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INACTIVATION 0F INFECTIOUS BRONCHITIS VIRUS

INFECTIVITY BY PARA-CHLOROMERCURIBENZGATE

by

Charles Donald Power:

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree 0!

MASTER OF SCIENCE

Department of Microbiology and Public Health

1965

ACKNOWLEDGMENTS

I wish to express my sincere gratitude to Dr. Charles
H. Cunningham, Professor of Microbiology and Public Health,
whose constant interest and support has made this investiga-
tion possible.

Warmest thanks are extended to Mrs. M. P. Spring,
Department of Microbiology and Public Health, whose techni-
cal assistance has contributed immeasurably to the comple-
tion of this work.

I also wish to acknowledge the advice and moral
support or my fellow graduate students and close friends,
Drs. M. J} Robinson and S. S. Tevethia.

This study was sponsored by the Agricultural Expertment
Station of Michigan State University.

11

ACKNOWLEDGMENTS .

LISTOFTABLES...............
LISTOFFIGURES

INTRODUCTION .

LITERATURE REVIEW .

Infectious Bronchitis Virus . . . . .

Sulfhydryl Groups in Protein Chemistry

TABLE OF CONTENTS

Sulfhydryl Groups in Virus Protein .

MATERIALS AND METHODS .

Virus . .

Cell Culture .

Buffer . . .

Reagents . .

Dialysis .

Filtration .

Storage .

O

Plaque Assay . . . . . . . . . . .

Experimental Procedures .

RESULTS . . .
DISCUSSION . .
SUMMARY . . .
BIBLIOGRAPHY .

111

RAGE

LIST OF TABLES

TABLE PAGE

1. Effect of OMB on IBV after one hour at
25°C 0 O O O O O O O O O I O O O O O O O O 19

2. Effect of different concentrations of
L-cysteine in arresting an /or reversing
inactivation by CUB (1x10' ) at 25°C . . . 21

3. Inactivation of IBV by CMB (1x10‘5M)
‘t 25°C 0 O 0 O O O O O O I O I O O O O O O 24

4. Inactivation of IBV by CHB (1x10'5m)
at 4° and 57°C . . . . . . . . . . . . . . 26

5. Inactivation of IBV by CIB (1x10'3n)
at 4° and 37°C 0 O O O O O O O O C O O O O . 27

6. Inactivatigfi of embryo-adapted IBV by
OMB (1110' ) at 25°C . . . . . . . . . . . 30

7. Effect of exposing IBV to various
concentrations of L-cysteine prior
to CMB (lxlO'5M) treatment at 25°C . . . . 32

8. Effect of exposing IBV to various

concentrations of L-cysteine prior
to CMB (1x10-3M) treatment at 2500 . . . . 33

iv

LIST OF FIGURES

FIGURE PAGE

1. Effect of OMB on IBV after one hour at
25°C 0 e e e e e e e e e e e e e e 0 e O O 20

2. Effect of different concentrations of
L-cysteine in arresting an /or reversing
inactivation by CHE (lxlO‘ ) at 25°C . . . 22

s. Inactivation of IBv by ems (1x10-3u)
at 25°C 0 O O O O C O O 0 O O O C O - C O O O 25

4. Inactivation of IBV by OMB (1x10'5u)
at 4°, 25°, and 37°C . . . . . . . . . . . 23

5. Inactivation by CMB (1x10‘5M) at 25°C
of IBV from two different sources
(CEKC and AF) 0 e e e a e e e e e e e e e e 31

6. Effect of exposing IBV to various
concentrations of L-cysteine prior to
OMB (lxlO'5M) treatment at 25°C . . . . . . 34

INTRODUCTION

Proteins possess an abundance of functional groups.
One of these is the sulfhydryl (SH) group to which much of
the biological activity of enzymes has been attributed.
When treated with SH reagents this group can be altered with
the effect that the catalytic properties of the enzyme are
vastly changed.

The same principles of protein chemistry apply to
viruses due to their high protein content. By eXposing
certain viruses to SH reagents, it has been possible to
alter the protein structure of the viruses and change their
biological activity. The viral response is indicative of
the presence of SH groups in the viral protein.

The main objective of this study was to determine the
effect of the SH reagent, para-chloromercuribenzoate (OMB),
upon infectious bronchitis virus (IBV) as an indication of
the possible presence of SH groups in the viral protein

coat.

LITERATURE REVIEW

Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is the causative
agent of an acute, contagious, respiratory disease of
chickens. According to electron microscopy, it is approxi-
mately spherical with a diameter of 60 to 155 mu, and bears
bulbous projections or "spikes" on its surface (Berry.g£.gl.,
1964; Nazerian, 1960; Reagan 33 51., 1950; Reagan and
Brueckner, 1952).

The virus can be readily propagated in embryonating
chicken eggs. The Beaudette chicken embryo-adapted culture
of IBV can also be cultivated in several types of cell
culture. Some of those in which the virus will multiply
without evidence of cytopathic effects (CPR) are chicken
embryo liver and heart (Fahey and Crawley, 1956), whole
embryo (Mahonty and Chang, 1963), isolated chorioallantoic
membrane (Ferguson, 1958; Ozawa, 1959), and de-embryonated
chicken eggs (Buthala, 1956). Others in which the virus
will multiply with the production of OPE are whole chicken
embryo (Mallmann and Cunningham, 1965), adult chicken kidney
(Kawamura 23.21,, 1961), and chicken embryo kidney (Chomiak
33.51., 1958; Mahonty and Chang, 1965; Spring, 1960; Wright
and Sagik, 1958).

The virus forms plaques in monolayers of chicken embryo
kidney cells overlaid with agar. These plaques may be used
for assay of viral infectivity (wright and Sagik, 1958;
Cunningham and Spring, unpublished data).

