J.
gen, Virol.(1969), 4, 77-88
77
Printed in Great Britain
The Kinetics of
Neutralization of Venezuelan Equine Encephalomyelitis Virus
by Antiserum and the Reversibility of the Reaction
By N. H A H O N
Aerobiology and Evaluation Laboratory, Fort Detrick, Frederick, Maryland, U.S.A.
(Accepted 3 July I968)
SUMMARY
The neutralization of Venezuelan equine encephalomyelitis virus followed
first-order kinetics, with the reaction rate dependent on the antibody concentration and the reaction temperature but independent of pH and virus
concentration within the prescribed limits. The linear relationship obtained
between the neutralization reaction rate constant (k) and antiserum dilution
showed that k increased fivefold for each tenfold dilution of antiserum. The
energy of activation, calculated from an Arrhenius plot of the data, was
approximately 9ooo cal./mole of virus. Each IO° change in temperature (Q10)
altered the k value by a factor of I'7. In the presence of excess antibody, a
small fraction of virus resisted neutralization. Neutralized virus could not
be appreciably reactivated by simple dilution under physiological conditions
but was dissociated at either acid or alkaline pH values. Re-neutralization
of virus occurred when the environmental medium was adjusted to neutrality,
indicating that reactivation was the result of dissociation and not denaturation. Neutralized virus could be attached to cell monolayers and reactivated
to an infectious state by treatment at acid pH.
INTRODUCTION
The advent and popularization of cell culture systems and their application in virus
research have made possible the performance of kinetic neutralization tests under
controlled environmental conditions (Dulbecco, Vogt & Strickland, I956). Recently,
a quantitative immunofluorescent assay of Venezuelan equine encephalomyelitis virus
was developed, based on the enumeration of fluorescent cells in cell monolayers
(Hahon & Cooke, I967). In the course of this investigation, a significant reduction of
infected cells was noted when Venezuelan equine encephalomyelitis antiserum mixed
with the specific agent was assayed for virus infectivity. This finding, coupled with the
advantages of the assay, provided a system highly suitable for the quantitative study
of the basic reactions of Venezuelan equine encephalomyelitis virus neutralization and
related phenomena.
This initial report of a series describes the kinetic interactions of Venezuelan equine
encephalomyelitis virus with neutralizing antibody and the reversibility of the reaction
under defined conditions.
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78
N. HAHON
METHODS
Virus. A stock suspension of Venezuelan equine encephalomyelitis virus (Trinidadian donkey brain strain) was prepared from infected McCoy cell cultures (Hahon
& Cooke, 1967) and used throughout this study. The suspension was distributed in
I ml. portions into glass vials and stored at - 6 0 °. It contained 5 x lO9 cell-infecting
units (c.i.u.) of virus/ml.
Cell line and cultivation. The established cell line, McCoy, derived from human
synovial tissue (Feruandes, 1959) was used for the assay of virus. Nutrient medium
for the cells consisted of medium 199, containing o.5 ~o lactalbumin hydrolysate, IO
heat-inactivated calf serum, and 5o/*g. of streptomycin and 75/*g. of kanamycin/ml.
Cells were cultured on circular coverslips (15 mm. diameter) inserted in flat-bottomed
glass vials (19 x 65 mm.). Each vial was inoculated with I ml. of cell suspension containing between lO5 and 3 x lO5 cells, and all vials were incubated at 35 ° from 24 to
48 hr. Complete cell monolayers on coverslips were used in the virus assay procedure.
Virus antiserum. Venezuelan equine encephalomyelitis antiserum was obtained from
rhesus monkeys exposed I month earlier to an aerosol dose of approximately IOOOc.i.u.
of virus. Two pools of antiserum, nos I and I A, were used in kinetic neutralization
studies. Each pool consisted of antiserum from two or three animals. Sera were
inactivated at 56o for 3o min. and stored at - 2 o °.
