(SANITIZED)UNCLASSIFIED CZECH PUBLICATION ON VIROL RESEARCH(SANITIZED)

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• Publication Date: May 8, 1963
• Content Type: REPORT

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VOLUME 7
JANUARY, 1963
1
0
NUMBER 1
International Journal
13S C610 .11
EDITORS
D. BLA8KOVI C, Bratislava
A. A. SMOR OD IN T S E V, Leningrad
V. VALENT A, secretary, Bratislava
EDITORIAL BOARD
S. ANGELOV, Sofia
N. CAJA L, Bucharest
CHEN SI UN. Fenjan
E. F ARK A S, Budapest
G A W H. ZANYI N, Wuhan
HUAN G, C. H., Peking
G Y. IVANOVICS, Szeged
H. MAK OWE R, Wroclaw
S. NICOLA U, Bucharest
F. PATOOK A, Prague
F. PRZESMYCKI, Warsaw
H. ROHRE R, Inset Riems
V. L. RYZHK 0 V, Moscow
L. STOJKOVI 0, Belgrade
A. L. TERZI N, Sarayevo
H.URBACH,Jena
E. YANE V, Plovdiv
V. M. ZHDANO V, Moscow
EE
cacgemom,ciwAm ACIDEAkOW COW S3C01%CMg3
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VOLUME 7
ACTA VIROLOGICA
ENGLISH EDITION
.JANUARY, 1963 NUMBER 1
CONTENTS
ZHDANOV, V. M., BUKRINSKAYA, A. G., & RAMENSKAYA, G. P.: Autoradiographic
Study of the Penetration of Sendai Virus into the Cell. III. Use of Virus Preparations La-
belled with Uracil-C", P32, Methionine-S" or Cysteine-S"
IVANIOOVA, 8., SKODA, R., MAYER, V., & SOKOL, F.: Inactivation of Aujeszky
Disease .(Pseudorabies) Virus by Nitrous Acid 7
GHENDON, YU. Z.: Mutations of Virulent and Attenuated Poliovirus Strains Induced by
Nitrous Acid 16
STYK, B., HANA, L., FRAN-PK, F., SOKOL, F., & MENMK, J.: Cofactor and Specific
Antibodies against Influenza Viruses. VII. The Nature of Cofactor and Influenza Anti-
bodies Studied by Density Gradient Zonal Cent ifugation 25
LEO, J., SZANTO, J., & ALBRECHT, P.: Mumps Virus Infection of HeLa Cells Studied
by the Fluorescent Antibody Method 37
NOSIK, N. N., & KLISENKO, G. 4.: Cytochemical Studies on Nucleic Acids in Cells from
Tissue Cultures Infected with Type 5 Adenovirus 42
ZALKIND, S. YA., ANDZHAPARIDZE, 0. G., BOG OMOLOVA, N. N., & FOKINA, A. M :
Morphological and Cytochemical Study of HEp-2 Cell Cultures Persistently Infected with
Tick-borne Encephalitis Virus 48
ILYENKO, V. I., & ZHILOVA, G. P.: Methods of Preparation and Immunogenic Properties
? of a Killed Tissue Culture Vaccine against Tick-borne Encephalitis 54------
SUPTEL, E. A.: Pathogenesis of Experimental Coxsackie Virus Infection. Distribution of
Coxsackie Virus in Mice after Air-borne Infection r 61
RUTTKAY-NEDECKIT, G., & 8PA.NIK, V.: Evaluation of the Efficiency of Tobacco Mosaic
Virus Purification Procedures by the Polarographic Method 67
NIKOLAYEV, V. P.: Virological and Serological Investigations of Sporadic Cases of Serous
Meningitis 76
CHUMAKOV, M. P., KARPOVICH, L. G., SARMANOVA, E. S., SERGEEVA, G. I ,
BYCHKOVA, M. B., TAPUPERE, V. 0., LIBIKOVA, H., MAYER, V., REHAOEK,
J., KOZUCH, 0. & ERNEK, E.: Report on the Isolation from Ixodes persulcatus Ticks
and from Patients in`Western Siberia of a Virus Differing from the Agnet of Tick-borne
Encephalitis 82
KORDOVA, N., & BREZINA, R.: Multiplication Dynamics of Phase I and II Coxiella
burneti in Different Cell Cultures 84
STYK, B.: Effect of Some Inhibitor-Destroying Substances on the Nonspecific Inhibitor of
C Influenza Virus Present in Normal Rat Serum 88
Letters to the Editor:
LINDE, K., & URBACH, H.: Complement-fixing Coxiella burneti Antigen Prepared from In-
fective Yolk Sacs by Trypsin Treatment
GEFT, R. A., & POLYAK, R. YA.: Removal of Thermostable Inhibitors against A2 Influenza
Virus from Immune Horse Sera by Rivanol
90
91
Reports (VIIIth International Congress for Microbiology, Montreal, August 19-24, 1962.
? A Jubilee of Czechoslovak Science) 92
Review (K. M. Smith: Viruses) 95
Erratum 96
For authors' addresses see cover p. iii
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Ada virol. 7: 1-6, 1963'
Autoradiographic Study of the Penetration of Sendai Virus into the
Cell
III. Use of Virus Preparations Labelled with Uracil-C", P32, Methionine-S35
or Cysteine-S35
V. M. ZI-IDANOV, A. C. BUKRINSKAYA, G. P. RAMENSKAYA
Ivanovsky Institute of Virology, U.S.S.R. Academy of Medical Sciences, Moscow and Severtsov
Institute of Animal Morphology, U.S.S.R. Academy of Sciences, MUscow
Received July 16, 1962
In a previous communication the dynamics of the displacement in the cell
of radioactive components of P32-labelled Sendai. virus were described (Buk-
rinskaya et al., 1961). In the present paper this process is characterized quanti-
tatively and the results of experiments with P32-labelled virus are compared
with those obtained with Sendai virus labelled with a specific precursor of
nucleic acids, namely uracil-C". To study the fate of viral protein, virus
preparations labelled with methionine-S35 or cysteine-S35 were used.
Materials and Methods
, The materials and methods used and the characteristics of the labelled virus preparations
were the same as those previously described (Bukrinskaya et al., 1961; Zhdanov and Bukrinskaya,
1961). The labelling of virus with uracil-C" was done by injecting 100 tiC of uracil-C" into each
ehick embryo simultaneously with virus; the virus was purified by a procedure similar to that
used previously. Preparations containing 105.?-107?0 1D55/ml. of virus were used throughout,
thus ensuring a high multiplicity of infection in view of the few thousand cells on the glass strips
used in the experiments.
Results
When virus preparations labelled with P32 or uracil-C" were used for in-
fection, 0.5-10% of the cells in the culture showed radioactive inclusions.
While in autoradiograms prepared 10 minutes after infection of the cells with
P32-labelled virus radioactive grains were located predominantly in the nucleolus
(Fig. 1), during further incubation the proportion of such cells decreased and
cells containing intranuclear and internucleolar grains appeared (Figs. 2 and 3).
Two hours after infection the proportion of cells showing grains in the cytoplasm
increased (Table 1), the grains being located in the form of conglomerates in
the perinuclear. part of the cytoplasm (Fig. 4).
To estimate the total radioactivity in the three groups of cells containing
cytoplasmic, nuclear or nucleolar grains, the mean number of radioactive
grains per cell was determined in an infected human amnion cell culture
(Table 2). To characterize the dynamics of incorporation of viral components
into cellular structures, the mean number of grains per cell, determined at
various intervals, was multiplied by the number of cells in the corresponding
group. The results obtained are presented in Figs 6 and 7. It can be seen that
the higher the interval elapsed after infection (10-180 minutes), the higher
the degree of incorporation of viral components into the nucleus. While during
1
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ZHDANOV, V. M. et at.
the first hour after infection the radioactivity of the cytoplasm did not change,
two hours after infection it increased rapidly. This increase coincided with
the decrease of the number of cells containing grains in the nucleolus. The
total radioactivity of the nucleoli however, did not change during incubation,
Table I. Distribution of radioactive grains in cells infected with P32-labelled Sendai virus
Per, cents of cells containing grains in the
Time after
infection
in mins
cytoplasm
nucleus
nucleolus
10
10
10
80
30
11
14
75
60
4
16
80
120
31
18
51
180
32
24
44
Table 2. Mean number of radioactive grains per human amnion cell after infection with
P32-labelled Sendai virus
Time after
infection
in mins
cytoplasm
Number of grains in the
nucleus
nucleolus
10
3.2 ? 0.5
11.0 ? 1.5
5.3 ? 0.8
30
5.8 + 0.7
13.0 ? 2.0
5.2 ? 0.4
60
8.8 ? 1.8
13.6 ? 2.0
6.6 ? 0.8
120
13.2 ? 1.2
16.1 ? 1.3
5.0 ? 0.3
180
11.1 ? 1.5
16.1 + 1.8
10.0 ? 1.9
this fact being caused by a parallel increase of the number of intranucleolar
grains during prolonged incubation of infected cells (Fig. 7).
