.2



El 2 Minutes 3.0x103 1.4x104 4.7

30 Minutes l.5x104 3.5x104 2.3




a BGM cell monolayers were treated with Q.1 ml of
PBS-2%FBS or 0.1 ml of PBS-2%FBS with 5.0 x 10 % PEI as
described in Table 4-1 except the cells were either
unwashed or washed three times with 1 ml of PBS two or
thirty minutes after infection with P1 or El. PBS-2%FBS-
treated, washed cells served as a control for PEI-treated
cells. The values indicate the mean of triplicate
determinations.

b PEI/UT represents the ratio of virus titers for
PEI-treated cells and untreated cells washed at after the
indicated period of incubation.















Table 4-7: Effects of adding PEI to cell monolayers after
infection with PI.a



PEI added
Time of before or after Fold increase
Infection infection washed in titer





30 min. before no 7.6 + 2.3

30 min after yes 2.8 + 0.4

10 min after yes 1.9 + 0.4

2 minutes after yes 1.0 + 0.3




a BGM cell monolayers on 6 well microtiter plated
were infected with 0.1 ml of P1 in PBS-2%FBS and allowed in
incubate at room temperature for in indicated time and
then washed three time- with 1 ml of PBS. Next, 0.1 ml of
PBS-2%FBS or 5.0 x 10 % PEI in PBS-2%FBS were added to
the infected cell monolayers and allowed incubate two
minutes at room temperature and then overlayed with MEM-MC
and incubated at 370C until plaques developed. BGM cell
monolayers pretreated with PBS-2%FBS or 5.0 x 10" PEI
in PBS-2%FBS served as the control. Valures represent the
mean and standard deviation of quadruplicate
determinations.













Table 4-8: Effects of processing of Poliovirus type-i cell
lysates on PEF mediated enhanced infectivity of
P1.a


P1 Processing


Freeze-Thaw

Centrifugation

Freon #1


Freon #2

Filtered


Fold Increase
in Titer


2.7 + 1.0

2.1 + 0.2

2.5 + 0.6

4.0 + 1.4

3.3 + 0.9


a BGM cell lysates from P1 infected cells were frozen
at -200C and thawed at room temperature three times
(freeze-thaw) and centrifuged at 14,000 x g for ten
minutes and the supernatant saved (centrifugation). The
supernatants were then treated twice with freon (freon #1
and #2) and the aqueous phase passed through a 0.2um pore
size filter (filtered). P1 titers were then determined for
each of these samples by pretreating BGM cell monolayers
in 6 well microtiter plates with 0.1 ml of PBS-2%FBS or
5.0 x 10- % PEI in PBS-2%FBS and were then infected with
0.1 ml of P1 diluted in PBS-2%FBS, incubated at room
temperature, overlayed with MEM-MC, and incubated at 370C
for 48 to 72 hours for plaques to develop. Values
represent the mean and standard deviation of quadruplicate
determinations.


_ ________ __C_ ______ ~_
















































Figure 4-1: Effects of PEI and washing of infected
monolayers of Poliovirus type-i infectivity.

BGM cell monolayers were treated with the cationic
polymer PEI as described in Table 4-1 except the cells
were unwashed (circle), washed three times with 1 ml of
PBS two minutes (triangles) or thirty minutes (squares)
after the cell monolayer was infected. Fold increase
referees to the ra io of P1 titers pretreated with PBS-
2%FBS or 5.0 x 10~ % PEI in PBS-2%FBS. Values represent
the mean and standard deviation of triplicate
determinations.


Il. l I ll-l i l'l l il 1 1[ .1 (



















o 7



S 6

o











0 2 3 4 5

Time (Days)




Figure 4-2: Effects of PEI on Poliovirus type-1
infectivity.