Virus assay. The technique based on immunofluorescent cell-counting was employed
for the assay of Venezuelan equine encephalomyelitis virus and is described in detail
elsewhere (Hahon & Cooke, 1967). Briefly, the procedure consisted of preparing virus
dilutions in phosphate-buffered saline (PBS), pH 7"I, free of calcium and magnesium
ions. Coverslip cell monolayers received o.2 ml. of inoculum each and were then
centrifuged at 19,642 to 29,432g for 15 min. in special containers (Hahon, 1966).
After incubation at 35° for 1 hr to ensure penetration of virus into host cells, cell
monolayers were held at 35° for 2o hr in the presence of a I]4O dilution of virus antiserum in medium 199 to preclude the formation of virus microplaques. Infected cell
cultures were fixed with cold ( - 6 0 °) acetone. When cell cultures were fixed at 12 hr,
the overlay was omitted. Assays were generally made in triplicate.
Immunofluorescence procedures. Monkey Venezuelan equine encephalomyelitis
antiserum conjugated with fluorescein isothiocyanate was used to demonstrate immunofluorescence of viral antigens in infected cells by the direct fluorescent-antibodymethod.
Immunofluorescence staining, counting of infected cells, and the fluorescence microscope equipment have been described previously (Hahon & Cooke, 1967).
Kinetic neutralization procedure. The concentration of reactants and reaction conditions varied in accord with the purpose of the neutralization experiments. In general,
however, a suspension containing 5 x IOs c.i.u, of virus/ml, was mixed with an equal
volume of appropriately diluted antiserum. Both reactants were prewarmed at the
temperature employed for incubation (usually 35°) before mixing. At designated times
during incubation I ml. of the mixture was withdrawn from the reaction tube and placed
immediately in a vial held in ice-water to arrest the neutralization reaction. Test samples
were then diluted in cold phosphate-buffered saline free of calcium and magnesium ions
and assayed for unneutralized virus. A control mixture consisting of virus and normal
serum in the same concentration as the test mixture was employed with each kinetic
neutralization determination and sampled in an identical manner. The surviving
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Neutralization o f VEE virus by antiserum
79
fraction of virus was determined from the ratio of unneutralized virus in the reaction
tube to the virus titre in the control tube at designated times during incubation.
Calculations. The neutralization reaction rate constant (k) was determined from
the equation:
k - 2"3Dlog Co~C,
t
where Co and C, are the concentration of virus at time zero and t (min.), respectively,
and D is the reciprocal of the final dilution of serum in the reaction mixture.
The energy of activation value (AE) was derived from the equation:
AE = 2"3RT1T~ log kJkl
T ~ - T1
'
where R is the gas constant, T is the absolute temperature, and k is the reaction-rate
constant.
The temperature coefficient of inactivation for an interval of Io ° was calculated
from any two values of k by the equation
log Q10 = Io(log k ~ - l o g k0
t 2 - tl
where t2 is the higher temperature (C), tl the lower, and k2 and kl the reaction rate
constants at the higher and lower temperature, respectively.
RESULTS
Neutralization kinetics with varied antiserum dilutions
The kinetics of virus neutralization were investigated with different dilutions of
antiserum at 35 °. The final dilutions of antiserum and of normal serum ranged from
I/2 to I/20oo after mixing with an equal volume of 5 x IOs c.i.u./ml, of virus suspension.
Virus neutralization curves show that the initial portion of the kinetic curves
was linear and first-order with all antiserum dilutions employed (Fig. I). The
absence of a shoulder or lag in the curves indicated that neutralization of virus began
immediately. The neutralization reaction rate was derived from the slope of the initial
part of each kinetic curve. A linear relationship between the neutralization reaction
rate constant (k) and antiserum dilution was obtained when each value was plotted
on a log. scale (Fig. 2). The neutralization rate increased approximately fivefold for
each tenfold dilution of antiserum.