Sulfhydryl Groups in Protein Chemistry

The sulfhydryl (SH) or mercapto group has perhaps
received more attention than any other reactive group of the
enzyme protein molecule due to its apparent importance in
protein structure and activity (Boyer, 1959). Another term
frequently encountered is "thio", a prefix denoting compounds
in which sulfur replaces oxygen. This prefix is often
expanded to "thiol," and usually refers to a complete SH-
containing compound rather than to the SH group itself.
Thiols, particularly aliphatic derivatives, are also known
as mercaptans.

Chinard and Hellerman (1954) have emphasized that many
enzymes must retain at least a fraction of their substituent
SH groups in the reduced form for normal catalytic activity.
Examples of this are urease and triosephosphate, succinic,
and alcohol dehydrogenases.

Two other enzymes require at least one intact SH group
each to be active (Cecil.and McPhee, 1959). Papain contains
one reactive SH group which is essential for activity but
the exact mechanism.of its activity is unknown. Glyceralde-
hyde-S-phosphate dehydrogenasc contains two to three reactive
SH groups which, through an S-acyl mechanism, are involved

in enzyme function.‘ In the case of both enzymes, the

remaining SH groups were somewhat less reactive with para-
chloromercuribenzoate (OMB), the reagent used. The precise
function of these SH groups is unknown.

Oxidation of SH groups results in the formation of
disulfide groups (88), a reaction which has a marked effect
on enzyme activity. Such interconversions of enzyme groups
in tissues may'be an important biological control mechanism
(Boyer, 1959). Rather than playing a direct role in the
biological action of the protein, the majority of native SS
groups seem merely to form part of the covalent structure
of the molecule. Consequently, their primary function seems
to be to aid in the maintenance of molecular structure
(Cecil and MoPhee, 1959).

A The willingness of SH groups to react with other
functional groups makes them more significant from.the stand-
point of biological activity than the 38 groups. ‘Iith the
exception of hydrogen ion associations, the SH group has
greater reactivity toward covalent bond formation than any
other group (Boyer, 1959).

Proteins have been experimentally exposed to three
types of SR reagent: (1) oxidizing, such as iodine and
ferricyanide; (2) alkylating, such as iodoacetate and
iodoacetamide; and (5) mercaptide-forming, such as OMB.
Both oxidizing and-alkylating reagents react as rapidly,
but not as specifically, with SH groups as do the mercap-
tide-forming compounds (Chinard and Hellerman, 1954).

Organic and inorganic mercurials have a high affinity
for anions or groups, specifically SH groups, with an avail-
able pair of electrons (Boyer, 19592. For biological
studies, aromatic derivatives are generally preferred over
the aliphatics because they are more soluble and less cyto-
toxic (Cecil, 1965). The meroaptide-forming mercurials
fulfill most of the requirements of an ideal protein
reagent in that they (1) have high specificity, (2) react
stoichiometrically under gentle conditions, and (5) can
readily be removed and the original SH group replaced
(Fraenkel-Conrat, 1957). Another advantage is that mercury
reacts essentially with one SH group per molecule when
linked through one covalent bond to an organic moiety
(Boyer, 1959; Chinard and Hellerman, 1954).

Hellerman at 21 (1945) introduced OMB as a specific
reagent for thiol enzymes. Boyer (1959) stated that of 154
reports on enzyme inhibition by SH group reagents, 101
involved the use of CHE. The following groups: imidazole,
indole, guanidyl, amide, amine, phenol, carboxyl, disulfide
and alcohol were negative to the action of OMB (Fraenkel-
Conrat, 1947). The reagent is not difficult to use since
many reactions can be performed near neutrality and at room
temperature. Such conditions are considered to be non-
denaturing for most enzymes (Fraenkel-Ccnrat, 1957).

Sulfhydryl groups in proteins are classified as

reactive or unreactive. The reactive groups respond to

SH reagents as do simple thiols. There is little or no
response by the unreactive groups in the native protein.
After denaturation, however, unreactive groups react
normally to SH reagents (Cecil, 1965). Such groups have
been classified as ”masked", "inaccessible," or "sluggish"
(Fraenkel-Conrat, 1957; Fraenkel-Conrat, 1959; Cecil and
McPhee, 1959). A

Cecil (1965) stated that it seems reasonable to assume
that these ‘masked" groups are involved in covalent bonding
of some nature. This may not be the case since the type of
covalent bond suggested would have to be one which is broken
by protein denaturation, thereby rendering the unreactive
groups fully reactive. Such treatment includes exposure to
urea, guanidine, detergents, or changes of pH, none of which
would be expected to cleave a covalent bond.

The phenomenon of increased reactivity accompanying
protein denaturation has been mentioned by Boyer (1959)
and Cecil and McPhee (1959), although it has never been
fully explained. It does, however, seem to be a reflection
of the unique structure of the native protein, and seemingly
indicates that chemical reactivity is sterically blocked by
the protein structure. Protein denaturation results in
structural changes whereupon the steric hindrance no longer
exists.

By causing structural changes in enzyme protein or by

removing an SH that plays a primary role in catalysis, a

chemical reaction involving OMB may alter enzyme function.
Boyer (1959) considers that there_are basically three types
of enzyme with respect to reaction with OMB: (1) enzymes
whose SH groups react rapidly with loss of activity; (2)
those whose SH groups react slowly with gradual loss of
activity; and (5) those with SH groups that react rapidly
without loss of activity‘but which subsequently lose
activity as more "sluggish” groups react.

