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ABSTRACT

GENETIC INFLUENCE IN ROUS

SARCOMA VIRUS INFECTION
by Richard H. Reamer

Genetic relationships of three Rous sarcoma virus
strains, Bryan standard (BS RSV), Harris (HA—RSV), and
Schmidt—Ruppin (SR-RSV) were studied by comparing the re—
sponse of individual backcross chicken embryos cell cul-
tures to the three viruses. Cell cultures were prepared
following modification of Rubin's technique (1960).

There were four patterns of response of the cells
to BS-RSV and HA-RSV: (l) resistance to both, (2) sensi—
tivity to both, (3) resistance to BS RSV only, and (4)
resistance to HA-RSV only.

Embryos of the original parent lines 6 and 7 re-
sponded differently. Line 6 was homozygous susceptible
while line 7, though uniformly resistant to BS-RSV, pro-

duced embryos some of which were susceptible to HAwRSV,

Richard H. Reamer

and BS-RSV appeared to be quite different in their host
range.

The Schmidt-Ruppin strain acted as a mixture of
viruses, one causing cellular response similar to that
by BS-RSV, the other similar to that of HA—RSV.

A cell phenotype was present which could have re-
sulted only through genetic recombination of the two par—
ent line chromosomes. This indicates that there are two
separate loci, one controlling infection by BS—RSV and

the other controlling infection by HA-RSV.

GENETIC INFLUENCE IN ROUS

SARCOMA VIRUS INFECTION

by

-i
. I “x ‘J
Richard Hi Reamer

A THESIS

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

MASTER OF SCIENCE
Department of Microbiology and Public Health

1967

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to
Dr. Charles H. Cunningham, Professor of Microbiology
and Public Health, and to Dr. Ben R. Burmester, Director
of the U. S. Regional Poultry Research Laboratory, for
their encouragement and assistance in the research work
and preparation of this thesis.

I acknowledge, with gratitude the advice and
guidance of Dr. Lyman B. Crittenden, Geneticist, and Dr.
William Okazaki, Microbiologist,of the U. 8. Regional

Poultry Research Laboratory.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . . .

LIST OF FIGURES. . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . .
LITERATURE REVIEW. . . . . . . . . . . .

Defectiveness of the Rous Virus. . .
PrOperties of the RSV. . . . . . . .
In Vitro Aspects of RSV Growth . . .
Importance of the Genetic Character

of the Host. . . . . . . . . . . .

MATERIAL AND METHODS . . . . . . . . . .

Bryan Standard RSV . . . . . . . . .
Harris RSV . . . . . . . . . . . . .
Schmidt-Ruppin . . . . . . . . . . .
Chicken Embryos. . . . . . . . . . .
Preparation of Cell Cultures . . . .
Quantitative Methods . . . . . . . .
Resistance Inducing Factor . . . . .
Absence of RSV in Resistant
Challenged Cells . . . . . . . . .

iii

Page

ii

vii

12
15

15
15
16
16
17
20
22

22

Table of Contents/cont.

RESULTS. . . . . . . . . . . . . . . . . . . . . . . . 23

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 35

LITERATURE CITED . . . . . . . . . . . . . . . . . . . 42

iv

LIST OF TABLES

Table Page

1. Challenge responses of embryos from several
individual pedigreed backcross dams . . . . . . 26

2. Challenge responses of embryos from two
pedigreed backcross dams. . . . . . . . . . . . 27

3. Challenge responses of embryos from a
single backcross dam. . . . . . . . . . . . . . 28

4. Challenge responses of embryos from a
single pen of line 7 dams . . . . . . . . . . . 29

5. Challenge responses of embryos from
several pedigreed line 7 dams . . . . . . . . . 3O

6. Challenge responses of embryos from
several pedigreed dams and a single
pen of line 6 dams. . . . . . . . . . . . . . . 31

7. Test for RIF activity of twelve day
old supernatant from cells resistant
to BS—RSV or HA—RSV . . . . . . . . . . . . . . 32

8. Test of supernatant and cell-free
extract for the presence of virus
in the challenged cells which were
resistant to transformation . . . . . . . . . . 32

9. Frequency of backcross embryos falling
in different categories as a result of

BS-RSV and SR—RSV challenge . . . . . . . . . . 33

V

List of Tables/cont.

