or.gen. ViroL (I968), 3, 87-96
Printed in Great Britain
87
The Neutralization of Rous Sarcoma Virus
By R. C. C H U B B AND P. M. B I G G S
Houghton Poultry Research Station, Houghton, Huntingdonshire
(Accepted 5 February I968)
SUMMARY
The neutralization of Bryan standard strain Rous sarcoma virus (Bs-RSV)
followed first-order kinetics after I5 min. incubation. No non-neutralizable
fraction was observed when about Io 5 focus-forming units of representatives
of subgroup A and subgroup B viruses were neutralized by homologous
sera.
The antigenicity of a strain of Bs-RSV was changed by growth on relatively
resistant cells. The antigenicity of an A subgroup Bs-RSV 'pseudotype' was
changed by semi-cloning the virus. Both changes of antigenicitywere expressed
by a change of slope and a reduction of titre on cross-neutralization by
the parent virus antiserum.
Antisera to a number of subgroup A leukosis viruses were examined for
neutralization of the homologous Bs-RSV 'pseudotype' and other subgroup
A 'pseudotypes'. The slopes of the neutralization curves for the heterologous
systems differed from those of the homologous system. Neutralization indices
derived from single points on the neutralization curves, because of the slope
differences, did not show consistent cross-neutralization relationships of the
viruses tested. When the slope differences were taken into account by comparing areas under the curves, the homologous system had an index at
least 2o % greater than the heterologous systems.
INTRODUCTION
The avian leukosis/sarcoma group of viruses can be divided into two subgroups,
A and B, on the basis of one or more of the following properties determined by the
viral envelope: host range, interference and antigenicity detected by neutralization
tests (Vogt & Ishizaki, I966a, b). The viral envelope of defective sarcoma viruses is
provided by an associated leukosis virus. The antigenicity of these viruses as detected
by neutralization is also provided by the associated leukosis virus (Hanafusa, Hanafusa & Rubin, I963, I964; Vogt & Ishizaki, I966b). Cross-neutralization is only
seen between viruses belonging to the same subgroup; however, there is clearly
antigenic heterogeneity amongst viruses of a single subgroup (Ishizaki & Vogt, I966;
Churchill, I968).
Early studies at this laboratory had noted that the slope of the neutralization
curve differed for the same antiserum against different preparations of the same
strain of Rous sarcoma virus. This paper presents the results of a study of the
neutralization of Rous sarcoma virus to investigate the cause of this phenomenon.
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R. C. C H U B B
AND
P. M. B I G G S
METHODS
Virus strains. Bryan standard strain Rous sarcoma virus (Bs-RSV) was kindly
supplied by Dr R. M. Dougherty as preparation RSV3 (Dougherty & Simons, 1962).
Wing-web tumours induced by this preparation in commercial Light Sussex chicks were
processed by the Moloney (I956) technique to give the Bs-RSV-R2B/2 preparation.
Similarly, the Bs-RSV-R3/2 preparation was prepared from tumours induced by the
Bs-RSV-R2B[2 preparation in Houghton Poultry Research Station L I5I White
Leghorn chicks (HPRS L ISI WL) (Biggs & Payne, 1967).
The lymphoid leukosis viruses HPRS-F45 and HPRS-F42 were isolated from field
outbreaks of the disease (Biggs & Payne, I964). HPRS-I lymphoid leukosis virus
was isolated from a canine distemper vaccine (Payne et al. 1966) and HPRS-2
lymphoid leukosis virus was isolated from Bs-RSV-R3/2 by end-point dilution by
the method of Rubin & Vogt (I962). These viruses have been shown to produce
predominantly lymphoid leukosis, but also other mesodermal tumours when inoculated into chickens (Biggs & Payne, I964; Biggs, 1967, unpublished).
' Pseudotypes" of these leukosis viruses were prepared by activating non-producing
cells of Bs-RSV-R3/2 by the method of Hanafusa et aL (1964). The term 'pseudotype' does not imply any previous cloning of the leukosis viruses used for activation.
All the virus strains were shown to consist predominantly of subgroup A viruses
by the criteria of host range, interference inducing properties and neutralization.
Cells. Chick embryo fibroblasts of specific phenotype were derived from embryos
of the Reaseheath C, I and W inbred lines of chicken which have been shown to
provide C/A, C/O and C/B cells respectively (Payne & Biggs, 1966). The designation
C/A cells indicates phenotypic resistance to infection with A subgroup viruses, C/B
resistance to B subgroup viruses and C]O susceptibility to both subgroup viruses
(Vogt & Ishizaki, 1965).