It is possible, in the case of many enzymes, to
reverse the effect of OMB through the addition of a slight
excess of a thiol compound. Chinard and Hellerman (1954)
illustrated the reaction as follows:

En-S-HgR - R'SH ------- En-SH - R'S-HgR
Cysteine and reduced glutathione are the mercapto substances
of choice (Boyer, 1959; Chinard and.Hellerman, 1954;
Hellerman gtugl, 1945). This reversal of inactivation by
low molecular weight thiols may be due to: (l) structural
changes brought about by rupturing an 8-3 bond; (2) recon-
struction of an SH group possessing primary enzymic
function; or (5) removal of a heavy metal inhibitor such as
mercury (Boyer, 1959). Hellerman‘gt.gl (1945) reported
that the activity subsequent to cysteine reactivation
frequently seemed to exceed the initial activity but no
explanation was offered.

Another widely used method of demonstrating reactive
groups in proteins is the titration of SH groups with OMB
using nitrOprusside as an indicator (Boyer, 1959; Chinard

and Hellerman, 1954; Fraenkel—Oonrat, 1957; Hellerman gt 21.,
1945). This procedure has been modified.with good results
by first adding an excess of standard CHB, then back-titra-
ting with cysteine to produce a positive nitroprusside test
(Chinard and Hellerman, 1954). Boyer (1954) has employed
spectrophotometric techniques in SH determination. The
interaction of OMB with SH groups leads to higher readings
at 255 mu (Boyer and Segal, 1955).

Bennett (1948, 1951) reported that mercury orange,
known commonly as RSR or red sulfhydryl reagent, is a
specific stain for thiol in tissues, and may be used to
locate SH groups in regions of tissue previously not known
to contain them as retinal rods and nerve cell bodies.

Dithizone reacts with OMB, and may be used in the
colorimetric determination of the reagent. Fridovich and
Handler (1957) noted a linear decrease in the absorption of
dithizone at 6250 A as the amount of mercurial in the system
increased.
Sulfhydryl (SH) Groups in Virus Protein

The SH group plays a major role in viral stability and
infectivity. One of the most consistent demonstrations of
the essential nature of the virus SH group is inactivation
of infectivity using appropriate cell cultures as the
indicator system. Hany sulfhydryl reagents have been used
but OMB is most frequently chosen.

Ohoppin and Philipson (1961) studied the inactivation
of enterovirus infectivity by OMB. The rate of inactivation

depended upon the ionic environment. Inactivation was rapid
im.most cases. With ECHO 7 and 9 and Ooxsackie B5 viruses,
there was a 50% inactivation within one to two minutes.

Allison gtflgl_(1962) concluded that most viruses, as
well as the pleuropneumonia and psittacosis-lymphogranulona
groups of organisms, are adversely affected when exposed to
OMB and iodoacetamide. Some arboviruses and enteroviruses
are resistant.

According to Allison and Valentine (1960b), there was
no detectable change in the rate of attachment of OHS-
treated fowl plague virus to host cells but viral infec-
tivity was reduced. The same pertains to other myxoviruses
(Knight and Stanley, 1944; Philipson and Choppin, 1960;
Philipson and Choppin, 1961).

Allison (1962) investigated the effects of 613, iodo-
acetamdde and HgOlg on partially purified vaccinia and fowl
plague viruses. Infectivity was considerably reduced. The
treated virus not only attached to the host cells but may
have penetrated th'flm Intracellular»multiplication of the
virus apparently did not occur.

Oxidized 2,5-dimercaptopropanol (BAL) inactivated
poliovirus types 1, 2 and 5, as well as ECHO 7 (Philipson
and Choppin, 1962). A mixed disulfide may have been formed
between the virus SH groups and BAL. I

Philipson and Choppin (1962‘) noted that the hemagglu-
tinating activity of ECHO 7 was inactivated by BAL. This

10

supported two earlier reports (Buckland, 1960; Philipson and
Choppin, 1960) that enterovirus hemagglutination (HA) could
be prevented by OMB and other similar reagents. Inactiva-
tion of viral HA was due to the inability of CID-treated
virus to adsorb to erythrocytes (Philipson and Choppin, 1960).
These results indicate a reaction between the reagent and SH
groups on the virus particle, and further suggest that viral
SH groups are involved in the attachment of enteroviruses to
erythrocytes. Buckland (1960) observed that the HA activity
of myxoviruses and arboviruses remained unaffected by OMB,
and Philipson and Ohoppin (1960) confirmed this with the
myxoviruses. All agreed that the SH reagents had no effect
on erythrocyte receptors.

Incubation of poliovirus type 1 with L-cystine enhanced
thermostability of the virus (Pohjanpelto, 1958). This
phenomenon was attributed to an exchange between cystine and
protein SH groups resulting in 8-8 bonds. If the virus were
exposed to OMB prior to chystine, there was no enhancement
of thermostability. The thiol reagent combined with the SH
groups of virus protein, thereby making them inaccessible to
cystine. Thermostability of the virus was not increased by
OMB treatment alone.

In 1961, Hirst reported that in addition to cystine,
several polysulfides such as NagSz and Na284 stabilized
poliovirus type 1 to high temperatures.

Inactivation of influenza virus by various mercurials

has been studied using mice and embryonating chicken eggs as

11

assay systems (Klein'gt.gl., 1948; Klein and Perez, 1948;
Knight and Stanley, 1944; and Perez 23.31., 1949).

Reversible inactivation of Rous sarcoma virus in vivo
by OMB has been reported by Schmidt (1957).

Reichmann and Hatt (1961) suggested that the action of
OMB on the viral particle results in dissociation of virus
into subunits. They postulated that substitution of SH
groups with a bulky radical, such as mercuribenzoate, might
interfere with subunit linkage sites, thereby resulting in
particle degradation.

Kaper and Houwing (1962), using spectrophotometry
(Boyer, 1954), determined that turnip yellow mosaic virus
(MW 5.5 x 106) possesses 650 SH groups per molecule.
Fridovich and Handler (1957) reported 5400 SH groups per
molecule of potato virus X.(MW 55 x 106), as measured by
the dithizone method.