When cells were treated for 10-60 minutes with noninfected allantoic
fluid labelled to the same degree as the virus preparations used for infection,
grains were not detected in the cells. After 2-3 hours' contact some of the
cells contained grains in the nucleus. P32 was not incorporated into the nucleoli
of such control cells.
The character of the incorporation of the virus into the structural components
of the cell was the same, regardless whether cultures of stable cell lines (Cyno-
molgus monkey heart or human amnion cells) or primary cultures of chick
embryo cells were used.
Experiments with uracil-C" were carried out in human amnion cell cultures.
The character of .the incorporation of viral components into cellular structures
was the same as with virus preparations labelled with P33 (Table 3), but the
proportion of cells containing grains in the nucleolus was lower duirng the
early period of incubation.
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SENDAI VIRUS PENETRATION INTO THE CELL
3
When human amnion cells were treated for 2 hours with noninfected allantoic
fluid labelled with uracil-C14 and diluted to the same degree as the virus pre-
parations used for infection, some of the cells contained grains in the nucleus.
After 3 hours an appreciable proportion of cells contained radioactive grains,
80
70
60
50
40
30
20
10
Fig. 6.
3
Fig. 7.
Proportion of cells (%) in culture infect- , The change of the total radioactivity of
ed with P"-labelled Sendai virus showing cells (proportion of cells in % showing
incorporation of radioactive viral corn- radioactive grains x Mean number of
ponents in relation to the incubation time grains per cell) infected with P"-labelled
ES cytoplasm, 0 nuclei, A nucleoli; Sendai virus during incubation
ordinate: % of cells; abscissa: hours of ordinate: cells x grains; for other expla-
incithation. nations see Fig. 5.
Table 3. Distribution of radioactive grains in cells infected with uraeil-C"-labelled Sendai virus
Time after
infection
in mins
Per cents
cytoplasm
of cells with grains in
nucleus
the
nucleolus
10
6
34
60
30
11
38
_
51
60
14
45
41
120
18
50
32
90% of them showing intranuclear and only 10% intranucleolar localisation
of the grains.
Using virus preparations labelled with methionine-S35 or cysteine-S35, faint
grains distributed uniformly over the nucleus and cytoplasm appeared on the
surface of most of the cells already 10 minutes after infection (Fig. 5). The
number of grains reached a maximum 60 minutes after infection. The uiform
distribution of grains suggest that they represented viral components adsorbed
onto, but which had not penetrated into the cell. To verify this assumption,
cells were inoculated in parallel experiments with methionine-S35-labelled
virus at 4 and 37? C, taking into account that the penetration of virus into
the cells is markedly suppressed at low temperatures. The number of grains
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ZIIDANOV, V. M. et al.
per cell was 21.0 ? 2.5 and 24.0 + 3.0 at 37 and 4? C, respectively, i.e. about
the same at both temperatures. Similar results were obtained when virus
preparations labelled with cysteine-S33 were used.
When cultures were inoculated at 4? C with uracil-C14-labelled virus, the
proportion of cells showing incorporation of viral components as well as the
occurrence of intranucleolar grains was decreased (Table 4).
Table 4. The effect of temperature on the incorporation of Components of uracil-C14-lab,elled
Sendai virus into human amnion cells
Temperature ,
.?C
Proportion of cells
containing grains
Percentage
cytoplasm
of cells with grains
nucleus
in the
nucleolus
37
4
10%
- 2%
5
36
(over the
whole cell)
44
44
51
20
Discussion
Analysis of virus preparations labelled with P2 or S33 has shown that the
distribution of the radioactivity between the viral components was not uniform.
While 44-58% of the activity was found in the S-antigen (internal ribo-
nucleoprotein) fraction of P32 labelled virus, only 16% was recovered in the
haemagglutinin and denatured protein fractions obtained after splitting of
the virus with ether. In virus preparation labelled with S23, 55.5% of the radio-
activity was bound to the viral protein and only 10.4% was found in the S-
antigen (Bukrinskaya et al., 1961). Thus the grains contained in cells infected
with 1332- or S33-labelled virus corresponded essentially to the nucleic acid
and protein fraction of the virus, respectively. Labelling with uracil-C1-4,
a specific precursor of nucleic acids, enabled a more reliable examination of the
fate of viral nucleic acid.
While 97% of the cells in the culture inoculated with methionine-S33 and
cysteine-S33-labelled virus showed grains on autoradiograms, only 0.5-10%
of the cells contained radioactive inclusions after inoculation with P32- or
? uracil-C14-labelled virus. In the former, case the grains were uniformly distribut-
ed over the whole cell, while in the latter a tendency for accumulation of the
grains in the nucleolus was evident. These findings can be readily explained
when it is assumed that the nucleic acid component of the virus penetrated
into the cell, while the viral protein remained on its surface. The results obtained
in experiments on the adsorption of S33-amino acid or uracil-C14-labelled viruses
at different temperatures supported this assumption. But the data obtained
do not exclude that a part of the virus protein penetrates into some cells.
This question could be solved by autoradiography of sectioned cells.
Taking into account the fact that one hours' adsorption was sufficient to
obtain optimum virus multiplication in the cells, it can be deduced that the
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A
SENDAI VIRUS PENETRATION INTO THE CELL
5
changes observed in the localization of grains during incubation of the infected
cultures were due to displacement of viral components already penetrated
into the cell rather than to the penetration of new virus particles, although
the latter factor cannot be completely excluded. The results of the present
experiments indicate that a part of the viral components which had penetrated
into the nucleolus were liberated during further incubation and concentrated
in the nucleus. Then they entered the cytoplasm and were located in the peri-
nuclear zone.
Recently autoradiographic studies on the synthesis of cellular ribonucleic
acids have been published (Errera, 1961; Perry et al., 1961). Their results
are in agreement with the data given in the present paper. Thus the fate of
viral ribonucleic acid which had penetrated into the cell is essentially the same
as that of cellular ribonucleic acid in a noninfected cell. Some hours after
the entry of viral ribonucleic acid from the nucleus into the cytoplasm an
intensive synthesis of viral antigen takes place (Zhdanov et al., 1961). In
agreement with other investigators (Brachet, 1960; Errera, 1961; Jacob and
Monod, 1961) we can asume that viral ribonucleic acid possibly plays the role
of messenger ribonucleic acid in the synthesis of specific protein in the cyto-
plasm.
Summary'
1. The dynamics of the displacement of the nucleic Acid component of P32-
or uracil-C1-4-labelled Sendai virus in the infected cell was studied by auto-
radiography. First radioactive grains appeared early after the infection mainly
in the nucleolus, then in the nucleus, and 2 hours after infection in the cyto-
plasm. The proportion of the cells in the culture showing incorporation of
viral components by autoradiography was 0.5-10%.
2. In autoradiograms of cells infected with methionine-S35 or cysteine-S35
labelled Sendai virus the radioactive grains were uniformly distributed on the
surface of 90-97% of the cells in the culture.
3. The interaction of methionine-S35 or cysteine-S35-labelled virus and cells
at 4? C instead of 37? C, did not lead to decreased labelling of the cells, while
the radioactivity, of the cells was markedly reduced when the cells were ino-
culated at 4? C with uracil-C14-labelled virus.
4. The results obtained indicate that after inoculation the ribonucleic acid
or the internal ribonucleoprotein (S-antigen) of Sendai virus penetrated into
the cells, while the protein coat of the virus remained on their surface.
Acknowledgement. The authors thank Dr. A. S. Konikova and her collaborators from the
Laboratory of Biochemistry of the Institute of Surgery, U.S.S.R. Acad. Med. Sci., and Prof.
V. K. Modestov and his collaborators from the Chair of Medical Radiology of the Central Institute
of Postgraduate Training of Physicians for the aid in this study.
References
Brachet, J. (196): Le role biologique d'acides nucleiques. R. C. Let. Sci. Camerino 1, 3.
Bukrinskaya, A. G., Zhdanov, V. M., and Ramenskaya, G. P. (1961): Autoradiographic study
of the penetration of Sendai virus into the cell. II. Use of virus preparations labelled in the P32.
V op. Virusol. 6, 547.
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ZIIDANOV, V. M. et al.
Errera, M. (1961): Biochemical processes in injured cells in relation to cell recovery. J. cell.
comp. Physiol. 58 (Supplem.), 209.
Jacob, F., and Monod, J. (1961): Genetic regulatory mechanisms in the synthesis of proteins.
J. Mol. Biol. 3, 318.
Perry, R. P., Hell, A., and Errera, M. (1961): The role of the nucleolus in RNA and proteins
synthesis. I. Incorporation of cytidine into normal and nucleolar inactivated HeLa cells.