A sharp decrease in the slope of the two highest antibody concentrations employed
was noted within Io rain. of the onset of the neutralization reaction. The horizontal
curves that eventually resulted indicated that a fraction of virus (o'o3 to 0"05) remained
unneutralized. This fraction has been termed the persistent virus fraction (Dulbecco
et al. I956).
Neutralization kinetics at varied reaction temperatures
The effect of temperature on virus neutralization kinetics was tested in the range
from 4 to 35 ° using I/Ioo antiserum dilution mixed in equal volume with 5 × Io8 c.i.u./
ml. The initial descending portion of the kinetic curves was linear, indicating a constant
change of the surviving virus fraction that was proportional, however, to the reaction
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80
N. H A H O N
1.0
I
I
I
I
12
;>.
0
0-1
I
I
/2OO0
_
1000
I
I IIIIIII
I
I IIIIIII
I
I IIIIIII
I
I IIIIII
-
20
-
O
100
G,
O
N
10
o
0.01
0
I
I
I
I
I
l\~..J
5
10
15
20
25
2;
I I I IIII
1
0-0
30"'40
- - I .0
--2.0
--3-0
Minutes
Serum dilution
Fig. I
Fig. 2
100
90
I
--4.0
I
I
I
I
3.3
3.4
3.5
3.6
80
70
60
1.0
,
_
f,
so
~
40
.~
.N
~
~g
2o
•-
o
30
20
0"1
0.07, [0
I
5
I
I
10
15
35°,C"
20
10
3.1
I
3'2
Minutes
I I T x 10 .3
Fig. 3
Fig. 4
Fig. I. Kinetics of Venezuelan equine encephalomyelitis virus neutralization at 35 ° with
different dilutions of antiserum, o, I/z; II, ]/2o;&, I/6OO; O, ]/zooo.
Fig. 2. The dependence of the neutralization reaction rate constant (k) of Venezuelan equine
encephalomyelitis virus on different antiserum dilutions.
Fig. 3. The kinetics of Venezuelan equine encephalomyelitis virus neutralization at different
reaction temperatures, e, 4°; A, 24°; II, 35 °.
Fig. 4. Dependence of the rate of Venezuelan equine encephalomyelitis virus neutralization
by antiserum on the absolute temperature.
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Neutralization of VEE virus by antiserum
8I
temperature (Fig. 3). The rate of neutralization was temperature-dependent. An
Arrhenius plot of these data demonstrated a linear relationship between the logarithm
of the neutralization reaction rate constant and the reciprocal of the absolute temperature of reaction (Fig. 4). The energy of activation was calculated to be approximately 9ooo calories/mole of virus, comparable to that cited for bacteriophage
(Bowman & Patnode, 1964). The effect of each IO° change in temperature (Q10) altered
the neutralization rate constant by a factor of 1.7. This value is within the range reported
for other animal virus antibody systems (Granoff, 1965; Mandel, 196o) and implies
that the reaction rate is not limited by diffusion.
1'0
"7C,
t~
0"1
0
I
I
I
5
10
15
Minutes
Fig. 5- Percentage law neutralization of different quantities of Venezuelan equine encephalomyelitis virus, each of which was mixed with a constant dilution (I/3oo) of antiserum and
reacted at 35 °. ©, 5 x io5; II, 5 × 1o% e, 5 × 1or c.i.u.
Demonstration of percentage law
Virus neutralization kinetics were investigated employing a constant antiserum
dilution (I/3ooo) mixed in equal volume with different concentrations of virus ranging
from 5 × IO5 to 5 x io 7 c.i.u./ml. Mixtures were incubated at 35° and sampled at
designated times. Neutralization curves followed first-order kinetics with the virus
concentrations used in the tests (Fig. 5)- The slopes were similar for each reaction
curve, which served to demonstrate the operation of the percentage law described by
Andrewes & Elford (I933); i.e. a constant percentage of virus is neutralized by a given
strength of antiserum irrespective of the quantity of virus present. This finding is
highly relevant to the development of a Venezuelan equine encephalomyelitis virus
neutralization test based on the 50 ~ serum-neutralizing endpoint.