Reactivation of virus inactivated by SH reagents has
occurred consistently subsequent to the addition of thiol
compounds (Allison, 1962; ChOppin and Philipson, 1961;
Hirst, 1961; Klein 23 31., Klein and Perez, 1948; Perez
23 51,, 1949; Philipson and Choppin, 1960; Philipson and
Choppin, 1962). Such reversal of inactivation by added
thiols is indicative of a previous reaction between SH
groups and reagents whether in viral protein or in an

isolated enzyme system.

MATERIAIS AND METHODS

Virus

The Beaudette chicken embryo-adapted culture of IBV,
designated as No. 42 in the Michigan State University
repository, was used. The virus had previously been
passed 111 times in chicken embryo kidney cells (CEKC) in
a cultural medium consisting of Hank's balanced salt solution
(B83), lactalbumin hydrolysate (LAB), and bovine serunm For
the experiments in the present study, medium 199 (General
Biochemicals), supplemented as described below, was used.

I Allantoic fluid from infected embryonating chicken eggs
was used in one experiment for comparison with cell-cultured
virus.

Virus was harvested 48 hours after inoculation of the
cultural system, dispensed into vials, and stored at -62°C
until used.

Cell Culture

Primary cell cultures were prepared true the kidneys
of 18- to 19-day—old chicken embryos. The kidneys were
removed from the embryos, minced, and washed in BSS
(Cunningham, 1963). The tissues were then treated with
0.25% trypsin (1:250, Nutritional Biochemical Corporation)
in BSS, pH 8.0 to 8.4, allowing approximately 10 ml of

12

13

enzyme solution per pair of kidneys. Trypsinization was
carried out at 37°C for 40 minutes. To facilitate digestion,
the flask was clamped in place above a magnetic stirrer, and
a sterile Teflon bar was placed in the tissue-trypsin mix-
ture.

Cells were washed free of trypsin and debris by centri-
fuging at 4373 at 4°C for five minutes, decanting the super-
natant, and resuspending the cells in fresh BSS. After the
third cycle of centrifugation, the supernatant fluid was
decanted, and the packed cells were resuspended in cultural
medium to a concentration of l to 1.5 x 107 cells per
milliliter.

The cultural medium.consisted of medium 199, and 1%
solutions each of vitamins, L-glutamine, and amino acids
(General Biochemicals); 5% newborn calf serum.(Crand Island
Biological Company); 100 units penicillin G potassiwm,

0.1 mg dihydrostreptomycin sulfate, and 100 units Mycostatin,
respectively, per ml (E. H. Squibb and Sons). The medium
was adjusted to pH 7.0 with 10% saturated NaHCOs (Cunningham,
1963). Plastic petri dishes (Falcon Plastics), 60 x 15 mm,
were seeded with 4 ml per dish of the cell-medium.l99
suspension.

All cultures were incubated at 57°C in an atmosphere
of 8% 002, two atmospheric changes per hour, and 85%

relative humidity.

14

Buffer

The buffer system employed was "Sigma 121" tris
(hydroxymethyl) aminomethane (Sigma Chemical Company),
0.01M, with no additional salts added. The pH was adjusted
from 9.7 (initial) to 7.0 since the reagent used (CMB) gives
optimum results at neutrality (Chinard and Hellerman, 1954).
For adjusting the pH, 5N HCl was used so as not to exten-
sively alter buffer molarity.
Reagents

p-Chloromercuribenzoate (OMB), Na salt and L-cysteine
HCl monohydrate, A grade (Calbiochem respectively) were
dissolved in 0.01M tris buffer, pH 7.0. The solutions were
freshly prepared prior to each experiment.
Dialysis

Dialysis tubing (Visking Company), average diameter of
4.8 mu, was used for dialyzing the virus samples against
0.01M tris buffer, pH 7.0. Individual tubes were prepared
in advance and soaked overnight in buffer. After virus
samples had been poured into their respective dialysis bags,
which were tied at one end, the Open end of each bag was
tied securely and a tag attached. The bags were then sus-
pended in a large beaker containing buffer. To increase
agitation, a Teflon bar was put in the buffer and the
beaker was placed on a magnetic stirrer. The sample was
dialyzed for 24 hours at 4°C,during which time the buffer

was changed three or four times.

15

Filtration

All virus samples were filtered subsequent to dialysis
using Type HA, 0.45 u pore size, 47 mm disc diameter Milli-
pore filters.
Storage

Samples which were not immediately used following
dialysis and filtration were dispensed in vials and stored
at -62°C.
Plaque Assay

The cultural medium was decanted from.72-hour-old cell
cultures. The monolayers were washed with 2 ml of BSS and
inoculated with 0.5 m1 of serial tenfold dilutions of virus
with BSS as the diluent. Two to four cultures were used
per dilution of virus. After incubation for 120 minutes,
the inoculum was poured off but the cells were not washed.
Agar medium overlay consisting of equal volumes of 1.8%
Special agar-Noble (Difco Laboratories) and two times con-
centrated medium 199 was added, 4 ml to each.monolayer.
After the agar had hardened, the cultures were returned to
the incubator. After three days, 0.5 m1 of a 0.1% neutral
red dye in phosphate-buffered saline solution was added to
each culture. The cultures were incubated at 57°C without
C02 for 15 to 50 minutes, and then for an hour at 4°C before
the plaques were counted.

The amount of infective virus was expressed in plaque-

forming units (PFU).

16

Experimental Procedures

The 0MB solutions were equilibrated at the reaction
temperature immediately prior to use. Virus was thawed and
mixed with an equal volume of twice concentrated CHE solu-
tion in a flask. A control consisted of equal volumes of
virus and tris buffer. In the single instance in which
virus in allantoic fluid was used, the sample was thawed,
centrifuged at 457g for 15 minutes at 4°C to remove cellular
debris, and the supernatant fluid was used.