Biochem. biophys. Acta 49, 47.
Zhdanov, V. M., and Bukrinskaya, A. G. (1961): Autoradiographic study of the penetration of
Sendai virus into the cell. I. Labelling bf Sendai virus with radioactive isotopes. Vop. Virusol. 6,
542.
Zhdanov, Y. M., Bukrinskaya, A. G., and Azadova, N. B. (1961): Fluorescent microscopic study
of incomplete Sendai virus formation in tissue culture cells. J. Immunol. 87, 641.
Explanation of Photomicrographs:
Fig. I. Autoradiogram of a human amnion cell infected'with P32-labelled Sendai virus.
Time after infection: 10 mins; grains are iodated on the cytoplasmic and nuclear membranes,
and in the nucleolus; x 900.
Fig. 2. Autoradiogram of human amnion cells prepared 1 hour after infection with P32-labelled
Sendai virus.
The upper left cell contains grains located in the internucleolar zone of the nucleus; x 600.
Fig. 3. Autoradiogram of human amnion cells prepared 1 hour after infection with P32-labelled
Sendai virus.
Isolated nuclei containing radioactive grains; x 600.
Fig. 4. Autoradiogram of human amnion cells infected with P"-labelled Sendai.virus. Two hours
after infection. The grains in the left cell are located in the perinuclear zone of the cytoplasm.
x 600.
Fig. 5. Autoradiogram of human arhnion cells prepared 1 hour after infection with methionine-
S35-labelled Sendai virus.
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Acta virol. 7 : 7-15, 1963
Inactivation of 'Aujeszky Disease (Pseudorabies) Virus by
Nitrous Acid
s. IVANI6OVA , R. 8KODA, V. MAYER, F. SOKOL
Institute of Virology, Czechoslovak Academy of Sciences, Bratislava
. Received August 17, 1942
Mundry and Gierer (1958) showed that desamination of purine and pyrimidine
bases of isolated viral ribonucleic acid (RNA), by treatment with nitrous acid
(HNO2), carried out as described by- Schuster and Schramm (1958), led in
addition to inactivation also to induction of mutations. They demonstrated,
too, that mutants can be obtained also when intact viruses are treated with
IIN02. Recently the kinetics of inactivation of various RNA- or deoxyribo-
nucleic acid (DNA)-containing viruses by IIN02, the properties of isolated
chemical mutants and those of inactivated virus were studied by several
investigators (Boeye, 1959; Tessman, 1959; Vielmetter and Wieder, 1959;
Schafer et al., 1959; Granoff, 1961; Bautz-Freese and Freese, 1961; Carp and
Koprowski, 1962; Restle et al., 1962; Wassermann, 1962). We studied the
reaction between 11NO2 and the Aujeszky disease virus, a DNA-containing
animal virus (Ben-Porat and Kaplan, 1962). In the present paper the kinetics
of inactivation of this virus at different pH, temperatures and HNO2 con-
centrations are described. The immunogenicity of the inactivated virus was
also investigated.
Materials and Methods
Virus. The Bucharest strain of Aujeszky disease virus in its 368th-372nd chick embryo cell
(CEO) culture passage was used throughout. Virus suspensions were prepared as follows. Bottles_
were seeded with 5 x 107 CEO in 100 ml. of growth medium (1: 1 mixture of medium 199 and
Earle's saline, containing 2% calf serum heated at 56? C for 30 minutes) (PEC) and incubated
at 37? C for 48 hours. The medium was then removed and the cells inoculated with about 108plaque
forming units (PFTJ) of virus. The virus was allowed to adsorb at 37? C for 90 minutes. Then
100 ml. of fresh PEC medium were added and the cultures incubated at 37? C for a further
48 hours. The harvested infectious culture fluids, containing about 5 x 107 PFU of virus per ml.,
were freed of cells and their debris by low speed centrifugation.
In some experiments virus partially purified by sedimentation at 40,000 x g for 30 minutes
was used. The pellet was resuspended in phosphate buffered saline pH 7.4 (0.007M phosphate,
0.14M NaCl).
Infectivity titration. Virus was titrated by the plaque method in CEO monolayers as described
by 8koda and Mayer (1961) using 2-4 plates for each serial tenfold dilution. After adsorption
of the virus at room temperature for 90 minutes the inoculum was removed. In some experiments
also the TCID? titres of the virus samples were estimated. Tube cultures containing 5 x 105 CEO
and 1 ml. of PEC medium were inoculated with 0.1 ml. of serial tenfold dilutions of the samples
using 4 tubes for each dilution. The cultures were incubated at 37? C and the cytopathic effect was
read after 5 days.
Neutralisation test. Serial twofold dilutions of rabbit immune serum heated at 56? C for 30 mi-
nutes were mixed with equal volumes of a suspension containing 103 TCID20 of virus per ml. and
incubated at 37? C for 60 minutes. Tube cultures of CEO were then inoculated with 0.2 ml. of
the mixture using 4 tubes for each dilution. The titres were taken as the reciprocal of the highest
initial dilution of serum showing complete inhibition of the viral cytopathic effect.
Treatment of virus by HNO2. Two parts of virus suspension were mixed with one part of 1M
acetate buffer of required pH and one part of a 4M NaNO, solution in distilled water. When
solutions of lower. NaNO2 concentration were used, NaCl was added to adjust the concentration
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8
IVANIOovA, et al.
of Na+ to 4M. All solutions were heated to the required temperature before mixing. The reaction
mixture was kept in a water bath of a constant temperature and stirred by a magnetic stirrer.
The pH of the mixture did not change during the reaction time. Samples were withdrawn at
intervals, diluted 1 : 10 with 0.25M phosphate buffer pH 7.85 to stop the reaction and immediately
assayed for infectivity. The final pH of the 1 : 10 diluted virus suspension was about 7.4. NaNO,
was not removed from the samples, because an appreciable drop in virus titre was observed
regularly after exhaustive dialysis against phosphate buffered saline pH 7.4. Controls set up in
parallel consisted of virus, acetate buffer and 4M Neel.
Several batches of inactivated virus for immunisation were prepared by treatment of crude
virus suspension for 30 minutes with 1M NaNO2 at pH 5.55 and 37? C. The suspension was then
dialysed exhaustively against tap water, the viral antigen sedimented at 40,000>< g for 30 minutes,
resuspended in 1/10 of the original volume of phosphate buffered saline pH 7.4 and freed of
insoluble material by low speed centrifugation. No live virus could be demonstrated in such
preparations by the plaque method, when 0.4 ml. of the suspension were plated on CEC mono-
layers.
Experimental
The character of the inactivation reaction
Most of the curves characterizing the decrease of infectivity with time showed
essentially an exponential rate of inactivation (Figs. 1 and 2) corresponding
to the equation
log (Io/I) =k X t +b, (1)
Io being the infectivity titre of control virus suspension at zero time, I that
of treated virus at time t and k the reaction rate constant (min. -1). However
Fig: 1.
Inactivation of Aujeszky disease virus by HNC/2
1M final concentration of NaNO2, pH 5.55, 20? C;
abscissa: time in minutes; 0 = controls; infectivity assayed by the plaque method;
k = 0.0397 min-1
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INACTIVATION OF PSEUDORABIES VIRUS BY HNO,
9
almost in all experiments the titre of virus suspension determined immediately
after mixing with NaNO2 solutions was significantly lower than the titre of
control virus suspension at zero time and therefore the constant b was usually
80
Fig. 2
Inactivation of Aujeszky disease virus by HNC),
Conditions as in Fig. 1; TCID? estimated in tube cultures; k = 0.0327 mm-'
120
higher than zero (Fig. 3). In some experiments, especially at low temperatures
and concentrations of HNO2, an initial shoulder was observed in the inactivation
curve (Fig. 4) followed by exponen-
tial inactivation according to equa-
tion (1). In such cases the rate con-
stant was calculated from the expo-
nential portion of the curve.
? Fig. 3.
Inactivation of Aujeszky disease virus by
IINO2 1M final concentration of NaNO2, pH
5.1, 20? C; for further explanations see Fig. 1;
? k = 0.378 min-l?
? Impurities of nonviral origin contained in crude virus preparations did not
affect the appearance of the shoulder or the rate of inactivation. At 1M final
concentration of NaNO2, pH 5.7 and 200.0 k was e.g. 0.0312 and 0.0310 for
crude and partially purified virus, respectively. When a pure virus line, obtained
by threefold plaque purification, was used instead of wild virus, the shoulder
did not disappear.
It was demonstrated that under identical conditions the cytopathic activity
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ivANIOOVA, 8. et al.
of virus and its ability to form plaques were inactivated at about the same
rate (compare Figs. 1 and 2). Therefore in further experiments infectivity
was assayed only by the more exact plaque method.
Fig. 4.