BGM cell monolayers either pretreated with 0.1 ml of
PBS-2%FBS (squares) or 0.1 ml of 5.0x10-3% PEI in PBS-
2%FBS (circles) prior to infection with increasing
dilutions of P1 in PBS-2%FBS. After infection, the
monolayers were incubated 30 minutes at room temperature
prior to overlay with a liquid medium (MEM). Cell
monolayers were observed for the cytopathic effect (CPE)
every 24 hours and positive samples were determined when
50% of the monolayer showed CPE. Values represent the mean
and standard deviation of triplicate determinations.
i-4


























and standard deviation of triplicate determinations.















































Time (Days)


Figure 4-3: Effects of PEI on Echovirus
infectivity.


type-i


The procedure described in Figure 4-2 was used except
that El was used in place of PI.


r I 1






















* 0-


0-0 0.


609


* 0 -*


Figure 4-4: Single step growth curve of P1 in the presence
and abscence of PEI.

BGM cell monolayers in 6 well microtiter plates were
pretreated with either 0.1 ml of PBS-2%FBS (open circles)
or 5.0 x 10 % PEI in PBS-2%FBS (closed circles) and
infected with P1 at a multiplicity of infection of 100 PFU
per cell and allowed to incubate at room temperature for
thirty minutes. The infected cell monolayers were then
washed three times with PBS, overlayed with three ml of
MEM-2%FBS, and incubated at 37C. At the indicated time
intervals, 0.1 ml samples were taken from the cell culture
medium, diluted in PBS-2%FBS, and assayed as PFU under a
methylcellulose overlay. Values represent the mean and
standard deviation of quadruplicate determinations.

















Table 4-9: Effects of PEI on P1 infectivity in competent
and uncompetent cell lines.a




Cell Cyto- Immunoperoxidase Final
Line Origin PEI pathology Staining Titer
(PFU/ml)

BGM Monkey
Kidney + + + 4.6 x 107

-+ + 4.5 x 107


HL3 Mouse
Macrophage
+ 0

0



a Competent (BGM) and uncompetent (HL3) cell
monglayers were either pretreated with PBS-2%FBS or 5.0 x
10" % PEI in PBS-2%FBS and infected with approximately
1000 PFU of P1 diluted in PBS-2%FBS and incubated for
thirty minutes at 240C and overlayed with MEM-2%FBS. After
72 hours, cell monolayers were observed for cytopathology
and were stained by the immunoperoxidase method of Payment
and Trudel (13) using pooled human serum. The culture
fluid was also assayed for viable viruses.












Discussion


The low numbers of enteroviruses found in

environmental samples has led to the developments of

several procedures for the concentration of viruses from

the environment (8). The concentrates are then normally

assayed for viable viruses using monkey kidney cells in

vitro by observing for viral cytopathology, plaques,

and/or in situ staining of viral antigens with fluorescent

antibodies for relatively non-cytopathic viruses such as

human rotaviruses (42). The cationic polymer DEAE-dextran

has been used in the tissue culture medium to enhance

viral infectivity in vito (42,84). Our results indicate

that DEAE-dextran lead to a 2.0 fold increase in P1

infectivity as assayed as PFU's under a methylcellulose

overlay, which is consistent with previous reports

(84,88). When several synthetic cationic polymers were

tested for their ability to enhance enteroviral

infectivity in vitro, it was discovered that cell

monolayers pretreated with a small volume of a relatively

low concentration of PEI showed a 5.5 fold increase in

poliovirus infectivity in terms of PFU's. It was also

noted that viral cytopathology was noted earlier when cell

monolayers were pretreated with PEI.

In an effort to determine at what point in the virus

replicating cycle that PEI exerted its effect, cell

monolayers were washed with PBS two or thirty minutes









after infection and were treated with PEI after infection.

The results of these studies indicate that PEI exerts an

effect within the first two minutes of the infection

cycle, probably during the attachment or penetration of

the virus into the host cell and has little effect when

added after virus replication has begun. A similar

phenomenon was noted by Wunner et al. (90) when

investigating the attachment kinetics of rabies virus to

host cells in the presence of DEAE-dextran. Kaplan et al.

(49) proposed that cationic polymers exerted their effect

early in the rabiesvirus replication cycle.