The flask was frequently shaken and, at appropriate
intervals, samples of the mixture were removed. Each
sample, including the control, was dialyzed, filtered, and
assayed for infective virus.

To arrest OMB-inactivation at specific times, equal
volumes of CMB—treated virus and L-cysteine were mixed.

The final proportions of virus:CMB:L-cysteine were 1:122.
These mixtures were held at room.temperature (25°C) for
varying periods of time, depending upon the experiment.
Controls consisted of OMB-treated virus in tris buffer,
untreated virus in tris, and untreated virus in Lecysteine.

The foregoing procedure was altered to establish the
effect of L-cysteine upon virus previously untreated and
then exposed to ONE. One part of untreated virus was mixed
with two parts of anysteine to match the ratio of respec-
tive components in other eXperhments. The mixture was

agitated at 25°C. Samples were removed at 15 and 50

17

minutes, pipetted into flasks containing a volume of OMB
'equal to that of the virus, then incubated at 25°C for 15
minutes. Controls consisted of untreated virus plus tris,
and untreated virus plus L-cysteine.

For reactivation of OMB-treated virus after 15 minutes
at 25°C, samples were added to flasks containing varying
concentrations of chysteine. The proportions of virus:CMB:
Lwcysteine were the same as in previous experiments.
Controls consisted of untreated virus plus tris, and virus
plus OMB. After 15 minutes at 25°C, all samples were

assayed for infective virus.

RESULTS

The concentrations of OMB and L-cysteine referred to
are the final concentrations in the mixture.

Determination of_0ptimum Concentration of CUB for
Inactivation of Infectivity

Inactivation of virus increased with the concentration
of OMB during the one hour reaction time (Table 1, Figure 1).
There was at least an 85% reduction of infectivity. Both
the 10-5 and 10'2M concentrations were adequate but, since
an excess of reagent in the system.would be of no advantage,
the former concentration was selected. All subsequent
experiments utilized 1x10'5m CHB.

Determination of Optimum Concentration of chysteine
for ArreSting OMB-inactivation

L-cysteine, 1x10‘3m, did not arrest inactivation within
15 minutes at 25°C (Table 2, Figure 2). At 2x10'5m, inacti-
vation was reversed. At 1.5x10‘5M, L-cysteine halted
inactivation but did not reverse it. During the subsequent
15 minutes to the termination of the experiment, there was
only an insignificant decrease in viral infectivity. For
subsequent studies, 1.5x10‘5M L-cysteine was employed.

There was no noticeable decrease of infectivity of the
untreated virus control.

The results of this experiment indicate that most of

the OMB-inactivation process occurs within 15 minutes.

18

19

 

 

“.mm Nomw.o- men a ~.H AzeIOHxHV mzu +.msna>
m.mm Nonm.H- can x N.H Azm-onHV mzo +.npna>
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20

Figure 1. Effect of OMB on IBV after One Hour at 25°C

 

\\\ v0 = 8.5 x 105

10‘

Log V/Vo \

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10

 

 

 

10'4 10'3 10'2
Molar Concentration of CMB

21

 

 

 

 

 

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HOHUGOO m5

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Houuaoo mnuw>

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GOfiumK/HuuwcH wafimugrmm Hobucm wswummufiw cw mcflmumkouq mo mcowumuuamoaoo ucmummmwa mo uumwmm .N “:an

22

Figure 2. Effect of Different Concentrations of L-Cysteine in
Arresting and/or Reversing Inactivation by CMB (1 x 10'3M)
at 25°C

 

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%

CONTROL
(Vines + TKO‘) /

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10'1 \
\\ /
Log V/Vo

     

 

 

 

 

 

10‘2
\
4' \
/' \
Vuaus + QMB \
\
\
\
\
—" — Z.OIOO.3M L‘CYSTEINE \ \
'.s‘ g. I.
._ __ ._ _ __ _. '00 u u N
0 15 30

Time (minutes)

25

Kinetics of Inactivation of IBV by CMB at 25°C

Inactivation by OMB was halted at desired time intervals
by the addition of L—cysteine. There was a 98% reduction in
viral infectivity within ten minutes after treatment with
the reagent (Table 5, Figure 5). A decrease in the rate of
effect was noticeable at about 15 minutes and extended to
the termination of the experiment. The virus was never
completely inactivated.

The titer of the controls remained fairly constant
throughout the experiment. At 60 minutes, there was a 78%
greater reduction in infectivity in the treated sample
than in the untreated sample. After 180 minutes, the un-
treated virus had lost only 40% of its infectivity.

The L-cysteine control indicated that the thiol com-
pound had no deleterious effect on the untreated virus.
Kinetics of Inactivation of IBV by OMB at 4° and 57°C

The inactivation rate of the virus was directly
related to temperature (Tables 4 and 5, Figure 4). The
curves from the data at 4° and 57°C have been superimposed
on those at 25°C.

The infectivity titer of the treated, 180 minute
sample at 4°C was no lower than that of the 60 minute
sample, offering another example of "tailing-off" after the
initial reaction. Total inactivation during the three hour
period was only a small fraction of that observed at the
other two reaction temperatures. The untreated control (4°C)

retained 74% infectivity after the three hour period.