Dissociation of the virus antibody complex
The effect of simple dilution on the dissociation of the Venezuelan equine encephalomyelitis virus antibody complex was investigated. After undiluted antiserum
6
J. Virol. 4
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82
N. H A H O N
mixed with an equal volume of 5 × lO8 c.i.u./ml, of virus had reacted at 35 ° for 3o min.,
the reaction mixture was diluted in neutral saline buffer and a portion assayed immediately. The remainder was held at 24 ° for 4 hr or at 4 ° for 2o hr and then assayed for
virus. After incubation for 4 hr, diluted reaction mixtures showed slight increases in
virus quantities, but these increases constituted only o'5 ~o or less dissociation of
neutralized virus (Table i). Moreover, although virus dissociation was slightly greater
when the mixtures were held for 24 hr, it ranged only from 1"7 to 2.o ~ . Thus, no
appreciable reversibility of the Venezuelan equine encephalomyelitis virus neutralization reaction occurred as a consequence of simple dilution. Dissociation of neutralized
virus m a y have occurred but free virus may have been re-neutralized by excess antibody
present in the medium. To test this hypothesis, the virus antibody complex was removed
from the reaction menstruum, held in an antibody-free medium for prescribed times,
and then assayed for virus. Neutralization tests were made with a I/IO dilution of
antiserum mixed with an equal volume of either 5 x i07 or 5 × IO~ c.i.u./ml, of virus
and incubated at 35 ° for 30 min. Assay of reaction mixtures at this time showed
approximately 99 ~ neutralization of virus. Neutralized virus was then sedimented
by centrifugation at 8 t,495 g for I hr, and the sediment was resuspended to the original
volume with phosphate-buffered saline, p H 7"0, or sodium borate + sodium carbonate
saline buffer, p H I I.O. After designated incubation periods, samples of the reaction
mixtures were diluted in neutral phosphate-buffered saline and assayed for virus.
Table I. Effect of dilution on the dissociation of Venezuelan equine
encephalomyelitis virusfrom neutralizing antibody
Dilutions of virus-antiserum mixture*
Test. n o .
I n c u b a t i o n (24 °)
o f dilutions
(hr)
I
0
4
% neutralized virus dissociated
2
o
4
2o~
% neutralized virus dissociated
(o to 24 hr.)
r
Undil.
( x ion)t
A
I/2
( x io 6)
I' 5
2'5
0-5
I/3
[/5
I/lO
( x loO
( x Io0
( x IoO
2"1
5'O
1"9
3"3
0"3
o'3
4"7
3"5
4-O
6-o
8.0
1.6
5'5
5-0
2.o
4'5
9"5
1"9
* Undiluted antiserum mixed with equal volume of 5 x 1o8 c.i.u./ml, of virus and incubated at 35°
for 30 rain., resulting in approximately 99% neutralization. Virus+antiserum mixtures diluted in
accord with above scheme and assayed for virus.
t C.i.u./ml.
:~Incubated at 24° for 4 hr and then at 4° for 20 hr.
Neutralized virus did not dissociate on incubation for as long as 3 days in an
antibody-free environment of neutral p H (Table z). That neutralized virus had been
sedimented and was available for dissociation was evident by the increase of virus in
reaction mixtures suspended in alkaline buffer for 3 hr. The decrease in virus quantity
that followed within 3 days of incubation is a consequence of virus inactivation in
the highly alkaline milieu. These findings indicate that neutralized virus does not
dissociate in an antibody-free environment.