Inactivation of Aujeszky disease virus by 11NO2
1M final concentration of NaNO2, pH 5.55, 4? C; for further explanations see Fig. 1; note the
initial shoulder in the inactivation curve; k ---- 0.00872 min-1
The relationship between temperature and the inactivation rate
The dependence of the inactivation rate on temperature was studied at
pH 5.55 and a 1M final concentration of NaNO2. When log k was plotted
against the reciprocal of absolute temperature (1/T), the experimental points
fitted well the Arrhenius equation
1 l\
log (k/k,)
2.303 x R \T, T)'
where R is the gas constant and q the activation energy. The latter was
15.2 kcal/mole, as determined from the slope of the line shown in Fig. 5.
(2)
3.2
3.
(//T) x /03
3.6
Fig. 5.
The temperature dependence of the inac-
tivation rate
1M final concentration of NEiNO? pH
5.55; temperatures: 4, 20, 30 and 37? C.
0.5
M NaNO2
Fig. 6.
Dependenceof the inactivation rate on the con-
centration of NaNO2
20 ?C, pH 5.7.
1.0
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INACTIVATION OF PSEUDORABIES VIRUS BY IINO,
11
E ffect of pH and NaNO2 concentration
According to the mass action law the dissociation of 11NO2 is characterized
by the equation
pH ? pK = log [NaNO2] ? log [HNO2]
(3)
As the rate of inactivation should be proportional to the concentration of
free IIN02, it was expected that k will be proportional to the concentration
of NaNO2 and that the plot of log k against
pH will give a linear relationship. As shown
in Figs 6 and 7, the experimental data
obtained fitted well the expected relation- 2.0
ships. Below pH 4.8 and above pH 5.8 the
rate of inactivation was, under the condi-
tions given in Fig. 7, too high and too low,
respectively, to be measured with sufficient
accuracy. < /0
b,
FIN. 7.
Dependence of the inactivation rate on pH
1M final concentration of NaNO2,20? C.
0
4.6 5.0 5.4
PH
Attempt to isolate infectious DNA from Aujeszky disease virus
It was suspected that the shoulder in some inactivation curves was caused
by the disturbing effect of viral protein, and possibly also of other substances
surrouding the viral DNA, on the interaction of HNO, and the virus nucleic
acid. Therefore it was attempted to isolate infectious DNA from the virus and
to study its inactivation by HNO2. However, all attempts to obtain infectious
nucleic acid by treatment of crude or partially purified and concentrated
virus preparations with cold or hot phenol were unsuccesful. The extracts
showed no infectivity when tested in CEO tube cultures or monolayers using
various isotonic and hypertonic NaCl and MgSO4 solutions as diluents.
5.8
Immunogenicity of HNO2-inactivated virus
Repeated intravenous injections of HNO2-inactivated virus to rabbits in
1-2 ml. amounts led regularly to the development of virus neutralizing
(VN) antibodies. The animals received 3-4 doses of inactivated virus at various
intervals (see Fig. 8). At intervals, samples of blood were collected and the
level of VN antibodies determined. The results of two representative immunis-
ation experiments are presented in Fig. 8. The level of antibodies increased
relatively slowly, reaching a maximum (VN titres 32-128) several weeks
or even months after the last injection. In 2 out of 6 immunized rabbits the
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IVANIOOVA, 8. et al.
antibodies persisted in relatively high levels for more than 3 months aft there
last injection of inactivated virus, while in remaining animals antibodies.
either disappeared within 2 months or their level decreased to a minimum.
7
6
5
4
3
2
0 20 40
60 80 100 120 160 ' 180 200 220
Fig. 8.
Immunisation of two rabbits with HNO, inactivated Aujeszky disease virus
abscissa: time in days; ordinate: VN titre (log, scale);
0 on the ordinate means no protection against the cytopathic effect of the virus; arrows indicate-
the time of injection of inactivated virus; * = intramuscular challenge with homologous live
virus; t = death of the animal.
Animals vaccinated with the inactivated virus were protected against intra-
muscular challenge with 300 TCID50 of homologous virus as long as antibodies
were preserved (se eFig. 8). All nonimmune control animals infected with
similar or even much lower doses of virus succumbed under typical symptoms
of the disease.
Even repeated subcutaneous administration of the inactivated virus did
not led to antibody formation in 4 rabbits. The fifth animal exhibited an
extremely low level of VN antibodies after the third and fourth injections_
Discussion
In general, the kinetics of inactivation of Aujeszky disease virus by HNO2
deviated from the strictly exponential inactivation observed with other
viruses (Schuster and Schramm, 1958; Tessman, 1959, Schafer et al., 1959;
Bautz-Freese and Freese, 1961; Restle et al., 1962): The lines characterizing
the dependence of log (Io/I) on the time of inactivation were regularly shifted
upwards (positive intercept). This fact can be interpreted in two different
ways. Either an extremely rapid inactivation of an? appreciable proportion
of virus particles occurred immediately after exposure to IIN02, which was
then slowed down and proceeded further at a much lower rate as a first order
reaction, or the reaction was not stopped completely immediately after dilution
of the sample with phosphate buffer pH 7.85 and HNO, bound by or penetrated
into the virus could react for an additional period of time with viral protein
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INACTIVATION OF PSEUDORABIES VIRUS BY HNO,
13
and DNA. The former explanation seems to be more probable, since at relati-
vely low temperatures or low HNO, concentrations this initial inactivation
became somewhat slower and was manifested by a shoulder in the inactivation
curve. Similar survival curves were observed in inactivation of poliovirus
(Boeye, 1959), bacterial transforming DNA (Litman and Ephrussi-Taylor,
1959; Stuy, 1962) and especially of type 1 adenovirus (Wassermann, 1962),
indicating that at least two different components are involved in the inactiv-
ation by HNO2. The possibility that the initial shoulder observed in some in-
activation curves of Aujeszky disease virus was caused by the heterogeneity
of virus population in respect to its sensitivity' to HNO, must be rejected,
because plaque purified virus displayed also under appropriate conditions
a shoulder in the inactivation curve. It is highly probable that the action of
HNO, on the infectivity of Aujeszky disease virus involved, as suggested also
for other viruses (Boeye, 1959, 1962; Restle et al., 1962; Wassermann, 1962),
both the viral DNA and protein. The fact that infectious RNA can be liberated
from HNOrinactivated poliovirus (Boeye, 1962) indicates that the alteration
of the viral protein coat only may cause inactivation. It was also found that
intact poliovirus was inactivated about three times faster by IINO, than its
isolated infectious RNA (Boeye, 1959). Thus the initial rapid inactivation
observed in the reaction of IINO, with Aujeszky disease virus may correspond
mainly to the alteration of viral protein and the slower exponential inactivation
mainly to that of viral DNA. Aujeszky disease virus particles have a diameter
of 1500-1800 A (Reissig and Kaplan, 1962) and a 1: 25 ratio of protein to
DNA (Ben-Porat and Kaplan, 1962). To reach the viral DNA located inside
of the virus particle, HNO, must penetrate trough the broad coat consisting
of protein and possibly also of lipids and saccharides. Unfortunately we did
not succeed in isolating infectious DNA from this virus and thus we could not
compare the kinetics of inactivation by IINO, of intact virus and its DNA.
The velocity constants, the activation energy, the dependence of k on pH
and NaNO, concentration determined in this study are all related to the second,
exponential stage of inactivation. A linear dependence of the inactivation rate
on FP- ion concentration and its proportionality to NaNO2 concentration was
also found with T2 bacteriophage (Vielmetter and Wieder, 1959; Vielmetter
and Schuster, 1960).
Aujeszky disease virus inactivated by HNO, retained at least a part of its
original immunogenicity as manifested by the fact that rabbits immunized
with killed virus developed specific antibodies and showed resistance against
challenge with homologous live virus. Our samples of inactivated virus did not
contain live virus in 2 ml. of suspension concentrated 10 times by sedimentation,
because all animals survived the injection of the first dose of inactivated virus.
Rabbits namely cannot survive infection even with extremely low doses of
the virus strain used in this study. Repeated intravenous injections of the
inactivated virus were necessary to obtain a relatively high level of antibodies
in the blood and to ensure its persistence for a prolonged period of time. Further
experiments are necessary to evaluate the efficiency of HNO2-inactivated virus
in vaccination. Poliovirus inactivated by IINO, to a sufficiently safe level was
found to be of little if any value for immunisation. Its immunogenicity was
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14
ivAmaovii, 8. et al.
lost almost completely (Boeye, 1962; Restle et at., 1962). The latter finding
cannot be generalized, however, for all viruses, as e.g. the composition and
the structure of Aujeszky disease virus is quite different from that of poliovirus.
In plaque titrations of 11NO2-treated virus the occurrence of small-plaque-
type mutants was regularly observed. The biological properties of the mutants
and their sensitivity to IIN02 treatment will be described later.