24

Table 3. inactivation of raw by can (1x10‘3M) at 25°C

 

Sample Infectivity Titer Log) V/Vo ' 1 Decrease
(Pfu/ml) in Infectivity

Untreated Controls:
(flrus + Tris)

0 minutes. 2.5 x 105 o 0
6o " 2.0 x 105 . -o.0969 21
180 " 1.5 x 105 -o.2218 40

(Eirus + L-cysteine)
60 minutes 2.1 x 10 -0.0757 16

Treated Virus:
(Virus + 6MB)

0 minutes 2.5 x 105 0 0

5 " 4.5 x 104 -o.7447 82

10 " 5.1 x 103 -1.6903 98

15 " 8.5 x 103 -l.4685 96.6
20 " 3.2 x 103 -1.8928 98.7
25 " 1.8 x 103 -2.1426 99.3
30 " 1.7 x 103 -2.1675 99.32
45 " 1.2 x 103 -2.3187 99.44
60 " 1.1 x 103 -2.3565 99.56
90 " - 3.8 x 102 -2.8181 99.85
120 " 2.3 x 102 -3.0362 99.91

180 " 3.0 x 101 53.9208 99.99

 

255

Figure 3. Inactivation of IBV by CMB (1 x 10'3!) at 25°C

 

 

 

   
 
 

 

 

 

0 \ ._______ If" CONTROL.
\ N \ (VIKUI + L- CYCTIHNL)
\; ~ ~ ‘ fl
Corn-act.
Lviaos + Tans)
Vo = 2.5 x 105
e
10'1
Log V/Vo .
10-2 ‘I‘\
Vines + M3
+ L- cars-rams.
10'3
- 1
1o 4
O 60 120 180

Time (minutes)

 

265

 

 

oooo sea a e.~ sea a m.m nos 4 o.H : owa
sea a 5.6 sea a o.~ sea a m.a --- = oo
--- sea a N.~ --- --- = we
NoH a o.s cos x 6.5 see a o.m --- e on
moa a ~.H sea a m.e sea a o.~ --- = ma
mes x s.m --- --- --- = oH
moa a o.o --- -u- --- = m
mes x m.~ mod n m.~ moH a m.~ moH x m.~ meadows o
ooam ooe comm . cos. ndmmmmwnmmmw_
. Ammo +.nonasu monam.+.nsna>v

pmummua roundups:

 

Aaaxsmmv noose sua>nooomoH

 

 

oonm one oe no AzmuonHv mzu so smH mo ooaoooaoooeH .e manna

27’

 

 

oooo aaHo.H- wmmn.o- HoHH.o- = one
Homm.m- owno.o- oemm.o- --- = oo
--- ammo.n- --- --- = we
“no“.m- Haan.o- moNd.o- --- = on
mem.~- aoaa.o- ommm.o- --- = we
mmoo.a- -1- --- --- = oH
omae.a- --- --- --- = m
o o o o mouaafia o
uoR cos 8on cos 3368 mean.

 

Ammo +.npna>v

raunchy

nanny +.oona>v

woucouusp

 

o>\> mos

 

 

 

comm one oe no Azw-oHaHv n28 an smH no oonuoonooooH .m manna

 

28

rigors 4. Inactivation of 13' by can (1x10'3na at 4°, 25°, and 37°C

 

 

 

 

 

 

104-

 

Los v/vo

_ r” — -— —- CONTROL
(Vines +T'ms)

_________.__._____———
VH‘lU‘l +- CM!

.. _._—__‘_..’___—.

_'-_——_.—-— “v —

10'3

2.5%..

 

 

 

 

 

o 120 180
Time (minutes)

29

At 57°C, the reaction reached completion sometime prior
to the final sampling. At 15 minutes, 0.5% infectivity re-
mained and, after one hour, 0.05% was detectable. Due to
the elevated temperature, the ”tailing-off" process also
was accelerated. .

The titer of the untreated control after three hours
at 57°C represented 14% of the original infectivity.

Kinetics of Inactivation of Embryo-adapted IBV by
DEB at’25°C

 

The CEKC-propagated IBV was used for all studies up to
this point.. The chicken embryo-cultured virus, later
adapted to CEKC, was tested.with OMB for comparison of
susceptibility. Although the rates of inactivation were
approximately the same for both strains, the embryo-cultured
virus was not as susceptible to CMB as was the tissue-
cultured virus (Table 6, Figure 5).

Effect of ExposingleV to.L-cysteine Prior to OMB
Treatment at 25°C

To further determine the effect of Lwcysteine, three
different molar solutions of the thiol compound were mixed
with untreated virus. After 15 and 50 minutes at 25°C,
OMB was added.

None of the concentrations fully protected the virus
from the effects of OMB (Tables 7 and 8, Figure 6).
Inactivation, however, was considerably less than that
observed with OMB alone. In contrast to previous results,

there was a marked decrease of infectivity of the controls

.‘50

Table 6. Inactivation of Embryo-adapted IBV by CMB (lxlO‘3M) at 25°C

 

Sample Infectivity Titer Log V/Vo 1 Decrease in
(Pfu/ml) Infectivity

Untreated Controls:
(Virus + Tris)

 

0 minutes 2.5 x 105 0 0
60 " 2.2 x 105 -0.0555 12
180 " 1.6 x 105 -0.1938 36
Treated Virus:
(girus + CMB)
0 minutes 2.5 x 105 O O
15 " 2.0 x 104 -1.0969 92
30 " 8.1 x 103 -1.4894 96.8
60 " 3.6 x 103 -1.8416 98.6

180 " 6.0 x 101 -3.6197 99.98

 

51

Figure 5. Inactivation by can (1x10‘3M) at 25°C of IBV from Two
Different Sources (CEIC and AF)

 

    

 

 

 

 

 

 

 

 

0
CONTROL:
\ LVmu: +Tan)
1
1
- vo - 2.5 x 105
\
\
10'1 ‘
——- — — — IIV41-AF
\
\ nv az-csxc
Log v/vo
10'2
10'3
10'“ 1
0 60 1 120 180 .

