311
J . gen. Microbiol. (1963), 31, 3114314
Printed in Great Britain
The Influence of Some Metal Ions and pH on the
Inactivation of Vaccinia Virus by Heat
BY C . KAPLAN
Lister Institute of Preventive Medicine, Elstree, Hertfordshire
(Received 24 September 1962)
SUMMARY
The addition of metal ions to the suspending medium of vaccinia virus
preparations made them more resistant to heat. Monovalent metals were
more effective than divalent, but a mixture of the two kinds was the most
effective. Heavy metals rapidly destroyed virus infectivity. The protective
effect is probably due to the formation of metal-protein complexes
with increased resistance to heat denaturation.
INTRODUCTION
Earlier work (Kaplan, 1958)showed that the inactivation by heat of vaccinia virus
does not follow first order kinetics to completion. The inactivation curve has a sharp
inflexion where the inactivation rate suddenly decreases. In those experiments the
virus preparations used were all suspended in dilute phosphate +citric acid buffer
(McIlvaine, 1921) pH = 7.2, Na,HPO, = 4 x 1 0 - 3 ~ . Kaplan & Micklem (1961)
reported that the stability of smallpox vaccine was enhanced by the addition to
the suspending fluid of 10-lM-Na,HPO,. I n the work reported here suspending
fluids of different compositions and various molarities were used, and the influence
of hydrogen ion concentration on the inactivation of vaccinia virus infectivity was
determined.
METHODS
Virus. The experiments were done with the Lister Institute vaccine strain adapted
to the chick embryo chorioallantoic membrane. A suspension (L. 13)was prepared
by inoculating the chorioallantoic membranes of ten 12-day chick embryos with
about lo6 pock forming infectious units (i.u.) of virus. After incubation for 2 days
at 35.5" the infected chorioallantoic membranes were harvested with sterile precautions, washed in cold sterile dilute McIlvaine buffer and extracted in 50 ml.
dilute buffer + 10 ml. Arcton 113 (trifluorotrichloroethane, I.C.I. ; Gessler, Bender
& Parkinson, 1956) by mechanical disruption in the chilled, stainless steel chamber
of a Servall 'Omnimix'. The extract was centrifuged at 2,000 rev./min. for 5 min.
in the 240 head of an International Centrifuge, size 1, type SBV. The supernatant
fluids of this centrifugation were re-extracted with another 10 ml. of Arcton and
centrifuged again. The second supernatant fluid of suspension L. 13 was kept a t
4' for a week, during the titration of potency, and then stored a t -70" in 1ml.
volumes in sealed ampoules.
Infectivity titrations were made by pock count in the chorioallantoic membranes
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312
C. KAPLAN
of 12-day chick embryos (Westwood, Phipps & Boulter, 1957). All dilutions were
made in dilute McIlvaine buffer.
Heat inactivation experiments. Virus was thawed, and diluted in the appropriate
suspending fluid to give an initial titre of about 107i.u./ml. at the pH value or
metal ion concentration required. One ml. volumes were then dispensed into small
test tubes (50 x12 mm.) which were placed in a water bath whose temperature
was maintained to within +0.05". Tubes were removed at intervals and chilled in
melting ice. Residual virus was then titrated and from the results the velocity constant
( K ) of the inactivation was calculated from the formula log VJV = Kt/2.3, where
Vo = initial virus titre, and V = residual virus titre a t any time t. Only the velocity
constants ( K p )from the fast inactivation portion of the curve were compared
(Kaplan, 1958) since differences in heat resistance were more obvious here than in
the slow inactivation part ( K J . Some of the experiments were done a t only one
temperature; where possible, however, three temperatures were used. Gard (1959)
emphasized the need for many repetitions of the same experiment to ensure a reliable
estimate of the mean inactivation rate of a virus under any given conditions. Most
of the tests were repeated four to ten times.
Suspendingj u i d s . In most of the experiments virus was suspended in McIlvaine's
phosphate + citric acid buffer, pH 7.2; Na2HP04= 1 0 - l ~1 ~
0 - 2 ~or 1 0 - 3 ~ ~In
.
some, solutions of other salts of sodium or solutions of salts of other metals were
used. Analar grade reagents were dissolved in deionized water.
RESULTS
Injuence of metal ion concentration
At pH 7.2 the reaction velocity of the inactivation decreased as the concentration
of Na,HP04 in the buffer increased (Table l(a)). To determine whether this effect
was attributable to the cation or the anion, the experiment was repeated at 50'
with virus suspended in sodium acetate and sodium chloride (Table l ( b ) ) . Clearly,
the cation was responsible for the stabilization of infectivity. Potassium (Table 1(c))
replaced sodium. Two experiments only were done with lithium; the results suggest
that this element, too, could replace sodium.