Summary
The kinetics of inactivation of Aujeszky disease virus by nitrous acid was
investigated at various concentrations of sodium nitrite, pH of the reaction
mixture and temperatures. After an initial short, but very rapid inactivation
of an appreciable proportion of virus particles the reaction was slowed down
and proceeded further with an exponential rate. For the exponential portion
of the inactivation curves, the rate of inactivation was proportional to NaNO2
concentration and linearly dependent on the hydrogen ion concentration.
The activation energy of the reaction was 15.2 kcal/mole. Attempts to isolate
infectious deoxyribonucleic acid (DNA) from the virus by phenol extraction
were unsuccessful and thus the kinetics of inactivation of intact virus could
not be compared with that of isolated DNA. It was -concluded that the action
of nitrous acid on the infectivity of the virus involved both viral DNA and
protein. Virus inactivated by nitrous acid retained its immunogenicity.
Rabbits immunized with the killed virus developed specific virus neutralizing
antibodies and showed resistance to challenge with homologous live virus.
References
Bautz-Freese, E., and Freese, E. (1961): Induction of reverse mutations and cross reactivation
of nitrous acid-treated phage T4. Virology 13, 19.
Ben-Porat, T., and Kaplan, A. S. (1962): The chemical composition of herpes simplex and pseudo-
rabies viruses. Virology 16, 261.
Boeye, A. (1959): Induction of a mutation in poliovirus by nitrous acid. Virology 9, 691.
Boeye, A. (1962): Inactivation of protein in poliovirus by nitrous acid. Nature (Lond.) 193, 601.
Carp, R. I., and Koprowski, H. (1962): Mutation of type 3 poliovirus with nitrous acid. Virology
17, 99.
Granoff, A. (1961): Induction of Newcastle disease virus mutants with nitrous acid. Virology 13,
402.
Litman, R. M., and Ephrussi-Taylor, 11. (1959): Inactivation et mutation des facteurs genetiques
de l'acide desoxyribonucleique du .pneumocoque par Fultraviolet et par l'acide nitreux.
C. R. Acad. Sci. (Paris) 249, 838.
Mundry, K. W., and Gierer, A. (1958): Die Erzeugung von Mutationen des Tabakmosaikvirus
durch chemische Behandlung seiner Nucleinstiure in vitro. Z. Vererbungslehre 89, 614.
Reissig, M., and Kaplan, A. S. (1962): The morphology of noninfective pseudorabies virus pro-
duced by cells treated with 5-fluorouracil. Virology 16, 1.
Restle, H., Hennessen, W., and Schafer, W. (1962): Verhalten der antigenen und sonstigen 1?io-
logischen Eigenschaften menschen- und tierpathogener Virusarten beim Behandeln mit
salpetriger Saure 1. Mitt.: Verhalten des Poliomyelitis-Virus. Z. Naturforsch. 17b, 228.
Schafer, W., Zimmermann, T., and Schuster, H. (1959): Inaktivierung verschied,ener menschen-
und tierpathogener Virusarten sowie des Tabakmosaik-Virus durch salpetrige Saure. Z-
Naturforsch. 14b, 632.
Schuster. H., and Schramm, G. (1958): Bestimmung der biologisch wichtigen Einheit in der
Ribosenucleins5,ure des TMV auf chemischem Wege. Z. Naturforsch. 13b, 697.
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INACTIVATION OF PSEUDORABIES VIRUS BY HNO,
15
Stuy, J. H. (1962): Inactivation of transforming deoxyribonucleic acid by nitrous acid. Biochem.
biophys. Res. Commun. 6, 328.
Skoda, R., and Mayer, V. (1961): fiber die ZUchtung des Virus der Aujeszkyschen Krankheit
in einschichtigen Hiihnerembryo-Fibroblastenkulturen mittels der Plaquemethode. Arch.
exp. Vet.-Med. 15, 391.
Tessman, I. (1959): Mutagenesis in phages OX174 and T4 and properties of the genetic material.
Virology 9, 375.
Vielmetter, W., and Schuster, H. (1960): Die Basenspezifitat bei der Induktion von Mutationen
durch salpetrige Shure im Phagen T2 Z. Naturforsch. 15b, 304.
Vielmetter, W., and Wieder, C. M. (1959): Mutagene und inaktivierende Wirkung salpetriger
Shure auf freie Partikel des Phagen T2. Z. Naturforsch. 14b, 312.
Wassermann, F. E. (1962): The inactivation of adenoviruses by ultraviolet irradiation and nitrous
acid. Virology 17, 335.
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Acta virol. 7 : 16-24, 1963
Mutations of Virulent and Attenuated Poliovirus Strains Induced
by Nitrous Acid
YU. Z. GHENDON
The Moscow Scientific Research Institute of Viral Preparations, Moscow, U.S.S.R.
Received September 4, 1962
Recent investigations have shown that mutants exhibiting new biological
properties can be obtained by direct treatment of viral nucleic acid with muta-
genic agents capable to alter the arrangement of nucleotides (Gierer and
Mundry, 1958; Schuster and Schramm, 1958; Siegel, 1960; Fraenkel-Conrat
and Tsugita, 1961).
In the present study mutations occurring in virulent and attenuated polio-
virus strains under the action of nitrous acid (IIN02), capable to cause des-
amination of purine (adenine, guanine) and pyrimidine (cytosine) bases of
viral ribonucleic acid (RNA) (Schuster and Schramm, 1958; Schuster et at.,
1960), were investigated.
Materials and Methods
Viruses. The virulent type 1, Mahoney, and type 2, MEF,, poliovirus strains and Sabin's
type 1, LSc 2ab, and type 2, P-712 Ch 2ab attenuated vaccine strains were used. Genetically
homogenous lines of these viruses, isolated from plaques formed after infection with viral RNA,
were used throughout. By this method virus lines with a high degree of homogeneity of the genetic
markers can be obtained (Ghendon et al., 1961a).
Tissue culture. Primary cultures of Macaca rhesus monkey kidney cells, obtained by dispersion
of the tissue by trypsin, were used.
Isolation of viral RNA. The phenol extraction method of Gierer and Schramm (1956) was used.
Treatment with HNO2. Both intact virus and isolated viral RNA were treated with HNO2.
Two volumes of the preparation examined were mixed with one volume of acetate buffer pH 3.3,
4.2, 4.7 or 5.2, and one volume of 4M NaNO2. The reaction was allowed to proceed in rubber
stoppered tubes at room temperature. The tubes were not agitated during the reaction. Separate
tubes were used for each time interval. The reaction was stopped by diluting the mixture 1 : 10
with 0.02 M phosphate buffered 1.2M saline pH 7.8, after which 0.2 ml. portions of the material
examined were inoculated into monkey kidney cell monolayers washed twice with phosphate
buffer. Viral RNA and intact virus were allowed to adsorb at 37? C for 30 or 60 minutes, respecti-
vely, and the monolayers were then overlaid with agar medium prepared as described by Hsiung
and Melnick (1957). In several experiments bottle cultures of monkey kidney cells in medium.
199 were inoculated with 1.0 ml of a 1 : 10 dilution of the sample examined.
Isolation of mutants. Mutations of virulent poliovirus strains were investigated in cultures
overlaid with agar medium and incubated at 36? C. Mutations of attenuated poliovirus strains
were studied in two types of culture: (1) infected bottle cultures in liquid medium 199 were in-
cubated in parallel at 36? and 40? C and examined for 7 days; cultures showing a characteristic
cytopathic effect were investigated further for the genetic markers of the virus; and (2) cultures
overlaid with agar were incubated at 36? C, while parallel bottles were incubated first for 30 hours
at 40? C and then at 36? C. This procedure facilitated the isolation of mutants with ST + genetis
markers. After plaques had developed in cultures infected with virulent or attenuated strains,
their size was determined and from all isolated plaques virus clones were subcultured by a method
described previously (Ghendon et al., 1961a) The genetic markers of the virus clones obtained
were then investigated.
Genetic markers. The folowing markers were studied: N ? neurovirulence for monkeys after
intracerebral inoculation; M ? the character of morphological changes in the central nervous
system of infected monkeys; mN ? neurovirulence for mice after intracerebral inoculation;
16
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POLIOVIRUS MUTANTS INDUCED BY NITROUS ACID
17
T (rot") ? ability to multiply at 40? C; d ? ability to multiply at low sodium bicarbonate
concentration; S ? plaque size. The methods used in studying these markers were described
previously (Ghendon et al., 1961b).
Experiments on attenuated and virulent strains were carried out in separate rooms isolated
from one another.
The data given in the figures and tables represent means from 3-4 experiments done at
different times.
Results
First, the kinetics of inactivation by 11NO2 of intact virulent or attenuated
polioviruses and of infectious RNA isolated from them were investigated.