The dissociation of Venezuelan equine encephalomyelitis virus antibody complexes
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Neutralization of VEE virus by antiserum
83
was tested at both low and high p H values. Neutralization reactions were carried out
with I / I o antiserum dilution mixed with an equal volume of virus (5 x I o 7 c.i.u./ml.) and
reacted at 35 ° for 30 min. The diluent for the reactants was phosphate-buffered saline, p H
7"o. Tests for dissociation of virus antibody complexes at alkaline and acid p H values
were made by diluting neutralized virus I / I o in sodium borate + sodium carbonate saline
buffer ( p H I I.o) or potassium biphthalate + tartaric acid saline buffer (pH 2.o). After
3o see. or 2o min. at 24 °, the reaction mixtures were diluted I/IO in neutral phosphatebuffered saline and assayed immediately or after further dilution. Virus may be dissociated from a neutralized condition by environmental medium of either acid or alkaline p H
(Table 3). In this experiment with Venezuelan equine encephalomyelitis virus, alkaline
p H appeared to be more effective in dissociating neutralized virus than acid pH.
Table 2. Test for dissociation of neutralized Venezuelan equine encephalomyelit&
virus in an antibody-free environment
pH of antibody-free medium
r
~
,
7"0
I x'o
( x ,o°)*
( x io ~)
Conditions of
incubation
o
3"3
2" 1
1"5
3 hr, 24 °
3 days, 4 °
0-2
2" I
o-I
* Cell-infecting units of virus per ml.
Table 3. Dissociation of Venezuelan equine encephalomyelitis virusfrom neutralizing
antibody at low and high pH values
Treatment (30 sec.)
Treatment
A
Dissociation
pH
c.i.u./ml,
after
dissociation
( x 1o5)
7"0
2"0
I I'O
4"6
6"9
I'o
(20
.&
% neutralized
virus
dissociated
--
0"9
I'4
Dissociation
pH
min.)
c.i.u./ml.
after
dissociation
( x xo5)
7"0
I'5
2.0
I I'O
15-o
64'5
% neutralized
virus
dissociated
--
3"0
I3.1
The effect of p H of virus antibody mixtures on the kinetics of neutralization and
on the reversibility of the reaction was investigated by preparing I/5O dilutions of
each of the reactants in one of the following o.I 5 M-saline buffers: potassium and
sodium phosphates (pH 5"o), potassium phosphate and sodium phosphate dibasic
(pH 7"o), sodium borate and potassium phosphate monobasic (pH 9.o), and sodium
borate and sodium carbonate (pH IPO). Equal volumes of reactants were mixed,
incubated at 35 °, and sampled at prescribed times. Samples were diluted in neutral
PBS for assay.
The rate of neutralization was constant and unaffected by p H during the first few
minutes of the reaction (Fig. 6). There was, however, a marked effect of p H on the
subsequent portion of the curve. A slight delay was first noted in both acid and alkaline
p H medium before virus recovery increased as a consequence of reaction reversibility.
The delay was shortest with mixtures in p H I I.o buffer, suggesting that neutralized
virus was rapidly dissociated under this conditioo. Virus recovery was complete at
6-2
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84
N. HAHON
both p H 5"0 and I I.O. Similar results were obtained with Japanese B encephalitis virus
at low p H (Hashimoto & Prince, I963), but, in contrast to the findings with Venezuelan
equine encephalomyelitis virus, Japanese B encephalitis virus exhibited increased
neutralization rather than increased dissociation at alkaline pH.
The possibility that high or low p H values exerted an effect on the chemical bonds
between virus and antibody, thus resulting in dissociation of the complex, or that
dissociation was a result of antibody denaturation was investigated. Employing a
saline buffer of p H I I.O, a I/25 dilution of antiserum was mixed with an equal volume
1.0
I
I
I
I
I
,H 5
.o
~H 9
0.1
pH 7
0.01
0
i
5
i
10
i
15
I
20
I
25
i
30
Minutes
Fig. 6. Effect of different pH values of virus + antibody mixtures on kinetic Venezuelan equine
encephalomyelitis virus neutralization curves. ©, pH 5; o, pH 7; II, pH 9; A, pH I I.
of I x IO8 c.i.u./ml, of virus and reacted at 35 ° for 3o min. Normal serum mixed with
virus was incubated similarly. Within the 30 min. interval, the reaction mixtures were
sampled periodically and then assayed for virus. The mixtures were neutralized with
I N-HC1, incubated at 35 °, and sampled again at intervals during the next 3o min.
incubation period. The neutralization curve was linear for the first few minutes and
then became horizontal for I o min. (Fig. 7)- The surviving fraction of virus thereupon
increased at a linear rate as a consequence of dissociation of neutralized virus. At
3o min. the neutralization reaction was almost completely reversed. When the p H of
the reaction medium was made neutral, reneutralization of dissociated virus occurred.