Divalent metal ions were also investigated (Table l(d)). At 50" 10-livx-Mg2+
and 10-lM-Ca2+were less effective than lO-lM-Na-l-.There was no indication of an
increased rate of inactivation. At 55" and 60" 10-lM-Mg2+was less effective than
lO-lM-Na+ but more effective than 10-3~-Na+.A mixture of lO-lM-Naf and
10-1M-Mg2f stabilized vaccinia virus infectivity more effectively than either ion
separately (Table 1 (e)).
A few experiments were done with salts of the heavy metals, copper, cobalt and
iron. These inactivated virus very rapidly. The "experiments were complicated,
however, by the toxicity of these heavy metals for the chick embryo chorioallantoic
membrane, so that the data were insufficient for calculation of reaction velocities.
Injuence of pH value
Virus was heated in McIlvaine buffer, Na2HP0, = 1 0 - l ~and l O - 3 ~ , pH = 6
and 7.2 at each molarity (Table 2). The inactivation rate was clearly increased in the
acid medium.
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Effect of cations and heat on vaccinia virus
313
Table 1. T h e injluence of various ions and temperatures on the reaction
velocities ( K , ) of heat inactivation of vaccinia virus
Kf(min.-l)*
Concentration
I
(4
Substance
10-1
Na,HP04
Deionized water
CH,COONa
NaCl
KCl
NaCl
CaCl,
MgSO4
MgSO4
NaCl
MgSO4
NaCl
MgSO,
55"
0.07
0.08
0.37
0.10
0.18
10-1
10-1
10-1
10-1
10-1
10-1
10-2
10-1
60"
2.3
n.t.
n.t.
n.t.
n.t.
n.t.
0.79
5.8
0.11
0.15
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
0.05
n.t.
n.t.
0.006
n.t.
n.t.
0.07
0.07
0.06
0.07
0.12
10-1
\
h
50"
0.42
0.48
0-46
3.2
* Each value is the mean of four to ten tests.
n.t. Not tested.
Table 2. T h e injluence of pH value and sodium concentration on the
reaction velocity ( K , ) of vaccinia virus inactivation
Sodium
conc.
K , (min.-l) at
(M)
Temp.
10-1
10-9
f
h
\
PH 6
pH7.2
50"
55"
0.2
0.07
0-37
50"
55"
0.24
0.61
1.1
0.1
0.79
DISCUSSION
Several authors (Burnet & McKie, 1930; Adams, 1949; Lark & Adams, 1953;
Northrop, 1955) have shown that the heat stability of bacteriophage suspensions
depends on the concentrations of cations in the suspending fluids. Stability was
generally increased when small concentrations of divalent cations were added to
a basic concentration of monovalent cation, although monovalent cations by
themselves conferred less protection than distilled water (Burnet & McKie, 1930).
Wallis, Yang & Melnick (1962)reported that 2 M-Na+ completely protected vaccinia
virus for 4 hr. a t 50" and at least 24 hr. at 37"; M-Na+ was less effective, while
>i-Mg2+ and M-Ca2+ increased the rate of destruction of infectivity. The results
of Wallis et al. (1962) are not comparable with ours, since the concentrations
of cations tested were so different. It is noteworthy, however, that the highest
concentrations of Mg2+( l o - l ~ )in our experiments had an appreciable protective
action at 55" and 60°, although a t 50" there was no significant difference between the
inactivation rates in 10-3~-Na+
and in 10-lM-Mg2+or Ca2+.Since Wallis et al. (1962)
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314
C. KAPLAN
did not record the provenance or mode of preparation of the vaccinia virus they used,
comparison with their results would in any case be unrewarding.
The findings in our experiments agree, in general, with those of workers with
bacteriophage. However, the mode of action of the cations is not .entirely clear.
Woese (1960) suggested that, at least at high temperatures, heat inactivation of
viruses might as well be due to denaturation of nucleic acid as to denaturation of
protein. Rice & Doty (1957) found that a highly polymerized preparation of deoxyribonucleic acid (DNA) began to change viscosity at 85' but not a t lower temperatures in the absence of substances (e.g. urea) effective in denaturing proteins.
If it be assumed that native DNA behaves similarly, then at the temperatures a t
which we were working, denaturation of DNA was probably not a factor in the destruction of infectivity. Eichhorn (1962) found Co2+to be a stabilizer of DNA. I n
our experiments Co2+and two other heavy metals Cu2+and Fe2+greatly accelerated
the rate of destruction of virus infectivity, suggesting that the most important
factor in heat inactivation is protein denaturation. The increased inactivation rates
a t pH 6 also indicate that the virus protein is the primary target in heat inactivation.
The protective action of some metal ions on the infectivity of vaccinia virus may,
therefore, be exerted by a bonding of the ions to polar groups of the protein with
consequent strengthening of its molecular structure against heat denaturationa familiar enough concept in prot,ein studies.
My thanks are due to Mr G. Hendy for skilful technical assistance.
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