All neutralizations were tested on C/O Houghton Poultry Research Station Brown
Leghorn (HPRS BrL) fibroblasts (Biggs & Payne, 1967).
Virus assay. Sarcoma viruses were assayed by the focus technique of Temin &
Rubin (I958) on chick embryo fibroblasts. Foci counts of whole plates were made
maeroseopically Io to 14 days after the cells had been challenged with virus, as
described by Dougherty & Simons (1962).
Neutralization. An appropriate dilution of the test virus was made with Puck's D I
phosphate buffered saline containing 2 ~/o calf serum, and 200 units penicillin and
20o #g. streptomycin/ml. Equal volumes of diluted virus and serial dilutions of
antisera were mixed and incubated at 3°0 for 2 hr (Dougherty, Stewart & Morgan,
I96O). As controls, equal volumes of diluted virus and diluent were mixed and
incubated for the same period. The final virus concentration was estimated to be
approximately 5oo focus-forming units (f.f.u.)/ml. The mixtures were then chilled
and o.2 ml. was inoculated on to each culture plate. At least two and usually three
plates were used per mixture. The mixture was allowed to adsorb for I hr at 37°
before removing excess fluid and overlaying with nutrient agar.
Kinetic and multiplicity curves. The rate of neutralization was measured by a
method similar to the normal neutralization procedure except that the volumes of
the neutralizing mixtures were sufficient for the withdrawal of several samples for
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Neutralization of Rous sarcoma virus
89
assay of surviving virus I5, 3o, 6o and I2o min. after the start of incubation. Control
virus was assayed immediately and at the stated time intervals.
The multiplicity of neutralization was also measured in a similar manner to the
normal neutralization procedure, except that higher concentrations of virus (Io 4 to
xo6 f.f.u./ml.) were used.
Antiserum. Antisera to sarcoma viruses were produced by inoculating 5 to zo f.f.u.
of virus into the wing webs of 6 week-old HPRS e 15 1 W L chickens. After tumour
regression the birds were rechallenged with Io 4 f.f.u, of virus and the serum collected
one week later. Antisera to leukosis viruses were induced by inoculation of IO5 tissue
culture infectious doses intravenously into adult H P R S L r 5 I W L cockerels. At least
one further dose at the same virus concentration was given intramuscularly I week
later. Sera were collected 4 weeks after the last virus inoculation. All sera were
inactivated at 56° for 30 min. before use. N o preinoculation sera neutralized the
viruses used in these experiments.
RESULTS
Neutralization
Thermal inactivation over the z hr period at 3°o was found to be negligible. The
results of two experiments were combined to construct kinetic curves of neutralization
of Bs-RSV-R3/2 (Dulbecco, Vogt & Strickland, I956) by several dilutions of an
homologous serum (Fig. I). Similar curves with an homologous serum were obtained
for Bs-RSV (HPRS-2). The curves show the normal pattern of an exponential rate of
neutralization which was greatest with the highest serum concentration. As the first
observation was at 15 min. (excluding adsorption time on the chick embryo fibroblast
monolayers) no observations were made as to the existence of a lag phase (Lafferty,
I963).
Table I. Rate constant values (k) at 30 min. incubation for Bs-RSV-R3/e
and an homologous serum (see Fig. 1)
Log. serum dilution
f
Expt i
Expt 2
where D = serum
t i m e t.
dilution,
3"0
3"3
3'5
3'6
123.6
113"5
-116-5
1o7"3
--
-l J3'2
k = l z ' 3 D l o g . (Co/Ct)]t
Co = i n i t i a l v i r u s c o n c e n t r a t i o n ,
Ct = virus survival at incubation
The rate constant values (k) of McBride 0959) calculated for different serum
dilutions at a fixed time of neutralization (30 min.) were similar within and between
experiments for each of the two virus preparations tested. The k values for the
kinetic curves of neutralization o f Bs-RSV-R3/2 are shown in Table I.
The neutralization curve obtained by plotting log. virus survival against log. serum
dilutions had the same slope at 6o min. as at I2o rain. incubation; however, the
former had a distinct flattening above 5° % virus survival which was not apparent
at I2o min.