Bailey et al. (2) had previously postulated that

DEAE-dextran exerted its effect on rabies virus

infectivity via an electrostatic-mediated, non-specific

attachment of virus to cell. We therefore used a competent

cell line (BGM-monkey) and an incompetent cell line (HL3-

mouse) for the replication of P1. Because P1 is a primate

specific virus, mouse cell so not have P1 reseptors. We

found that PEI enhances the adsorption of P1 to cells.

One possible mechanism of cationic polymer-mediated

enhanced infectivity that has not been investigated is the

dissagregation of multiviral complexes by cationic

polymers. We sequentially treated P1 cell lysates by

freeze-thawing to disrupt cell membranes, centrifuged at

14,000 x g to remove cell debris, treated the supernatant

two times with freon to remove contaminating membranes,

proteins, and to promote a monodispersed virus suspension,








and finally passed the freon treated aqueous phase through

a 0.2 um membrane filter to remove large viral aggragates.

The results of these studies indicate that PEI has a

maximum effect on PEI titer when cell lysates have been

treated twice with freon and/or passed through a

microporous membrane filter, indicating that PEI exerts

its maximum effect with monodispersed virus suspensions.

We therefore propose that PEI enhances the

infectivity of enteroviruses in vitro by enhancing the

attachment of virus to cell in a specific manner.

Regardless of the mechanism of PEI-mediated enhanced

infectivity of enteroviruses, it could be important to the

study of environmental virology by reducing the problems

associated with toxicity of samples to cell monolayers by

allowing toxic elements to be washed away from cell

monolayers infected with environmental samples. It is

likely that shorter incubation periods can be used with

environmental samples without decreasing the number of

viruses detected, but with a reduction of toxicity to

cells.

















CHAPTER 5

RAPID DETECTION OF ENTEROVIRUSES
USING IMMUNOLOGICAL METHODS



Introduction


In an effort to enhance the speed and sensitivity of

enteroviral assays, researchers have investigated

immunological techniques (22,45,62) for the rapid

detection of enteroviruses in water. Non-tissue culture-

based immunological assays such as solid-phase enzyme-

linked immunosorbent assay (ELISA) have given

sensitivities of approximately 106 plaque-forming-units

(PFU) of enteroviruses (22). These assays have not

received significant attention for the direct detection of

viruses from water due to this lack of sensitivity. The

use of immunological techniques for the detection of

viruses in clinical samples has been recently reviewed

(43). Although numerous monoclonal antibodies directed

against polioviruses have been produced and used to

differentiate poliovirus strains (12,34,56,58) and to

study the nature of neutralizing epitopes









(11,25,26,35,44,48), monoclonal antibodies have not been

reported for the direct detection of enteroviruses from

water.

Recently, Payment and Trudel (62) reported a rapid

and sensitive method for detecting virus infected cells in

vitro by in situ immunoperoxidase staining of infected

cell monolayers using pooled human serum or rabbit.

polyclonal serum against rotavirus as the primary

antibody. Payment's immunoperoxidase method using the MA-

104 cell line gave approximately a 100-fold increase in

the sensitivity of tissue culture-based assays for the

detection of indigenous viruses from water samples (62).

This increase in sensitivity of the tissue culture based

assay is most likely attributed to the ability of several

human enteric viruses to infect but not replicate

efficiently in the cell monolayers used for enteroviial

assays (62). This situation leads to singly infected cells

that are not readily distinguishable as cytopathic and do

not form plaques on host cell monolayers. A most notable

example of a non-cytopathic human enterovirus which can be

detected by an in situ radioimmunoassay is the hepatitis

type-A virus which has been implicated in numerous

outbreaks of infectious hepatitis in the United States

(65). Other viruses which may be detected by the

immunoperoxidase method would include adenoviruses (47)

and rotaviruses (62).