The results obtained showed that the rate of inactivation increased with
decreasing the pH of the reaction mixture. At any pH the intact virus was
inactivated more rapidly than the corresponding infectious RNA. There was
no difference between attenuated and virulent polioviruses in the rate of
their inactivation by 11NO2, irrespective of whether intact virus or infectious
RNA were subjected to 11NO2 treatment. In control experiments the prepara-
tions examined were exposed to acetate buffers without the addition of NaNO2.
Neither intact virus nor infectious RNA were inactivated under these conditions
within the reaction time used (2-5 minutes at pH 3.3 or 4.2; 20 minutes at
pH 4.7; and 60 minutes at pH 5.2).
As next, we studied the appearance of mutants in virulent type 1 poliovirus,
strain Mahoney, following treatment of intact virus or infectious RNA with
HNO2. The alteration of the T(rct40) genetic marker served as the basic criterion
of mutation in these experiments. (Virulent poliovirus strains with a T+ genetic
marker multiply equally well at 36? and 40? C, while attenuated strains with
a T- genetic marker do not multiply at 40? C.)
Data presented in Fig. 1 show that the number of mutants increased with
prolonging the time of treatment and, as the mutation rate is closely connected
with the rate of deamination of nucleotides, with lowering the pH of the re-
action mixture. No T- mutants were observed in control preparations of
18
16
14
12
10
8
6
4
pH 4.2 ? pH 4.7 pH 53
?X
Ax
??XX
X
?X x
-x{
. 2 x
8 12 16 20 30 40 50 60
Fig. 1.
Induction of T- mutants by treatment of the Mahoney virulent poliovirus strain with HNO2
in dependence on the reaction time and pH of the medium
0 - - -? isolated viral RNA; X x intact virus
Abscissa: time of treatment in minutes; ordinate: % of mutants among survivors.
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GHENDON, YU. Z.
ntact virus or infectious RNA exposed only to acetate buffer pH 4.2, 4.7 or
5.3 for 4, 20 and 60 minutes, respectively.
Further experiments were devoted to mutations of attenuated type 1
Fig. 2;
mutants induced by treatment of the attenuated LSc 2ab strain of type 1 poliovirus with HNO2
Sor,e the csingle large 'S+ plaques on the background of small S- plaques.
10 -
9-
8 -
7 -
6-
?lc) 5-
. 4 -
3 -
2 -
1 -
7
x X
?/
*.x x
'?
:XXX
?X
p1-14.2'
XX
??
?
"XX
?XX
II
II 109 _
8 -
7 - .
6 -
?
oh, 5-
/ x
I.
/ x
poliovirus strain LSc 2ab, induced
by treatment with IIN02. First,
monkey kidney cell cultures were
inoculated with nitrous acid-treated
virus and, after adding liquid nutri-
ent medium, incubated at 40? C. In
this way optimal conditions for the
growth of T+ mutants were ensured,
if such mutants were formed follow-
ing treatment with HNO2. Results
of these experiments showed that
treatment of attenuated poliovirus
for only a few minutes was suffi-
cient to induce mutants capable to
multiply at 40? C (T+ mutants).
.x
,?Zx
"x-P-
. x
Fin. 3.
?s:xx
?xx
x x
3
2 3 14
Frequency of S+ and T+ mutants among
surviving virus following treatment of at-
tenuated poliovirus with HNC),
I ? isolated viral RNA; II ? intact virus!
? ? S+ mutants; x x T+ 'mu-
tants. Abscissae and ordinates as in Fig. 1.
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POLIOVIRUS MUTANTS INDUCED BY NITROUS ACID
19
Then a quantitative study of the formation of S+ and T mutuants follow-
ing treatment of attenuated poliovirus with HNO, was carried out. Tissue cul-
tures were inoculated with nitrous acid-treated preparations of intact virus or
infectious RNA, overlaid with agar medium and incubated first for 30 hours
at 40? C and then at 36? C. These conditions favoured the multiplication of S+
clones, which could be clearly differentiated from S- clones (see Fig. 2).
It is evident from the results summarized in Fig. 3 that treatment of attenuated
virus with IINO, led regularly to the appearance of S+ and T+ mutants.
As with virulent poliovirus strains, the number of these mutants increased by
prolonging the time of treatment or by lowering the pH of the reaction mixture.
The genetic markers of several mutants obtained were investigated in
detail in special experiments. The properties of the mutants showing various
combinations of genetic markers are shown in Table 1. In addition to changes
of the S and T genetic markers also other properties changed of both virulent
and attenuated strains. With several mutants there occurred a separation of
Table 1. Genetic markers- Of poliovirus mutants induced by nitrous acid
Virus
strain
Mutant
Genetic markers
N 1M
T d J
S
mN
Mahoney,
type 1
,
Original strain
M/4.2/3
M/4.7/4
M/4.2/6
M/4.2/2
?
?
?
?
?
_t_
?
?
?
+.
?
?
?
?
?
?
?
+
?
MEFI,
type 2
Original strain
MF/4.2/1 -
MF/4.2/9
ME/4.7/6
MF/4.2/2
MF/4.2/5
MF/4.2/3
+
?
?
?
?
?
+
?
?
?
?
_
?
?
?
?
?
?
+
?
?
?
?
_
?
?
?
?
?
?
?
?
?
?
?
LSc 2ab,
type 1
Original strain
L/3.3/2
L/3.2/5
L/3.3/3
?
?
?
?
?
?
?
?
+
?
?
?
?
?
+ and ? = genetic markers characteristic of virulent and attenuated strains, respectively
very closely related genetic markers, as of the virulence for mice and monkeys
(mutants MF/4.7/6 and MF/4.2/2), or of the T, d, and S genetic markers
(mutants M/4.2/3, MF/4.2/1 and L/3.3/2). Other mutants exhibited changes
in all the genetic markers -examined. Mutants with genetic characters of
attenuated strains, including -apathogenicity for mon-keys (mutants M/4.2/2
and MF/4.2/3),. were obtained from virulent poliovirus strains, and a mutant
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Table 2. The stability of genetic markers of poliovirus mutants In the course of passaging In tissue cultures
Mutant
Changes of genetic markers in the course of passaging
1st passage
2nd passage
3rd passage
4th passage
M/4.2/1
M/4.2/2
? M/4.2/4 ,
M/4.718
M/4.7/12
M/5.2/40
MI5.2/50
NMTdS
NMTdS
N-1VI?T?d?S?
N?M?T?d?S?
N?M?T?d?S?
NMTdS
NMTdS
N M T?d S
N+M+T+d+S+
N?M?T?d?S?
N?M?T?d?S?
N?M?T?d?S?
N?M?T?d?S?
NMTdS
N+M+T+d+S+
NMTdS
NMTDS
N?M?T ?d?S?
N?M?T?d?S?
N?M?T?d?S---
N M T cl S
N+111+T+d+S+
NMTdS
NMTdS
NMTdS
MEF1
N+M+
T+d+
S+mN+
MF/4.2/3 N?M?T?d?S?mN?
MF/4.2/9 N+M+T?d?S?mN+
MF/4.2/5 N?M?T+d+S+mN?
N?M?T?d?S?mN
N+M+T?d?S?mN+
N?M?T+d+S - mN?
NMTcl SmN
N+M+T?d?S?mN+
N?M?T+d+mN?
NMTdSmN
N+M+T?d?S?mN+
N?M?T+d+S+mN?
L/4.2/1
L/4.2/2
L/4.2/31
L/4.2/32
L/4.2/61
L/4.2/62
L/4.7/20
T?d?S?
T?d?S?
T?d?S?
T?d?S?
T?d?S?
T?d?S?
T?d?S?
T?d?S?
T?d?S?
'T?d?S?
T?d?S?
T?? d?s?
T?d?S?
T?d?S?
T?d?S?
T?d--s?
T?d?s?
T?d?s?
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POLIOVIRUS MUTANTS INDUCED BY NITROUS ACID
21
with characters of a virulent virus (L/3.3/3) was obtained from the attenuated
strain.
The stability of the newly acquired genetic markers of several mutants
during passaging in tissue cultures was also investigated. The results obtained
(Table 2) showed that the genetic markers of the prevailing proportion of the
mutants examined remained completely stable on passaging in tissue culture.
The results obtained by Mundry (1959), Boeye (1959), Vielmetter and Wieder
(1959) and Siegel (1960) ruled out the suggestion by Bawden (1959) that
treatment of viruses with HNO2 does not induce mutations, but leads to
selection of particles less sensitive to the inactivating action of 11NO2. Never-
Table 3. Isolation of virus clones from mixtures of virulent and attenuated poliovirus strains
treated with nitrous acid
Mixture
Treatment
Number
of clones
examined
Number of clones
with genetic
markers
Significance
T+ S+
T.?S-
97% Mahoney
untreated
252
244
8(3.2%)
, i - 0.024
+ 3% LSc 2ab,
intact virus
pH 4.2, 2 mins.
/
174 ?