Time (minutes)

 

o S x mic. sen e2 4 an eon .82 x we nuns. + sons 08
m camoumhong +.m:a«>

 

 

e18 e2 4 ea oxen e3 4 we one e2 x 9m 88 + peas 83
mdwoum%019 +.mDHH>
see 2 x m4 «.8 2 x ~.N 2mm eS a 64 use + Acne m3
m d maweumzolfl +.m:ufi>
a. we}: .e as}: a. as}:
cam-Sam cam-Sam. H zm-ou: nouosem

 

32

oawcum%01q mo nowumuucmoaoo

 

 

on e2 x 0.6 .. me
o moH x m.N - mouaafia o
bazoooufi 9335 Ania + eongfi
cw museuoma N uwuHH kuw>wuoomaH mHouunou veuwcuudb

 

comm um uncaumoua
mamuoaxav mzu ou uoaum ccwcum%014 mo mcowumuuacocoo msowum> ou >mH wswmomxm mo uocmmm .n caan

35

 

 

 

 

 

 

0 sea 28.? {on Echo- on; + cons 83
cfiw0umh01fl +.m:ufi>
.26 see 886. one wowed- 88 + cons 83
mafioummouq +.msuw>
wee «.3 33.7 6.2 82.7 88 + eons m3
ocamummonq +.m:u«>
a. o>\> won a. o>\> mos .N. oi» m3
. x moamamm
SnuonN Smuoaxm H EMIOH H
odwouw%01a mo dowumuucmoaoo
#, a w w w w w w w
on mmao.on = me
o o wouszE_o
hufi>wuoomaH
uH mmmouoon.N H>\> moA AmHHH +.mDHH>v

mHouusoo pmumcuucn

 

comm no essence:
AZMIOHxHV mzc ou uoHHm camoumhong mo msowumuunoocoo mDOHum> ou >mH wwwmomxm mo uoommm .w manna

:54

Figure 6. Effect of Exposing IBV to Various Concentrations of
L-Cysteine Prior to CMB (1x10' Treatment at 25°C

0— — —-o thus CONTROL
o———————o Loago'3M L‘CYSTEINL
0 as 1.: .-

 

°__———'—OZ.OI n u

 

% CMB Annan

 

 

 

 

 

 

01\
Log V/Vo
10'1
10"2 g - ¢ . ; - A -
0 15 30 4'5 0 15 30 45 0 15 50 4‘5

 

 

Time (minutes)

55

with one exception. When compared with these controls, it
is seen that inactivation due to OMB was not extensive.
The exceptional control consisted of virus and 2x10'5M
L-cysteine. This sample was stable to whatever environ-
mental factors were responsible for decreasing the titers
of the other controls. It also confirmed that L-cysteine,
under these circumstances, did not inactivate the virus.
There was no consistent difference between a 15 and 50
minute eXposure of virus to L-cysteine.
Reactivation of OMB-treated Virus
By adding 2x10'3M L-cysteine to OMB-inactivated virus,
it was possible to reactivate viral infectivity (Table 2,
Figure 2). This indicated that previous inactivation was

due to a reaction between OMB and viral protein SH groups.

DISCUSSION

The susceptibility of the OEKC- and embryo-adapted IBV
42 to OMB indicates that the reagent reacts with SH groups
in the viral protein. The inactivation reaction with OMB
appears to be first-order for the first ten minutes during
which there was a 98% decrease of infectivity. At this
point, there was a break in the curve and a "tailing effect"
which extended to the termination of the experiment. A
decrease such as this is quite common in virus survival
curves (Hiatt, 1964). The initial slope may be abrupt but
more often is gradual, as was the case here.

The problem.arose as to whether the reaction was first-
order followed by a "tailing effect“, or second-order
involving two p0pulations of the virus. The former seemed
to apply here because the curve was linear until 98% of the
virus had been inactivated. Hiatt (1964) states that
"thermal inactivation of viruses has often been observed to
proceed as a first-order reaction until about’90% or more
of the infective particles have succumbed.” Had a hetero-
geneous population been involved, an earlier break in the
curve would have been expected.

Since only three points.were available for the first
ten minutes, wherein the greatest inactivation occurred,
no attempt was made to determine the velocity constant (k)

or energy of activation (E).

56

37

One experiment (Figure 5) compared the inactivation
rates of viruses from two different sources, CEKC and
infected AF. The regression curves for inactivation of
OEKO-cultured virus and viral-infected AF were relatively
parallel, although the AF virus was somewhat less suscep-
tible to the reagent. There are two possible explanations
for this differential response: (1) The tissue culture (TC)
virus, originally derived from.AF virus, may have undergone
a modification of its protein coat as a result of propaga-
tion in kidney cells. This implies the possible acquisition
of more SH groups essential to infection than the ”parent"
strain possesses; (2) The AF virus, in the highly protein?
aceous embryonic fluid, may not have been in as intimate
contact with as many OMB molecules as was the TC virus. It
is possible that much of the reagent united with SH groups
in the AF proteins. The,TC medium contains proteins, also,
but perhaps not as many with SH groups as may be found in
AF. It is possible that both alternatives are involved.

The regression curves for inactivation at 4°, 25° and
57°C were linear for initial inactivation of virus with two
exceptions which occurred at 15 minutes at 25°C, and 50
minutes at 4°C. No explanation can be offered for this
other than a possible disruption of viral clusters due to
temperature effects and agitation of the reaction flasks.
At 57°C, such a disaggregation may have occurred before the

first sampling so was not reflected in the curve. The time

58

required for this phenomenon to occur was directly related
to temperature in all cases.

The presence of OMB in the sample appeared to accele-
rate dispersal of viral clusters, as indicated by the
delayed response of the control at 57°C. Reichmann and Hatt
(1961) reported that OMB caused the virus particle to disso-
ciate into subunits. Control samples at 4° and 25°C were
removed late in the experiment and disruption, if it
occurred, was not detected.