Table Page
10. Frequency of backcross embryos falling into
different categories as a result of
response to HA-RSV and SR-RSV challenge . . . . 33
11. Frequency of backcross embryos falling into
different categories as a result of
34

response to BS-RSV and HA-RSV challenge . . . .

vi

Figure
l.

2.

LIST OF FIGURES

Origin of Rous sarcoma virus strains

Photograph of focus against normal
cell background. . . . . . . . . .

vii

INTRODUCTION

The objective of the present investigation was to
determine the relationships among three strains of the
Rous sarcoma virus based on the response of cell cultures
prepared from chicken embryos sensitive or resistant to
Bryan standard Rous sarcoma virus (BS-RSV). The criterion
of infection was the foci of transformed cells in response

to the virus strains.

L IT ERATURE REVIEW

Rous in 1911 described a sarcoma in the subcutaneous
tissue of the breast of an adult hen as clusters of spindle—
shaped fibroblasts, with vacuoles at the periphery. The
tumor was first transmitted with cellular suspensions and
later with cell—free filtrates. Cell division was most fre-
quently amitotic, but mitosis did occur (56).

The origin and history of Rous sarcoma virus (RSV)
is presented in Figure l. The term strain refers to the
origin and passage history of the viruses. Many strains
can be antigenically differentiated (67, 45, 66). Recent
work indicates that some of these strains contain two or
more antigenically different viruses (78). Some strains
are infective for mammals (1). There are also differences
in the morphological type of transformation induced by
these viruses in cell cultures (54, 74).

The amount of infectious virus recoverable from
Rous sarcomas is highly variable and at times no viruses

2

Figure l.--Origin of Rous Sarcoma Virus Strains

'P . ROUS (1911)

/
// (before 1924)
/ W. E. GYE
A. CLAUDE/ w. J. PURDY (1929)
| RSV (29)
(1941) (1935)
w. R. BRYAN c. R. AMIES
(BS-RSV & BH-RSV)
(1957) J G CARR
R. M. DOUGHERTY \\\\‘\\\\\\\\
RSV (BRYAN) ZILBER
RSV (ZILBER)
(1948) cz (RSV)
R. J. c. HARRIS ’
HA—RSV
| (1963)
mmmm.so
P. J. SIMONS SOUTHAM
HA-RSV \\\‘\\\\\\\a
BANG
OBERLING ENGELBRETH-HOLM MILL HILL
STRAINS
MURRAY & BEGG
SVOBODA ANDREWS
(1959) PR-RSV (1932. 1933)
SCHMIDT-RUPPIN MH2
sR—Rsv
AHISTROM
HUEBNER
P. SARMA
Adapted from Simons P. J. and Dougherty R. M. (1963).

 

can be recovered even from highly malignant tumors (57,
64). The absence of virus in sarcomas is related to the
dose of virus, the age of the tumor, and the age of the
host (15, 18, 26, 50). Recent experiments have confirmed
the dual origin of non-infective Rous sarcomas; (1) a low
initiating dose results in the formation of antibodies,
(2) in the case of the high initiating dose of RSV, the
immunologically competent cells within the tumor suppress
viral synthesis in the sarcoma cells (64). There is no
correlation between neutralizing antibody and recovery of

virus from a tumor (50).

Defectiveness of the Rous Virus

 

The Bryan high titer Rous sarcoma virus (EH-RSV)
contains a Rous associated virus (RAV) which is several
times the concentration of RSV and can induce a cellular
resistance to the neoplastic transformation of RSV. The
RAV is closely related antigenically to RSV and produces
erythroblastosis in chickens when inoculated intravenously

in embryos (63).

Single foci of transformed cells picked from RSV
infected cell cultures containing anti—RAV sera, multiplied
indefinitely without morphological differences and failed
to produce either RSV or RAV. When RAV was added to such
cells, they quickly produced large amounts of both RSV and
RAV. It was concluded that this strain was a defective
virus which could produce mature virus only in the presence
of a helper virus such as RAV (35).