This study reports the use of Payment and Trudel's

technique (62) for the in situ immunoperoxidase staining

of virus infected cells in conjunction with pretreating

host cell monolayers with the cationic polymer

polyethylenimine (PEI) to enhance virus infectivity and

to give a rapid assay of greater sensitivity than that of

Payment's (62). In these studies, a neutralizing

monoclonal antibody of poliovirus type-i (PINutMAb),

pooled whole human serum (PHS), and rabbit anti PI (RP1)

were used as the primary antibody for the

immunoperoxidase staining of virus infected cells.


Materials and Methods


Viruses and Viral Assays

Poliovirus type-1 (P1) Mahoney strain, coxsackievirus

type-B5 (CB5), and echovirus types-i and -5 (El and E5)

were determined as plaque-forming-units (PFU) on Buffalo

Green Monkey kidney cells (BGM) under a methylceilulose

overlay as previously described (71).


Chemicals and Tissue Culture Media

The following chemicals with their sources were used

in this study: bovine serum albumin (BSA) 3,3-

diaminobenzadine (DAB), Freund's complete and incomplete

adjuvant, polyethylenimine (PEI), pooled whole human male

serum (PHS), protein-A horseradish-peroxidase conjugate

(PAHRP), sodium phosphate dibasic, and Tween 80 were from

Sigma Chemical Co. (St. Louis, MO.). Citric acid, dimethyl








sulfoxide (DMSO), granular agar, 50% hydrogen peroxide,

methyl alcohol, sodium bicarbonate and trichloro-

trifluoro-ethane (Freon) were from Fisher Scientific Co.

(Fair Lawn, NJ.). DEAE Sepharose CL-6B was from Pharmacia

Chemical Co. (Upplala, Sweden). Polyethylene glycol 1500

was from Boehringer Mannheim (Indianapolis, IN). The

following tissue culture media for hybridoma cell lines

were used in this study. Amphotericin B (450 mg/ml),

Dulbecco's modified Eagle's minimal Medium (DEM), HT media

supplement (5.0 x 10-5 M hypoxanthenine and 8.0 x 10-4 M

thymidine final concentrations), HAT media supplement (5.0

x 10-5 M hypoxanthenine, 8.0 x 10-4 M thymidine, and 2.0 x

10-5 M aminopterine final concentrations), gentamycin

sulfate, horse serum, and hybrimax supplement CPSR-3 were

from Sigma Chemical Co. (St. Louis, MO.). The following

tissue culture media for the BGM cell line were used in

this study. Minimum essential medium Eagle (MEM) was

front; MA Bioproducts (Walkersville, MD). Cellect Gold Fetal

Bovine Serum (FBS) and L-glutamine were from Flow Labs,

Inc. (McClean, VA).


Production of Hybridoma Cell Lines Secreting Poliovirus
Type-1 Monospecific Neutralizing Antibody (PINutMAb)

Purification of poliovirus type-1. P! was grown on BGM

cell monolayers at an infection rate of 100 PFU per 125

cm2 of cell monolayer in flasks containing 50 ml of Eagles

minimal essential medium supplemented with 2% fetal bovine

serum (MEM-2%FBS). After 48 to 72 hours the cell

monolayers showed complete cytopathology and the flasks









were frozen at -200C and thawed at 250C three times to

disrupt cells. The culture fluids were combined (200 ml

total volume) and centrifuged at 14,000 x g for 10

minutes. The supernatant was extracted twice with freon

and the aqueous phase was centrifuged at 40,000 rpm in a

Ti60 rotor for 24 hours. The pellets were suspended in

phosphate buffer saline (PBS) and the volume reduced to 5

mil using a 30 kDal ultrafiltration membrane (Centriprep

30, Amicon Corp., Lexington, Mass). Virions were further

purified by adsorption onto DEAE-CL6B Sepharose column and

eluted from the column with a 0.1 to 1.0 M NaCl step

gradient in 3.2 mM sodium phosphate buffer at pH 8.0.