These findings indicated that reactivation of the virus antibody complex was the
result of dissociation at high p H and the high alkalinity of the environment exerted
little effect on the abilities of virus and antibody to reform a neutralized complex.
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Neutralization 03" VEE virus by antiserum
85
Reactivation of neutralized virus attached to cell monolayers
To determine whether neutralized Venezuelan equine encephalomyelitis virus could
attach to host cells, cell monolayers were exposed to an acid environment after the
addition of inoculum in an attempt to induce reactivation of the attached virus +
antibody+cell complex. It should be noted that, in these experiments, the term
'attachment' rather than the general term 'adsorption' is used to describe the union
1.0
o~O
I
I
I
I
i
I
0.1
._=
5
t"~pH
0'01
10
11"0
20
pH 7"0
~-1<
30
40
50
)l
60
Minutes
Fig. 7- Neutralization of Venezuelan equine encephalomyelitis virus by antiserum at alkaline
pH followed by dissociation of neutralized virus and reneutralization at neutral pH.
of neutralized virus and cells for the reasons cited elsewhere (Hahon & Cooke, I967).
A neutralization test was performed using I/Io dilution of antiserum mixed with an
equal volume of virus (5 x Io 7 c.i.u./ml.) and reacted at 35 ° for 30 min. A control
mixture of normal serum and virus was treated similarly. Inocula from appropriate
dilutions of the reaction mixtures were centrifuged on to cell monolayers to promote
efficient attachment of neutralized and unneutralized virus. Cell monolayers were
washed with neutral PBS followed by the addition of either p H 7"o or pH 2.0 saline
buffers. After zo min. the buffer solutions were withdrawn and replaced with neutral
buffer. Cell monolayers were centrifuged again to attach any reactivated virus. Buffer
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86
N. HAHON
fluids were brought to neutrality and added to new cell monolayers for assay of
reactivated virus.
No reduction or dissociation of virus occurred when inoculum from virus plus
normal serum mixtures was attached to cell monolayers and then exposed to acid pH
(Table 4). From cell monolayers inoculated with 98 ~o neutralized virus, approximately
38 ~ more virus was dissociated from the attached virus antibody complex at acid
pH compared to treatment at neutral pH. Greater virus recovery might have been
obtained if cells had been exposed for longer periods to acid pH. These observations
indicate that neutralized virus can be attached to cell monolayers and reactivated by
treatment at acid pH.
Table 4- Reactivation of neutralized Venezuelan equine encephalomyelitis
virus attached to McCoy cell monolayers
Cells exposed
to buffer p H
c.i.u./o-z ml.
( × io a)
c.i.u, recovered
f r o m buffer
( x io 3)
% neutralized
virus
reactivated
Virus+normal serum
7"0
2.0
9"0
io.o
o.o
o.o
o.o
o.o
Virus + a n t i s e r u m
7"o
2.o
o.2
o.2
o'o4
3"8
o'45
38"7
Inoculum
DISCUSSION
The rate of Venezuelan equine encephalomyelitis virus interaction with neutralizing
antibody is dependent on the antibody concentration and the reaction temperature
but independent of pH and virus concentration within prescribed limits. With all
variables tested, the initial slope of kinetic curves is constant and characteristic of a
first-order reaction. The kinetic curves pass as straight lines through the origin without
any lag, indicating no delay in establishing the maximum rate of neutralization. The
horizontal reaction curve manifested even in the presence of excess antibody is indicative of a virus fraction that resists neutralization. The phenomenon has been noted
with a number of animal viruses and has been attributed to a variety of factors: a
nonsusceptible virus population (Dulbecco et al. I956), dissociation of the virus
antibody complex (Fazekas de St Groth, Watson & Reid, 1958), non-avid antibody
(Lafferty, 1963), or virus aggregation (Wallis & Melnick, I967). In a report to follow,
attempts to elucidate the nature of the non-neutralizable or persistent virus fraction
are described.