The failure of the replicating RSV genome to mature
into infectious virus suggests that the RSV is defective
and is not capable of stimulating the cells to synthesize
the specific portion of the outer coat of the virus. Trans—
formed cells which do not produce measurable virus are des-
ignated non—producer (NP) cells. The NP cells, when implanted
in chicks, do not produce detectable neutralizing antibodies.
The failure of chickens with NP tumors to resist RSV infec—
tion reinforces the conclusion of the absence of an outer
coat of the virus (35).

Viruses of the leukosis group such as RAV, avian
myeloblastosis, and Rubin's isolate designated Resistance
Inducing Factor (RIF), can serve as helpers for activation

of NP cells (35). Viruses which are structurally similar

but biologically distinct such as Newcastle Disease Virus
(NDV) are ineffective as helpers (37).

There are runnerous evidences of a serological re—
lationship between RSV and viruses of the avian leukosis
group (43, 11, 30, 27, 35, 63). The leukosis viruses
cause a proliferation of blood—forming cells resulting in
visceral lymphomatosis, erythroblastosis, myeloblastosis,
and osteopetrosis. Neutralizing antibodies formed against
myeloblastosis virus also neutralize erythroblastosis and
visceral lymphomatosis viruses. These viruses are related
to RSV virus because their antisera neutralize RSV. The
RSV antiserum neutralizes visceral—lymphomatosis and myelo—
blastosis virus but not erythroblastosis virus (11). The
RAV isolated by Rubin (63) is non—cytOpathic microsc0pically
but does produce leukosis in chickens. The RAV is indis-
tinguishable from RSV in thermal stability, growth rate,
site of cellular maturation and immunological specificity.

The RSV bears the antigenic imprint of the par—
ticular helper virus associated with it. When two anti-
genically distinguishable leukosis viruses, such as RIF
(36) and RAV, are used for activation of NP cells, the

resulting viruses are designated RSV(RIF) and RSV(RAV);

the RIF and RAV indicating the helper protein coat. When
anti-RAV serum is mixed with RIF, all the neutralizing
antibody against RSV(RIF) but not against RSV(RAV) is ab-
sorbed. When RSV(RAV) is mixed with anti-RAV serum,
neutralizing antibody against both viruses is absorbed (36).

A second helper virus, RAV-2, has recently been
isolated from BH—RSV (37). It is antigenically unrelated
to RAV—l although both are found in the same virus prepa—
ration. The RAV—2 does not grow in some embryos in which
RAV—1 multiplies. The original studies were conducted
with cell cultures prepared from embryos from Kimber Farms,
Niles, California. The cells resistant to RAV—2 were des—
ignated K/2 cells. All the cell cultures from these embryos
were sensitive to RAV-l. A RSV obtained by activating an
NP with RAV-2 is insusceptible to interference by RAV-l.
These experiments lead to the conclusion that the helper
virus is responsible for two important characteristics of
RSV: (l) the host range and (2) susceptibility to viral
interference. These are prOperties conferred by the virus
coat (37).

It is probable that all chickens reared under usual

conditions become infected with avian leukosis viruses, and

when they are used as host for propagation of RSV strains
many antigencially different progeny may result. This
probably is the main reason for the evolution of antigeni-

cally distinct strains of RSV.

Properties of the RSV

 

According to electron microscopy particles 67-80
mu. in diameter are present in cytoplasmic vacuoles in
tumor cells but not in normal cells (19). In cross sec—
tion, the particles are round, contain a dense nucleoid
about 34—40nu1. and are surrounded by a thin, limiting
membrane (31). The particles are released by a budding
process at the cell membrane (40).

Filtration of RSV indicates it to be from 75—lOOmu.
in diameter (29, 30). The specific gravity is 1.16—1.19
in rubidium chloride (20) and the sedimentation constant
in sucrose is from 600—6553 which indicates a molecular
weight of about 107 (42).