Column fractions were assayed for PFU on BGM cell

monolayers and as total protein by the method of Bradford

(13). Column fractions containing greater than 103 PFU/ml

and less than 1 ug protein/ml were combined, concentrated,

and dialyzed against PBS by ultrafiltration and titered as

PFU on BGM cell monolayers.

Immunization of Balb/C mice. Five BALB/C mice were

immunized intraparitanealy with 108 PFU of purified P1 in

Freund's complete adjuvent. These mice were then immunized

intrapritanealy four more times at weekly intervals with

10 PFU of purified P1 in Freund's incomplete adjuvent.

Production of hybridomas secreting P1 neutralizing
monoclonal antibodies (PINutMAb). After immuniz-

ation, one mouse was sacrificed and the serum used as a

positive control. The hyperimmunized mouse spleen was

macerated and the recovered spenocytes fused with an SP2/O









Agl4 myaloma cell line using 50% polyethylene glycol 1500

in 75 mM HEPES buffer at pH 8.0 following the

manufacture's suggested protocol. The fussed cells were

plated out in 96 well microtiter tissue culture plates

(Corning Glass Works, Corning, NY) in DME-10% hybrimax

supplement at a volume of 50 ul per well and incubated

for 1 day at 370C in 5% CO2 and 100% humidity. At this

time, cells were fed 50 ul of 2 X HAT-DME-10% hybrimax

supplement. Cells were then fed 50 ul of 1 X HAT-DME-10%

hybrimax supplirement every five days thereafter for two

weeks. During this time, wells with hybridoma colonies

were transferred to 24 well microtiter tissue culture

plates and maintained in HT-DME-10% hybrimax supplement.

The hybridoma supernatants were assayed for P1

neutralizing antibody by adding approximately 100 PFU of

P1 to 0.5 ml of hybridoma supernatant, incubating for 2

hours at 370C, and then assaying for infectious virus

particles as PFU on BGM cell monolayers. Those hybridoma

cultures whcih showed greater than 50% neutralization of

P1 were further cultured and then cloned twice by the

method of limiting dilution. Positive hybridoma cell lines

were frozen in DME with 10% DMSO or horse serum with 10%

DMSO at -80C in 1 ml aliquots and labeled as.


Production of Rabbit Anti-Poliovirus Type-1 (RP1)

Rabbits were immunized with P1 as described

previously for the immunization of Balb/C mice for the

production of monoclonal antibodies except that P1 tissue









culture lysates grown on BGM cell monolayers were first

frozen -200C and thawed at 240C three times, centrifuged

at 14,000 x g for ten minutes, and then extracted twice

with freon prior to immunization. Rabbits were bled by the

ear vein one week after boosting. Serum was frozen at -

200C.


Determinations of Neutralizing Titers of Antibody
Preparations

One-half milliliter of PHS, RP1, or P1NutMAb diluted

in PBS supplemented with 2% FBS (PBS-2%FBS) was added to

100 PFU of PI, El, E5, or CB5 diluted in PBS-2%FBS and

incubated at 370C for one hour. Infectious virus were

determined as PFU on BGM cell monolayers under a

methylcellulose overlay.


Detection of BGM Cells Infected With P1 Using an In Situ
Enzyme-Linked Assay

Laboratory virus strains. The procedure of Payment

and Trudel (17) with minor modifications was used to

detect virus infected cells. Briefly, BGM cell monolayers

grown in 24 well microtiter plates (Corning Glass works,

Corning NY) were infected with virus diluted in PBS-2%FBS.