There is much evidence for the irreversibility of the neutralization reaction under
physiological conditions (Granoff, 1965). Critical tests of dissociation showed that the
Venezuelan equine encephalomyelitis virus antibody complex on simple dilution did
not dissociate. Virus was dissociated, however, from a neutralized condition by
environmental medium of either acid or alkaline pH. Reneutralization of virus was
demonstrated by adjusting the pH to neutrality. These findings indicate that the
integrity of both virus and antibody was unaffected by the neutralization or reactivation process (Mandel, 196I; Granoff, 1965).
Since neutralized Venezuelan equine encephalomyelitis virus attaches to host cells
and may be reactivated to an infectious state, prevention of virus attachment by
antibody is not, therefore, the sole mechanism involved in neutralization (Dales &
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Neutralization of VEE virus by antiserum
87
Kajioka, I964; Granoff, I965). Mandel (I967) recently showed that neutralized
poliovirus may penetrate and uncoat within host cells, but little intact viral RNA
could be demonstrated. This has led to the suggestion, supported by evidence of
unusual degradative reactions of neutralized virus in cells (Dales & Kajioka, I964;
Silverstein & Marcus, I964) , that the presence of antibody either suppresses the
release of intact RNA or stimulates a reaction which degrades RNA, resulting in the
inhibition of viral synthesis. The mechanism underlying loss of viral infectivity as a
consequence of interaction with neutralizing antibody remains to be elucidated.
The use of a fluorescent cell-counting viral assay, demonstrated in this study, to
follow the course of neutralization reactions offers several advantages over other
procedures used for the purpose. The assay is quantitative and direct (each infected
cell is the result of infection by one viable virus particle). Signs of virus infectivity
are not dependent on several cycles of infection (i.e. viral plaques) but can be observed
within 12 hr of a primary cycle of infection. The procedure surpasses available methods
in approaching the ideal assay from the standpoint of measuring the instantaneous
state of a virus antibody reaction by employing centrifugal force to ensure efficient
contact of virus antibody complexes and free virus with host cells. By this technique,
approximately 97 ~o of the inoculum is attached to host cells within minutes (Hahon &
Cooke, 1967). The short time required to bring the virus antibody complex and
unneutralized virus in proximity with host cells and the subsequent removal ofinoculum
fluid diminishes the time of exposure of the reactants to the influence of excess antibody
in the inoculum during the attachment process. The use of centrifugal force also
provides a greater latitude in the volume of inoculum that may be employed in fluorescent cell-counting assays because a proportionality has been demonstrated between
the number of fluorescent cells and the volume of inoculum (Hahon, I966).
Additional considerations for the employment of the fluorescent cell-counting viral
assay in conjunction with the Venezuelan equine encephalomyelitis virus antibody
host cell system for studies of neutralization reactions are: (I) the virus antibody
complex on dilution does not undergo dissociation (dilution is often necessary to
obtain countable numbers of infected cells), (2) minimal elution of attached virus
from cells (< o.I ~o in 2 hr), (3) almost complete susceptibility of cells to viral infection,
and (4) the extracellular rather than the lateral spread of virus from cell to cell (Hahon
& Cooke, x967). The former may be controlled by an antiserum overlay. Together,
these advantages provide a highly suitable system for accurately characterizing the
in vitro state of reactions between virus and neutralizing antibody and investigating
the mechanisms of neutralization.
REFERENCES
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