The multiplicity curve (Dulbecco et al. I956 ) for two homologous sera of Bs-RSV
(HPRS-z) (Fig. 2) did not show any non-neutralizable virus. All the virus in the most
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90
R.C. CHUBB AND V. M. BIGGS
concentrated preparations available (about IoS f.Lu./o.2 ml.) was neutralized; the
serum concentration range being at least two dilution steps beyond where detectable
virus was found. Any non-neutralizable fraction must be below o.ooI % virus survival.
A similar multiplicity curve was obtained with a subgroup B virus (Schmidt-Ruppin
RSV) and its homologous serum.
1.0
100.0
10-0
F
I
L"
I
1
"
0.1
I
•
"~ 0.01
1.0
0,001
\
0.1
I
I
15 30
I
I
60
120
I--i
--3.0
I
I
--2.0
--1.0
Time (min.)
Log. serum dilution
Fig. I
Fig. 2
Fig. i. Kinetics of neutralization of Bs-RSV-R3/2 with a homologous serum. The adsorption
period of I hr was not included in the time base. The log. antiserum dilution is given on
the curves, t---i, Expt I ; O--©, Expt 2.
Fig. 2. Multiplicity curves of neutralization of Bs-RSV (HPRS-2) by two homologous
antisera.
Factors affecting the slope of the neutralization curve
The heterogeneity of the virus
Preliminary experiments indicated that sera gave different neutralization curves
with two different preparations of Bs-RSV (R2B/2 and R3/2). Sera prepared against
R2B/2 in HPRS L I 5 I W L birds neutralized the R3/2 preparation better than the
homologous preparation (Fig. 3). It was considered that this may have been caused
by the change of host to HPRS LI5 1 W L for the preparation of R3/2 and for the
induction of antisera to R2B/2. This principle could be tested by culturing the virus
on chick fibroblasts relatively resistant to the subgroup of sarcoma virus forming the
main component of the R3/2 preparation of Bs-RSV.
Reaseheath C line (C/A) chick embryo fibroblasts were challenged with 5o f.f.u, of
Bs-RSV-R3/2 and grown in liquid medium for Io days. The supernatant fluid was
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Neutralization o f Rous sarcoma virus
91
Table 2. Log. titres/ml, of Bs-RSV on genetically different cell lines
Cell type
C
(C/A)
I
(C/O)
W
(C/B)
2"32
3"55
6.2
< 2'0
< 2"0
6.6
8"z5
6"z
5"9
7"I
6-2
8-27
4"5
57
69
Virus
Bs-RSV-RzB2
Bs-RSV-R 3/2
Bs-RSV-R 3/2/x
Bs-RSV-(HPRS F42)/I
Bs-RSV-(HPRS F42)/Io
The phenotyped cells were from the highly inbred Reaseheath C, I and W lines of chickens.
I
100
I
I
I
100
I
0
O
"~ 10
O
I0 "i
"a---
1.0
--1.
1.0
--2.0
--3.0
Log. serum dilution
Fig. 3
--4.0
I
I
I
--1.0
--2.0
--3..0
--4.0
Log. serum dilution
Fig. 4
Fig. 3. Comparison of neutralization of Bs-RSV-R2B/2 ( I - - I ) , and Bs-RSV-R3/2 (CY--O)
virus preparations by Bs-RSV-R2B/2 homologous antiserum I I26/4. V o, initial focus count;
V, surviving focus count.
Fig. 4. Comparison of neutralization of Bs-RSV preparation R 3 / 2 (O----©), and the
preparation R3[2/I ( I - - I ) derived from the former virus preparation by growth on
resistant cells, using the same antiserum as in Fig. 3-
harvested and used to produce confluent lesions on the chorioallantoic membrane of
fully susceptible I i-day-old C/O HPRS BrL embryos. The chorioallantoic membranes
were harvested after 8 days and used to prepare a Moloney Tz preparation (Moloney,
1956). This virus Bs-RSV-R 3/2/I was compared with its parent virus by neutralization
with the same serum used to show the difference between the R2B/2 and R3/z
preparations of Bs-RSV (Fig. 4). As can be seen a markedly similar effect to the
former neutralization was observed. The R3/2/I preparation was poorly neutralized
and the neutralization curve had a fiat slope. However, the growth characterics of
the R3/2/I preparation on genetically phenotyped cells showed that it was different
from the other two strains (Table 2) and probably consisted predominantly of a B
subgroup virus.