Cell monolayers were pretreated with 20 ul of PBS-2%FBS or

5.0 x 10-3% PEI in PBS-2%FBS. Infected cell monolayers

were incubated at room temperature for thirty minutes,

overlayed with 2 ml of MEM-2%FBS, and incubated 24 or 48

hours at 370C. After these incubations, cell monolayers

were washed three times with PBS and then fixed in








mo 'th~lhol with 2 % ihydroq(ef n pcroxide at_ roIom.(P1 temp:e'ra t ure for

t rn mlinutes. Fi xot el monlloayes were trhen washed twice-

with PF ar Mon i ric2 te d at room tKnsyerature Eor 15

mino rte- withr P;2 suipp3lrlemeted with is (wv), bovine serurf,

Th Kui:,ljn and 0.51 (v/v) rleen RG (L[-PSA-K80). NKt 0.3

cj o PHlL ui EP diilcutedK! inTBS-13A-Ti1U ur addjed to ~c

:1: o' ja!ndnurated for 1 h''-r at 37"C. RI 1uts t reateK

with BOM cell 3monolai a s for ar hou o 370C to absor)rh

out ciii ti hodle5 d'irected o'ainst t he BEGfl ce1 l ine. Lotfh

P11 io PD' T w e rac x m V,!t KT C :J ':-i 0LC f 2 thi rt y

P102l (' > 11t. `cwre waniiu'U three Lor'oo with tePiSA-TBIi3O

ad ov erae w~ 0.1 m iiti of PAHR"P 3 3 9 to 0.5U un /Fl. 11-

pFr--;L7r nc ACU artciei at 3-4 C for 4, hoz. Wells W1ere

vhn trc: timo w1 PW-SA-J'-I' I M "YvAvcc with M0.



, .1 ;)t I i cc. U&B !n 06 .0 11 sna NPt V ha ardr.

a r:2( jl jo,--J t( ircunat' at room, t1)&'rat'yru fi t on

i Be ll wr w th'en iep ~ed, wCshr e once wit!,,

C i zeId Yn and t 1 Cd with .1 ml1 of 0.1% [lrypcar

blue for- f ivu rin e an 'S c d vO whe with'Ki' deiro r












arV goat anti mu PC' lnt i-I and aiCfi-gcM lCight and CU

chain s pcc tor-' tIiu i I e-'t' 1e ru ( K li U n 1 gat di lu i'rA 1








1/100 in PBS-BSA-T80 following the manufacturers

instructions was used in place of PAHRP.


Indigenous viruses from raw wastewater. The procedure

described above for laboratory virus strains was used

except that PHS diluted 1/50 in PBS-2%FBS was used for

these experiments. Indigenous viruses were concentrated

100-fold from raw wastewater from the University of

Florida and Ocala wastewater treatment plants by aluminum

flocculation. Briefly, 4 liters of raw wastewater were

made 0.003 M AlC13 and adjusted to pH 6.3 by the addition

of 1 M sodium bicarbonate. The resulting flock was allowed

to settle and the supernatant decanted and discarded. The

flock was centrifuged at 7,000 x g for 5 minutes, the

supernatant discarded, and the pellet resuspended in 3%

beef extract with 0.5% Tween 80 at pH 9 and shaken on a

rotary platform shaker at room temperature for 15 minutes.

The samples were centrifuged at 14,000 x g for 10 minutes,

the supernatant adjusted to neutrality with 2 N HC1, and

filtered through a 0.2 um pore size membrane filter.

Samples were inoculated onto BGM cell monolayers and

stained by the modified immunoperoxidase method of Payment

and Trudel (62) as previously described after five days of

incubation at 370C.










Results


In order to determine the specificity and cross

reactivity of PINutMAb, PHS, and RP1 antibody

preparations, neutralizing titers were determined for P1,

El, E5, and CB5 (Table 5-1). PiNutMAb could neutralize

only P1, whereas PHS showed significant neutralizing

titers for all viruses tested. RPI showed strong

neutralization of P1 and a very weak cross reactivity with

Fl but not with E5 and CB5. Due to the lack of cross

reactivity of RP1 and PINutMAb, immunoperoxidase staining

of BGM cell monolayers infected with P El, E5, and CB5

were determined (Table 5-2). RP1 was found to be specific

for P1 and P1NutMAb was found to be unsuitable for this

method. Therefore; PHS was used for subsequent

environmental samples due to the sensitivity and scope of

the viruses detected using this antibody preparation.