The half-life of the Bryan standard Rous virus

(BS—RSV) in 0.01M phosphate buffered saline containing

1% horse serum is 4 hours at 370C. However, the half-life
of the virus at 370C varies from two to six hours depending
on the strain, source of tumor, and the diluent (13, 51, 55).
At —50 to -76OC. in potassium citrate, RSV remains infective
for one to two years (14). The RSV can survive many years
when dried by sublimation (25) and it is ten times more
resistant to inactivation by ultraviolet light than NDV and
animal virus of similar size and composition (58). The RSV
is ether sensitive (30) and contains Ribonucleic acid (RNA)
as determined by fluorescent microscopy, enzymatic digestion
(48) and paper chromatography (9). Between 24-60% of the
virus is lipid and O.62—l.84% is RNA.

In turkeys, tolerance to RSV can be produced by
inoculating turkey embryos or one-day—old poults intrave-
nously with whole blood from the chicken in which the tumor
was propagated (69). However, blood from different strains
of chicken, pigeons, guinea pigs, sheep, and human group A
(Rh+) also confer tolerance, thus indicating that the RSV
tumor and its causative agent have Forssmann antigens in
common (38, 39). This particular relationship is question—

able. The ability of fresh anti—chicken embryo cell rabbit

10

antiserum to suppress neoplastic properties of RSV on
susceptible cells is due to the anti—cell antibody which
damages the cell and suppresses cell division so that tumors
cannot form. About 40% of this cell division inhibition
is due to the Forssmann type antibody as indicated by
removal of that amount of activity by adsorption of the
anti-cell serum with sheep red blood cells. However, the
virus itself is not neutralized. All the apparent RSV
antibody of the anti—cell sera can be removed by adsorp-
tion with chicken embryo cells (12, 61).

The Schmidt—Ruppin strain of RSV (SR—RSV) induces
in hamsters a specific complement-fixing antibody which
is reactive with the homologous virus and with the soluble
antigens of the leukosis viruses (41). This seems to be
a group specific antigen common to all the members of the

avain sarcoma leukosis group (2, 53).

In Vitro ASpects of RSV Growth

Infection of chicken embryo cell cultures by RSV

results in the production of discrete foci of neoplastic

11

cells, which provides a simple method for investigations
using RSV and Rous sarcoma cells (46). During the replica-
tion cycle of the virus, there is an eclipse period of
about two days. Although viral antigen can be detected by
fluorescent antibody microscopy in 24 hours, virus cannot
be detected by electron microsc0py until the second day
after infection of the cell. The number of fluorescent
particles increases rapidly and by the fourth day they
are concentrated in patches along the cell membrance (40,
75, 76). The number of sarcoma cells within a focus
doubles every 15-20 hours. All cells release virus when
there is a confluent layer of tumors. There is 40—70%
more RNA in infected cells than in noninfected cells.
Several morphological types of foci are produced by dif—
ferent strains of RSV (54). One strain produces cytOpathic
effects in rat, guinea pig, and mouse cell cultures (10).
Resistance of cell cultures from normal chicken
embryos to infection with RSV is reported to be due to
RIF. The RIF infected cell causes a reduction of the
number of infected RSV viral receptor sites (60, 62, 70).

Interferon can also account for an apparent interference

12

with RSV foci, but to be effective it must be available
to the cells during the early stages of the cell virus
interaction (7).

Recently it was reported that the group specific
antigen of the sarcoma—leukosis viruses is synthesized
in the nucleus, moves to the cytOplasm, and then can be

detected on the cell surface (53).

Importance of the Genetic Character
of the Host

The heritability of resistance to RSV in fowls
has been demonstrated by the matingtxfan RSV resistant
male to several close—relative females. Progeny showing
resistance to RSV tumor growth were selected for mating
and resistant offspring were consistently produced (33).
Genetic resistance to RSV cultivation on the chorio—
allantoic membrane (CAM) is controlled by a single pair
of autosomal genes and susceptibility to the RSV is
dominant (49). Intra—cranial inoculation of day old
chicks confirmed the dominance of susceptibility and the

control by a single pair of autosomal genes (79, 80).

13

Cell cultures from embryos of genetically resistant chick-
ens resist transformation by RSV. This resistance is con-
trolled by a single autosomal recessive gene pair (22, 21).