A similar antigenic change to that seen with the Bs-RSV-R3/2 preparation grown
on C-line cells could be obtained by a cloning method. The 'pseudotype' Bs-RSV
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92
R . C . CHUBB AND P. M. BIGGS
( H P R S F42)/I was semi-cloned by picking an isolated focus f r o m an overlaid plate
o f fully susceptible cells with ten to twenty foci and plating the selected focus on
to susceptible cells to produce sufficient virus to repeat the procedure. After repeating
nine consecutive times, the tenth such clone (Bs-RSV ( H P R S r42)/Io) was grown up
and used to produce antisera in H P R S L I5 1 W L birds. The neutralization characteristics o f this serum are shown in Fig. 5. The neutralization o f the h o m o l o g o u s
Bs-RSV ( H P R S F42)/IO virus was o f high titre and g o o d slope. The neutralization
o f the parent virus Bs-RSV ( H P R S F42)/r was p o o r with fiat slope. Similarly, with the
reverse neutralization, the h o m o l o g o u s ( H P R S v42)/I virus preparation was neutralized
well with steep slope, whereas the heterologous virus preparation ( H P R S F42)/IO was
100
=~ 1o
I
I
-
•
100
I
i
")
10
•
II
m\m
o
1.0
\
I
--1.0
1.0 - 1
•
I
--2.0
--3-0
Log. serum dilution
--4.0
--2-0
•
m-J
I
--3.0
--4.
Log. serum dilution
Fig. 6
Fig. 5
Fig. 5. Comparison of neutralization of as-RSV (HPRS F42)/IO ( I - - I ) , and the parent
virus Bs-RSV (HPRS P42)/I (O--O) from which it was cloned using serum prepared
against Bs-RSV (HPRS F42)/io.
Fig. 6. Comparison of neutralization of Bs-RSV (HPRS r4z)/I (O--O) and (HPRS F42)/Io
(B--m) with homologous serum prepared against Bs-RSV (HPRS F42)/I.
Table 3. Neutralization indices calculated in various ways for
antiserum HPRS- I lymphoid leukosis virus ~333/7
Virus Bs-RSV
r
Neutralization index
Extrapolation to X axis
(log. serum dilution)
Extrapolation to Y axis
(log. virus neutralized
by undiluted serum)
Serum dilution to give
5o ~ virus survival
virus survival at Io-1
serum dilution
Area value (A~/B)
Normalized area ~
Slope of curve (B)
(HPRS-I)*
(HPRS-2)
(HPRS F42)/I
(HPRS F45)
6.0
3"9
4"0
5"0
2.6
2"4
2'7
I- l
5"4
3"4
3"6
3"7
< i.o
1.6
< i.o
1.2
I5'9
Ioo
--0"43
9"4
59"1
--o-62
1o.8
68.I
--0.68
* Homologous virus.
All values were calculated from the regression lines in Fig. 7.
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5"7
35"9
-0'23
Neutralization of Rous sarcoma virus
93
hardly neutralized at all (Fig. 6). This change of antigenicity, however, did not affect
the host range of the virus, its characteristics still being of the A subgroup (Table 2).
Cross-neutralization
In order to assess whether change of slope is the important factor for differences
o f antigenicity, the Rous sarcoma virus 'pseudotypes" of the leukosis viruses listed
in methods were compared by cross-neutralization. Neutralization with any one
antiserum against all the 'pseudotypes' was carried out at the same time. An example
of the neutralization curves for the various 'pseudotypes' against a single serum is
shown in Fig. 7. Unless the slopes of the neutralization curves are taken into account
an assessment of the degree of cross-neutralization is difficult. Some of the values
3.0
i
i
i
--1-0
--2"0
--3-0
w
i
i
--5"0
--6"(
2-0
1.0
--4.0
Log. serum dilution
Fig. 7. Neutralization curves o f anti H P R S - I lymphoid leukosis virus serum against its own
pseudotype and three other pseudotypes o f Bs-RSV. The points on the individual curves
were regressed to the straight-line equation Y = A + B X . Y = the log. decrease in virus
titre and X the log. reciprocal o f the antiserum dilution. ~ - - I ,
( H P R S - t ) ; O--C),
(HPRS-F42); A - - ~ , (HPRS-2); O - - O , (HPRS-F45).
normally given for neutralizing indices derived from the neutralization curves shown
in Fig. 7 are shown in Table 3. The use of the area under the curves after extrapolation to the axes takes the slope of the curves into account (Westaway, 1965).