168
6(3.5%)
P> 1.0
97% Mahoney
untreated
116
112
4(3.5%)
x2 ..-- 0.12
+ 3% LSc 2ab
RNA
pH 4.2, 4 mins.
112
109
3(2.7%)
P> 1.0
97% LSc 2ab
untreated
133
4(3.0%)
129
x2 --- 0.018
+ 3% Mahoney
intact virus
pH 4.2, 2 mins.
108
3(2.8%)
105
P> 1.0
97% LSc gab
untreated
121
4(3.3%)
117
x2 ,--- 0.067
+ 3% Mahoney
RNA
pH 4.2, 4 mins.
127
5(4.1%)
122
P> 1.0
theless we carried out experiments to prove once more that the appearance
of virus clones with unusual genetic markers after treatment of poliovirus
with 11NO2 is a result of mutation and not of selection. Known amounts of
virulent and attenuated polioviruses were mixed and treated with HNO2,
and the S and T markers of the viruses before and after treatment were de-
termined. The data presented in Table 3 show that the ratio of virulent to
attenuated viruses did not change after treatment of the mixture with 11NO2,
as the differences observed were not statistically significant. The results of
this experiment, together with the aforementioned data on the same rate of
inactivation by 11NO2 of both virulent and attenuated poliovirus strains,
confirmed the finding by other investigators that the appearance of virus
clones with altered genetic markers in the viral population surviving after
11NO2 treatment is a result of mutation and not of selection resulting from
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GHENDON, YU. Z.
a different sensitivity to HNO2 of virus particles showing different genetic
markers.
Discussion
In the present experiments mainly the virulent strain Mahoney and the
attenuated strain LSc 2ab of type 1 poliovirus were used in studying mutations
induced by the action of IIN02. The virus strains used were found previously
to be genetically highly homogeneous (Ghendon et al., 1961b; Ghendon and
Diskina, 1962). The present investigations showed that treatment with HNO2
of both virulent and attenuated poliovirus strains led to the appearance of
mutants. Bautz-Freese and Freese (1961) reported that reversion of genetic
markers may occur on treatment of some mutants with HNO2. However,
as shown by Wittmann (1961), such reversion can take place only with mutants
obtained by the action of mutagens other than HNO2. In agreement with the
latter finding we did not observe reversion of genetic markers following treat-
ment with HNO2 of mutants induced by HNO2 and possessing properties
of attenuated strains (Ghendon, unpublished). On the other hand, treatment
with HNO2 regularly induced mutations of attenuated strains selected by
Sabin. These findings suggest that changes of biological properties of poliovirus
may be related with different mechanisms affecting the nucleotide composition
of the viral nucleic acid.
Studies on Newcastle disease virus (Granoff, 1961) and bacteriophages
(Freese, 1959; Vielmetter and Schuster, 1960) showed that the frequency of
nitrous acid-induced mutants increased with lowering the pH of the reaction
mixture. Vielmetter and Schuster (1960) found with T2 bacteriophage that
increasing the pH of the medium up to 5.0 decreased more markedly the rate
of mutation than that of inactivation by HNO2. The results of our experiments
on poliovirus confirmed that the frequency of mutants induced by 11NO2
increased with lowering the pH of the reaction mixture. However, the pH
dependence of the inactivation rate was the same as that of mutation rate,
i.e. both processes had a parallel course at any pH examined. This fact sug-
gested that the ratios of the deamination rates of the nucleotides in poliovirus
RNA, in contrast to the phage DNA (Vielmetter and Schuster, 1960), remained
constant and were independent of the pH at which RNA was treated with
HNO2.
Our experiments showed that, if using HNO2 as mutagen, mutants can be
obtained on treatment of both infectious RNA and intact virus. But the pro-
portion of mutants among survivors was much higher with isolated viral RNA
than with intact virus. This was apparently caused by the fact that, in addition
to deamination of bases of the viral RNA, also alterations of the protein coat
may lead to the inactivation of intact virus. Thus in experiments aimed at
obtaining mutants it is more advantageous to treat directly the carrier of
genetic informations, the naked viral RNA, than the corresponding intact
virus. When exposing isolated RNA to the action of HNO2, inactivation
due to deamination of the viral protein coat is avoided and optimal conditions
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POLIOVIRUS MUTANTS INDUCED BY NITROUS ACID
23
for the induction of mutations can be achieved, offering the possibility of
obtaining great numbers of mutants.
Summary
1. The rate of poliovirus inactivation by nitrous acid increased with lowering
the pH of the reaction mixture. Intact viruses were inactivated at a higher
rate than the corresponding infectious viral ribonucleic acids (RNA). There
was no difference between virulent and attenuated poliovirus strains in the
rate of their inactivation by nitrous acid.
? 2. Treatment with nitrous acid of infectious RNA derived from virulent
poliovirus strains led regularly to the appearance of T- mutants among surviving
virus. The number of mutants increased with prolonging the time of treatment
or with lowering the pH of the reaction mixture. The ratio of the inactivation
rate to the rate of mutation was independent of the pH at which the reaction
proceeded.
3. A part of the mutants obtained by treatment of attenuated poliovirus
strains with nitrous acid possessed all genetic markers typical of virulent
strains. As with virulent strains, the frequency of mutation increased with
prolonging the reaction time or with lowering the pH of the medium.
4. When treating attenuated or virulent intact viruses instead of isolated
viral RNA with HNO2, mutants were also obtained, but the ratio of the rate
of mutation to the inactivation rate was considerably less than in experiments
on viral RNA.
5. Mutants obtained after treatment of virulent and attenuated poliovirus
strains with nitrous acid exhibited various combinations of genetic markers.
Mutants obtained after treatment of virulent strains and possessing all the
genetic markers characteristic of attenuated strains could be of practical
importance.
References
Bawden, F. (1959): Effect of nitrous acid on tobacco mosaic virus: mutation or selection? Nature
(Lond.) 184, 27-29.
Bautz-Freese, E., and Freese, E. (1961): Induction of reverse mutations and cross reactivation
of nitrous acid-treated phage T 4. Virology 13, 19-30.
Boeye, A. (1959): Induction of a mutation in poliovirus by nitrous acid. Virology 9, 691-700.
Fraenkel-Conrat, H., and Tsugita, A. (1961): Effect of chemical alteration of RNA of tobacco
mosaic virus on the structure of protein and biological properties. 5th Int. Congr. Biochem.,
Moscow, 1961, Symposium 3, Evolutionary biochemistry 3, 19-20.
Freese, E. (1959): On the molecular explanation of spontaneous and induced mutations. Brook-
haven Symposia in Biology 12, 63-75.
Ghendon, Yu. Z., and Diskina, B. S. (1962): Vaccine strains of poliovirus obtained by different
physical treatments of ribonucleic acid isolated from virulent strains. Acta virol. 6, 289-296
Ghend on, Yu. Z., Diskina, B. S., and Marchenko, A. T. (1961a): Infection of tissue cultures with
viral RNA as a method for isolation of virus clones with stable genetic markers. Vop. Virusol. 6,
651-656 (in Russian).
Ghendon, Yu. Z., Khesin, Ya. E., and Marchenko, A. T. (1961b): Studies on the stability of genetic
markers of Sabin's attenuated poliovirus strains. In: Oral live vaccine against poliomyelitis,
461-483 (in Russian).
Gierer, A., and Mundry, K. (1958): Production of mutants of tobacco mosaic virus by chemical
alteration of its ribonucleic acid in vitro. Nature (Lone!.) 182, 1457-1458.
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24
GHENDON, YU. Z.
Gierer, A., and Schramm, G. (1956): Infectivity of ribonucleic acid from tobacco mosaic virus.
Nature (Lond.) 177, 702-703.
Granoff, A. (1961): Induction of Newcastle disease virus mutants with nitrous acid. Virology 13,
402-408.
Hsiung, G., and Melnick, J. (1957): Morphologic characteristics of plaques produced on monkey
kidney monolayer cultures by enteric viruses (poliomyelitis, coxsackie and ECHO groups).
J. Immunol. 78, 128-136.
Mundry, K. (1959): The effect nitrous acid on tobacco mosaic virus: mutation, not selection.
Virology 9, 722-726.
Schuster, G., Gierer, A., and Mundry, K. (1960): Inaktivierende und mutagene Wirkung der
chemischen Veranderung von Nucleotiden in Virus Nucleinsaure. Abhandl. Dtsch. Ak. Wigs.
Berlin. Kl. Med. 1, 76-85.
Schuster, H., and Schramm, G. (1958): Bestimmung der biologisch wirksamen Einheit in der Ribose-
nucleinsaure des Tabakmosaikvirus aur chemischem Wege. Z. Naturforsch. 13b, 697-704.
Siegel, A. (1960): Studies on the induction of tobacco mosaic virus mutants with nitrous acid.
Virology 11, 156-167.
Vielmetter, W., and Schuster, H. (1960): The base specificity of mutation induced by nitrous
acid in phage T2. Bioch. Bioph. Res. Corn. 2, 324-328.