The susceptibility or resistance of an anti-
genically related avian leukosis virus designated RPL—12
is influenced by the same locus as that controlling BS-RSV
resistance or susceptibility (17, 22, ll, 27). The RPL~12
virus causes no cytOpathic changes in cell culture but
does interfere with the transformation of the BS—RSV (60).
An allel of the BS—RSV gene or an altogether different
gene was suggested in recent work where a RSV(RAV—2) was
used to challenge cells. There was no apparent effect of
the gene controlling BS—RSV on the RSV(RAV-Z). The results
also indicated that susceptibility to RSV(RAV—Z) was domi-
nant. The expression of the gene as a component, or lack
of a component, on the cell surface determines whether or
not adsorption or penetration takes place (65).

Two subgroups of the avian tumor viruses are
distinguished on the basis of their host range. The
first, referred to as subgroup A, consists of RAV—l and
viruses having similar antigenic enve10pes. The second

group is designated subgroup B and is represented by RAV—2

14

and its immunological relatives. These A and B subgroup
viruses react with different cellular receptors during the
initiation of infection. Selective resistance of chicken
embryo cultures to one subgroup is probably correlated
with the absence of a corresponding cellular receptor site.
Helper viruses of each subgroup will induce resistance

only to the RSV strain which are within its group (78).

MATERIALS AND METHODS

Bryan Standard Rous Sarcoma Virus

 

The Bryan standard RSV (BS—RSV) was supplied by
Dr. Ray Bryan, National Cancer Institute, and designated
by him as C.T.—750. The BS—RSV used in cell culture was
a 20% tumor suspension, twice clarified by centrifugation
at 2,000g for 60 minutes at 40C, and filtered through a
0.02 Selas candle. The virus was propagated by one wing
web passage and two passages in the breast muscle of line

151 chickens.

Harris Rous Virus

 

This strain was obtained from Dr. F. Bang of
Johns Hopkins University. The preparation was a 10%
extract of 15I CAM pocks, twice clarified by centrifu—
gation at 2,000g for 60 minutes at 40C, and filtered

through a 0.02 Selas candle.

15

l6
Schmidt—Ruppin Rous Virus

This strain was obtained from Dr. Padman Sarma at
the National Institutes of Health. The preparation was a
10% extract of line 7 CAM pocks. The extract was twice
clarified by centrifugation at 2,000g for 60 minutes at

40C and filtered through a 0.02 Selas candle.

Chicken Embryos

 

Since 1939, close inbred lines of Single Comb White

Leghorn chickens have been separately maintained at the
U.S.D.A. Regional Poultry Laboratory, East Lansing, Michigan
(81). Embryos used were from chickens of the second back
cross of line 6 by line 7. This means that the F1 (6X7)

was mated back to line 7; then the resulting progeny were
mated to line 7 again. On the basis of intra—cranial inocu-
lation of the second back cross (BX—2) one day old chicks,

a random sample of progeny was available which had an

equal probability of being either resistant or susceptible

17

to BS—RSV. The line 6 and line 7 progeny were also used.
Line 6 chickens are susceptible to and line 7 are resistant

to BS-RSV (22).

Preparation of Cell Cultures

 

Cell cultures were prepared from 9 day old embryos
by a modification of the procedure described by Rubin (60).
Decapitated embryos were drOpped into 252(150 mm. test
tubes containing approximately 20, 3/16 diameter perforated
glass beads and 5ml of phosphate buffered saline (PBS).
The embryos were fragmented when the tube was inserted in
revolving rubber cup of a Vortex mixer. The fragments
were washed with 20ml of PBS, and after the cells settled
by gravity, the supernatant fluid was decanted. This
procedure was then repeated. A 0.25% solution of trypsin
(Nutritional Biochemical Company) was diluted 1/5 with
PBS and 20ml was added to each tube.

The tubes were placed in a 370C water bath and
shaken by hand every ten minutes. After one hour, the

supernatant fluid was carefully removed by a pipette and

18

placed in similar tubes, and centrifuged at 2,000g for
five minutes. The supernatant fluid was then poured off.
The pellet was resuspended in growth medium to contain
1 X 106 cells per ml. Ten ml. of the cell suspension was
added to 100mm diameter tissue culture petri plates (Fal-

con Plastics), and incubated at 370C in an atmosphere of

5% C02.
Growth Medium ml.
10 X 100 (Microbiological Associates) 100
Tryptose Phosphate Broth (Difco) 100
Calf Serum (Colorado Serum Co.) 40
Sodium Bicarbonate 2.8% solution 20