PHS was then used in conjunction with PEI to enhance

the speed and sensitivity of the immunoperoxidase staining

of cell monolayers infected with P1, El, E5, or CB5 (Table

5-3, Figure 5-1, and Figure 5-2). In general, after 48

hours of incubation the immunoperoxidase method was more

sensitive than the PFU assay for the detection of El, E5,

and CB5, but less sensitive for P1. In the presence of

PEI, all viruses were detected at less than 1 PFU after 48

hours.

The immrnunoperoxidase-PET method was then used to

quantitate viruses concentrated from raw wastewater (Table





87



5-4). The results of these studies indicate that this

method may be useful to increase the speed of the MPN

assay for indigenous enteroviruses in environmental

concentrates. The background associated with environmental

samples was much greater that that observed when

laboratory strains were used, making results obtained

using this technique difficult to interpret.














Table 5-1: Enteroviral neutralization titers of antibody
preparations used for In Situ immunoperoxidase
staining of virus infected cells. a




Neutralizing Titers for Virus


Antibody
Type P1 El E5 CB5



P1 Monoclonal 1/1 NNb NNb NNb
from hybridoma
culture fluid

Polyclonal
from Human 1/512 1/16 1/64 1/64
Serum


P1 Polyclonal
Rabbit anti- 1/1000 1/2 NNb NNb
P1



a Approximately 100 PFU of the indicated virus
diluted in PBS-2%FBS were added to 0.5ml of the indicated
antibody diluted in PBS-2%FBS, incubated at 370C for one
hour and then assayed as PFU on BGM cell monolayers under
a methylcellulose overlay. Antibody dilutions showing
greater than 80% neutralization of virus were scored as
neutralizing. Values represent the average of triplicate
determinations.

b NN = Undiluted antibody preparation is non-
neutralizing.















Table 5-2: Immunoperoxidase staining of virus infected
cells using RP1 and PINutMAb antibody
preparations. a


logl0 PFU detected


RP1


3

NDb

NDb

NDb


P1NutMAb


NDb

NDb

NDb

NDb


a Twenty-four well microtiter plates contain BGM
cell monolayers were infected with the indicated virus as
described in the materials and methods. After 24 or 48
hours, monolayers were stained by ine immunoperoxidase
method described in the materials and methods using PHS
diluted 1/50 in PBS-BSA-2%FBS. Values represent the
average and standard deviation of quadruplicate
determinations.


b ND = None detected


Virus


P1

El

E5

CB5













Table 5-3: Effects of PEI and Time of incubation for the
sensitivity of In Situ immunoperoxidase
detection of BGM monolayers infected with
enteroviruses using PHS.a


log10


Virus PEI


24 hr.


PFUdetected


48 hr.


Hours to
develop
PFU


0.75 + 0.5

1.75 + 0.5



1.3 + 1.0

1.5 + 1.3



0.75 + 1.0

1.8 + 1.0



0.75 + 0.5

1.8 + 0.5


CB5


-0.75 + 0.75

0.75 + 0.75



-0.5 + 0.5

-0.25 + 0.5



-0.25 + 0.5

-0.25 + 0.5



-1.5 + 0.6

-0.5 + 0.6


a The procedure described in footnote a of
was used for these experiments.


Table 5-2


-- ----~------I-------





















Table 5-4: Immunoperoxidase stainging of BGM cell
monolayers infected with concentrates from raw
wastewater.



MPN per liter of raw wastewater


Sample PEI CPE Immunoperoxidaseb


Belle + 920 >2,400
Glade
180 >2,400



a The procedrue described in footnote a of Table 5-2
was used for these experiments except that environmental
concentrates were used in place of laboratory virus
strains diluted in PBS-2%FBS.

b After five days of incubation at 370C, those flasks
showing no CPE were stained by the immunoperoxidase method
of Payment and Trudel (62) using pooled human serum
diluted 1/50.

























































Figure 5-1: Immunoperoxidase staining of uninfected BGM
cell monolayers.


























































Figure 5-2: Immunoperoxidase staining of BGM cell
monolayers infected with P1 for 24 hours.




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