Vielmetter, W., and Wieder, C. (1959): Mutagene und inaktivierende Wirkung Salpetriger Siture
auf freie Partikel des Phagen T2. Z. Naturforsch. 14b, 312.
Wittmaim, H. G. (1961): Studies on the nucleic acid-protein correlation in tobacco mosaic virus.
6th Int. Congr. Biochem., Moscow 1961, Symposium 1, Biological structure and fractions
at the molecular level 6, 39-44.
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Acta virol. 7 : 25-36, 1963
Cofactor and Specifle Antibodies against Influenza Viruses
VII. The Nature of Cofactor and Influenza Antibodies Studied
by Density Gradient Zonal Centrifugation
B. STYK, L. HA-NA, F. FRANEK*, F. SOKOL, J. MENSTK**
Institute of Virology, Czechoslovak Academy of Sciences, Bratislava; *Institute of Microbiology,
Czechoslovak Academy of Sciences, Prague; and **Research Institute of Veterinary Medicine,
Brno
Received July 10, 1962
Based on pilot immunoelectrophoretic investigations we suggested that
cofactor ? a component of normal animal sera potentiating the effect of in
antibodies ? is of macroglobulin nature (Hama et al., 1961). This
assumption has been supported by the results of rivanol precipitation of mouse
sera (Styk et al., 1962b).
But we also showed (Styk et at., 1962b) that cofactor occurs in sera from
newborn pigs which had not yet been suckled. As pig placenta is impermeable
for serum proteins originating from the mother (Brambell et al., 1951; Bram -
bell, 1958), serum from newborn unsuckled pigglets contains only proteins
synthetized by the newborn organism. It was found (Sterzl et at., 1960; Franek
et al., 1961) that sera from newborn pigglets do not contain either 19S gamma-
globulin (beta2m-globulin) or 7S gamma-globulin. Such sera contain in the
gamma-globulin region only components with a sedimentation coefficient
of 3-6S (Fran& et al., 1961) showing no antibody activity even against
antigens to which adult pigs regularly possess the so-called natural antibodies.
This fact has been confirmed by the extremely sensitive bactericidal test
(Sterzl et al., 1962). Thus the question arose as to the validity of our assumption
concerning the macroglobulin character of cofactor. Two explanations of the
discrepancy mentioned appeared possible: either sera from newborn pigs do
contain macroglobulins or the nature of cofactor from such sera differs from
the nature of cofactor from other animal sera.
? In order to elucidate this question as well as the nature of cofactor from
different animal sera we used density gradient zonal centrifugation, which
method has been suggested for differentiating serum proteins of a different
molecular weight (Edelman et al., 1958). In a part of the experiments this
method was supplemented by our modification of rivanol precipitation of
serum (liana and Styk, 1962a) which makes possible a partial separation of
macroglobulins from other serum proteins.
? These two methods were also used in investigating another problem. In
a previous paper we reported about the different character of influenza anti-
bodies from early immune and hyperimmune sera. The principal criterion for
their differentiation ? in addition to different Sensitivities to inhibitor-
destroying substances and to heating ? was their different ability to be po-
tentiated by cofactor (Styk, 1962). We are reporting the results of experiments,
in which we studied by density gradient zonal centrifugation (Edelman et al.,
1958) whether the two kinds of antibody differ in their molecular weihgt.
25
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26
STYK, B. et al.
Antibodies of different molecular weights are namely known to occur in man,
rabbit, horse and pig (Kuhns, 1955; Stelos and Talmage, 1957; Kabat,
1943; Bauer and Stavitski, 1961; Fran& et al., 1962; and others). In mice,
different molecular weight antibodies have not yet been reported.
A brief preliminary account has been published (Styk et al., 1962a).
Materials and Methods
Normal animal sera were kept frozen until used. Sera from white mice were pooled, those
from other animals were examined individually. In the case of pigglets occasionally serum pools
from two animals of the same litter were used. For details on the sera from newborn pigs see
Styk et al. (1962b).
Early immune and hyperimmune sera were prepared in white mice as described previously
(Styk, 1962). As antigens for immunization and in serological tests We used influenza virus
strains A2/Bratislava/4/57 and B-Lee, both in the form of infective allantoic fluids. (In a part
of the experiments viruses partially purified by adsorption onto and elution from formolized
erythrocytes were used.)
Saccharose gradient zonal centrifugation. The method of Edelman et al. (1958) as modified by
fliha (1963) was used. A discontinuous saccharose gradient in 0.15 M NaC1 was prepared. in
lusteroid tubes of the 40.2 rotor of model L Spinco ultracentrifuge. The saccharose concentration
was 40 and 10% at the bottom and top, respectively. The sera examined were mixed with an
equal volume of 0.15 M NaCl solution and layered on the top of the gradient. After centrifugation
at 32 000 rev/min. for 16 hours at 10? C the contents of the tubes were consecutively withdrawn
by means of a hypodermic needle and syringe in six equal portions, starting from. the top. The,
individual fractions were numbered 1?VI from the top to the bottom. The cofactor contents
of, the fractions were determined after removing saccharose by dialysis. In a part of the experi-
ments also the protein content of the fractions was estimated. Under these conditions 7S gamma-
globulins were located in fractions II and III, macroglobulins in fractions IV and V, occasion-
ally VI.
Rivanol precipitation was done as described by liana and Styk (1962a). A. 0.06% rivanol
concentration was used which, according to our experiences, leads to the most selective precipi-
tation of cofactor from mouse serum.
Cofactor titration was carried out by the method of Styk (1961) using detection sera anti-A2
(with non-avid influenza virus A2/Bratislava/4/57) or anti-B (with influenza virus B-Lee). The
distribution of cofactor activity determined by these two sera was the same (with the exception
of guinea pig serum in Table 1), though the absolute titre values determined using the anti-B
serum were regularly lower. If not stated otherwise, the results given in the Tables are those
obtained in titrations using anti-A2 detection serum.
Results
I. The nature of cofactor in different- normal animal l sera as revealed by
saccharose density gradient zonal centrifugation,
Table 1 presents the results concerning the distribution of cofactor activity
in different normal animal sera after centrifugation in saccharose gradients.
In mouse, bovine and rat sera maximal cofactor activity occurred in fractions
IV and V. With two guinea pig sera the cofactor activity was found in several
fractions when anti-A2 detection serum was used; when using anti-B dectection
serum, the distribution of cofactor activity was narrower, but the titres were
lower. With rat serum, the level of cofactor in fraction III could not be exactly
determined because of haemolysins occurring in this fraction.
Sera from adult pigs (mothers) also showed maximal cofactor activities in
fractions IV and V. A different behaviour was shown by cofactor in sera from
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27
newborn unsuckled pigglets (Table 1). In all these sera examined maximal
cofactor activity occurred in fractions I and II, which suggested that the
molecular weight of cofactor in such sera was lower than in sera from adult
Table 1. Distribution of cofactor activity in different normal animal sera after saceharose gradient
centrifugation
' Fraction
Kind of serum
Mouse (Pobra
Voda breed)
Mouse (Dean
breed)
Mouse (3 weeks
old mice)
a)
Guinea
pig
Pig (mother
No.1)
Pigglet of mo-
ther No. 1, befo-
re 1st feeding
Pig (mother
No. 2)
Pigglet of mo-
ther No. 4, be-
fore 1st feeding
0
?--,
? DS
DS
1
A2
B
4
rg
I
0
0
0
0
0
0
0
0
4
0
+2
II
0
0
0
?2
0
?4 ?
0
1
16
0
4
III
0
0
0
+2
4
?4
0
2
4
0
+1
(Haem)
IV
1
2
4
. 8
132
4
2
8
2
+4
?2
V
?8
4
4
4
?16
+4
1
4
1
+2
0
VI
1
+1
1
2
8
1
0
1
0
0
0.
Whole
serum
?80
40
40
160
+320
+160
20
+160
80
+80
20
0 means < 1
pigs or other animals. Sedimentation analysis of fractions I and II obtained
after centrifugation of sera from newborn unsuckled pigglets revealed a single
component, the s20, w of which varied in the individual experiments from 3.01 S
to 4.03 S. The protein content of these fractions varied from 2.4-6.0 mg./ml.
After taking food (maternal milk) the distribution of cofactor activity after
centrifugation in the density gradient changed (Table 2). In ten-days-old
Table 2. Distribution of cofactor activity in sera from young pigs and from their mother (on the day
- of parturition) after saccharose gradient centrifugation
Fraction
Newborn pig
(before 1st
feeding)
10 days'
pig
20 days'
pig
Mother
1
4
+1
0
0
II
8
2
1
1
III
?2
2
?1
?2
IV
?1
?16
8
+8
V
o
2
?-4
4
VI
0
o
+1
?1
Whole serum
?80
+160
?80
+160
0 means