L-cysteine added to the reaction mixture arrested
inactivation of the virus by OMB. However, none of the
concentrations of L-cysteine employed completely protected
the virus from inactivation by subsequent treatment with
OMB. This indicates that even though excessive thiol com-
pound was present, at least a few OMB molecules altered
some virus particles. This seems logical, although Allison
(1962) stated that SH reagents react with cysteine much more
rapidly than with virus. On this basis, one might expect
nearly 100% protection by the excess cysteine. The present
results indicate that OMB is not necessarily so very
selective in its.choice of SB groups and will react almost
as readily with SH groups of viral origin as with those in
cysteine.

The question arose as to why the 2x10'5M L-cysteine
permitted some inactivation by OMB with no evidence of

reactivation, when the same concentration had reversed

59

inactivation in an earlier experiment. It might be assumed
that a certain amount of excess cysteine remained free in
the reaction mixture following the interaction of OMB with
virus and cysteine molecules. Such free cysteine might be
capable of reversing whatever viral inactivation had
occurred if given sufficient time. The samples for assay
may have been removed too soon for reactivation to occur.

An attempt at reactivation using a slightly modified
technique proved unsuccessful. Each sample was dialyzed
immediately after OMB treatment, and again after exposure
to L-cysteine but there was no significant reversal of
inactivation. Perhaps some protective substance was lost
during initial dialysis, thereby rendering the virus sus-
ceptible to the low pH of the cysteine solutions. Adjusting
the cysteine solutions to pH 7.0 resulted in a limited
renewal of infectivity but this was not interpreted as
positive reactivation.

During the initial dialysis, more than the removal of
excess OMB may have taken place. Certain ions or substances
essential for reversal by L-cysteine might also have been
removed; or possibly some factor was involved which made
the virus-OMB union more stable.

In a previous experiment, reactivation occurred after
only single dialysis, that following the addition of
L-cysteine to the virus-OMB mixture. Essential ions or

substances, if they exist, must have been present in that

4O

mixture as well as in the mixture that was dialyzed twice.
It could be assumed that such substances would have demon-
strated the same tendency to dialyze out, and yet reactiva-
tion occurred. Perhaps their egression was sufficiently
gradual to enable them to assist cysteine in reactivation
prior to being dialyzed out with excess OMB and thiol
compound.

Second dialysis had no deleterious effect on the virus,
as both controls and inactivated samples produced anticipa-
ted titers.

The technique of ridding the sample of excess OMB prior
to thiol exposure has been employed with success by others
(Allison, 1962; Oh0ppin and Philipson, 1961). A possible
improvement was suggested by Fraenkel—Oonrat (1957) invol-
ving single dialysis of OMB-inactivated virus against a
cysteine solution. The author claimed that this reversed
the reaction and simultaneously removed the SH reagent.

The virus was more stable in tris buffer than in
cultural medium, and the incorporation of L—cysteine into
the buffer further enhanced viral stability. Hirst (1961)
and Pohjanpelto (1958) stabilized certain enteroviruses to
heat by exposure to cystine. It is possible that a similar
phenomenon occurs with IBV but this aspect of SH involve-

ment was not explored in the present study.

l.

2.

5.

4.

SUMMARY

The Beaudette culture of infectious bronchitis virus
(IBV 42) is susceptible to inactivation of infectivity
by the sulfhydryl (SH) reagent, p-chlcromercuribensoate
(OMB). This reaction indicates the presence of 3H-
ccntaining components in the viral protein.

Both the embryo-adapted and chicken embryo kidney cell
cultured IBV 42 responded to OMB treatment. Differ-
ences in reaction rates of the two samples suggest that
minor changes in protein structure may have accompanied
adaptation of the virus to tissue culture from.the
chicken embryo.

Inactivation is typical of a firsteorder reaction, and
proceeds rapidly at 25° and 57°C. A "tailing effect"
is noticeable at all three reaction temperatures

(4°, 25° and 37°C) but is most obvious at 25°C.

The possibility exists of a disruption of viral clusters
in the early minutes of experimentation. The presence
of OMB may have an accelerating effect but the same
phenomenon was observed in an untreated control. This
implies that such a-breakup of particles is due prime
arily to factors such as temperature, pH, and agitation

of the reaction flask.

41

5.

6.

7.

42

Addition of anysteine to a OMB-virus mixture arrests or
reverses the inactivation reaction depending upon the
concentration of cysteine used. Reactivation by a thiol
compound indicates the nature of the preceding inactiva-
tion process, and confirms that SH groups were involved.
Exposure to L-cysteine prior to OMB treatment affords
the virus a certain degree of protection from the
inactivating properties of the reagent. L-cysteine, as
used in these experiments, had no deleterious effects

on the virus.

IBV 42, when suspended in tris buffer, 0.01M and pH 7.0,
is extremely stable to temperatures up to 57°C. This
stability appears to be enhanced by the presence of

certain concentrations of chysteine.

BIBLIOGRAPHY

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viruses by sulfhydryl reagents. Virology,‘lz: 176-183.

Allison, A. G., Buckland, F. E., and Andrewes, 0. H. 1962.
Effects of sulfhydryl reagents on infectivity of some
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Allison, A. O. and Valentine, R. O. 1960b. Virus particle
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Bennett, H. S. 1951. The demonstration of thiol groups in
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Bennett, H. S. and‘thantis, P. A. 1948. Under "New Comp
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44

Boyer, P. Du 1959. Sulfhydryl and disulfide groups of
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Choppin, P. W. and Philipson, L. 1961. The inactivation
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”

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48

Reagan, R. L., Brueckner, A. L., and‘Delaplane, J. P. 1950.
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