These areas can be normalized (McBride, 1959) by calculating the heterologous areas
as a percentage of the homologous area (Table 4). Using the normalized area values
shows that the homologous systems have area values at least 20 % greater than the
heterologous systems. This difference may not be so apparent when neutralization
indices are derived from a single point on the dose response curve (Table 3).
DISCUSSION
Several viruses have a non-neutralizable fraction, e.g. Newcastle disease virus
(Granoff, I965), poliomyelitis, (Dulbecco et aL 1956). We were unable to demonstrate
a non-neutralizable fraction for representatives of subgroup A and subgroup B
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3"43 --0'83
N o neutralization
-0"55
-0'43
Slope
76
43'3
xoo
59'I
Normalized
area (700)
5"5
3'5
23"9
9"4
Area
(HPRS-2)
-- t'o
- I.I
- I"45
-0"62
Slope
xoo
74'2
37'5
68"1
Normalized
area ( ~ )
7'1
6'0
8"9
1o'8
Area
(HPRS r42)/I
--0"95
-o'84
-o-31
-o'68
Slope
50"0
xoo
6'8
35"9
Normalized
area (%)
3"5
8"I
1"65
5"7
Area
(HPRS F45)
--o"5I
-o'68
-o"31
-0'23
Slope
Areas were calculated from the regression line equation Y = A + B X as (A*/B)/2. The factor x/2 is common to all values and has been omitted from the
calculations (see Fig. 7).
Normalized area ~ is the heterologous area calculated as a percentage of the homologous area for each antiserum.
48"0
HPRS F42/I
HPRS F45
7"9
I5'8
ioo
33"2
HPRS-I
Area
HPRS-2
Antiserum
Normalized
area (9/oo)
(HPRS-t)
Virus Bs-RSV
T a b l e 4. Area values f o r 'pseudotype' cross-neutralizations
>
e0
(3
N e u t r a l i z a t i o n o f R o u s s a r c o m a virus
95
Rous sarcoma virus, and have shown that if present it forms less than o.ooI % of the
infectious virus.
The cross-neutralization results between Bs-RSV and 'pseudotypes' formed from
subgroup A field isolates of leukosis viruses show, in agreement with Ishizaki & Vogt
(I966) and Churchill (I968), that antigenic heterogeneity exists within this subgroup.
However, this antigenic heterogeneity expresses itself in cross-neutralization tests
not only by differences in titre, but also by differences in slope of the neutralization
curves. In this respect our results closely resemble those of Westaway (I965) with
arboviruses.
We have shown that the antigenicity of two strains of sarcoma virus (Bs-RSV and
8s-RSV (HPRS F42)/I) can be changed by subjecting them to selection pressures, i.e.
changing host susceptibility and cloning respectively. The change in antigenicity
was shown in cross-neutralization tests in which the slope of the neutralization curves
and titre of each serum for the homologous virus was different from those for the
heterologous virus. This suggests that these strains are mixtures of viruses which are
not antigenically identical. These findings suggest an explanation for the different
slopes of neutralization curves noted in the homologous systems, and the occasional
occurrence of steeper slopes in heterologous tests compared with the homologous
test. We suggest that virus mixtures are responsible for these results. The antigenicity
of a strain of virus will vary from stock to stock if there are changes in the proportions
of the components of the virus mixture or loss or addition of some components, and
changes in the degree of phenotypic mixing. Phenotypic mixing is a phenomenon
which is likely to occur with virus mixtures (Vogt, I967). High degrees of phenotypic
mixing occur only when relatively high multiplicities of infection are used, whereas
changes in the components of the virus mixture are more likely to occur at low multiplicities of infection and where selection pressures, such as changes in host susceptibility, are present. The same factors could explain the difference in neutralizing
properties of different sera for the same stock of virus.
It is clear from these studies that because of differences in slope of neutralization
curves, estimates of antigenic relationships between strains of virus based on crossneutralization tests are inadequate where titres are expressed in the conventional way
as a single point on the neutralization curve. We suggest that area values are more
satisfactory as a method of fully describing the neutralization properties of antisera
to viruses of the leukosis/sarcoma group.
We are grateful to Mr B. S. Milne, Miss M. Brand, Mr E. G. Carrington and
Mr P. Sewter who gave valuable technical assistance during the course of this work.
We are indebted to Dr H. G. Purchase for cloning Bs-RSV (HPRS F42)/I.
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