Penicillin and Streptomycin
(10,000 units/ml.) 10

Water (deionized and sterilized) 750

Phosphate Buffered Saline:

NaCl 8.0gm
KCl 0.2gm

.2
Na2HP04 1 gm
KH2P04 0.2gm
H 0 1 liter

2

19

After four days, the cells were treated with 5 ml.
of a 0.05% trypsin solution for ten minutes at 370C and
then centrifuged and resuspended in growth medium to con-
tain 2 X 105 cells per ml. Five ml. of the cell suspension
was added to 60mm diameter petrie plates.

Each of the three viruses was appropriately diluted
and 0.1 ml.inoculum containing 103 focus forming units was
added to 2 plates of each individual embryo cell culture.
After 24 hours, when the cells had formed a monolayer, the
supernatant fluid was poured off and the cells were over—

layed with 5 m1. of agar medium.

Agar Medium ml.
2 X 199 50
Tryptose Phosphate Broth 14
1.8% Agar 50
Calf Serum 6
Sodium Bicarbonate (2.8% solution) 2.6
Stock Antibiotics 1.0

Three days later, 3 m1. of growth medium was added
to enhance visual recognition of the foci. 0n the 4th or

5th day after infection, the number of foci on l/lO of

20

the area of a plate was counted. If no foci were observed
the entire plate was examined.

The transformed areas are made of round refractile
cells in grapemlike clusters while the normal cells are
flat and diamond shaped. Idealy the foci are discrete and

easily seen against the normal cell background (Fig.2).

Quantitative Methods

The criterion for resistance was the absense of
foci on the cell monolayer in response to a standard
challenge virus dose which would normally produce 1,000
foci. This is a relatively rigid criterion and has been
previously used with the BS-RSV (22,21).

To test the significance of the data, an X2 test,

using the 2 X 2 table method, was calculated according to:

 

 

2 2 '
X : ngad-bc) k=(a+b) (c+d) (c+d) (b+d)
k .
n = total number of observations.
X Virus
Sensitive Resistant

Sensitive a b a+b
Y Virus

Resistant c d c+d

 

a+c b+d n

21

 

Figure 2.--Neop1astic foci in background of normal
chicken embryo fibroblasts.

'22

Resistance Inducing Factor

 

Supernatant fluids were collected from 12 day old
primary cultures of individual embryos which were resistant
to BS-RSV or HA—RSV or to both viruses. Two ml of these
fluids were inoculated on line 6 cells and after 6 days the
cells were trypsinized, replated, and challenged with BS—RSV

to determine presence of RIF.

-Absence of RSV in ResiStant
Challenged Cells

 

 

Supernatant fluids plus cell free extracts of
resistant challenged cells were clarified by 2,000g for
60 minutes and 2 ml were inoculated on line 6 cell cultures

to test for foci producing ability.

RESULTS

The response of secondary embryo cells of 10 dif—
ferent pedigreed matings to challenge by BS-RSV and HA—RSV
places each BX embryo into one of four categories; (1)
Resistant to HA-RSV and sensitive to BS-RSV, (2) Sensitive
to both viruses, (3) Resistant to both viruses and, (4)
Sensitive to HA—RSV and resistant to BS—RSV (Tables 1-3).
One dam (111) supplied 23 embryos which provides a model
for patterns of resistance.

The parent lines 6 and 7 produce progeny which
respond differently to the virus challenges. Individual
embryo cell cultures from line 7 were either in category
3 or 4 (Table 4 and 5). These data support the hypothesis
that line 7 is homozygous resistant to BS-RSV and indi-
cates that this line also is segregating genes for resis—
tance to HA—RSV. All embryos from line 6 (Table 6) were
in category 2 suggesting that this line is homozygous

susceptible to both viruses.

23

24

Supernatant fluid from twelve day old primary cul—
tures of cells, which were resistant to one of the viruses
or to both of them, were inoculated on sensitive line 6
cultures. The RIF was apparently not responsible for the
resistance because there was no difference in the virus
titer of the control and that of the test challenge (Table
7).

Supernatant fluids and the cell free extracts of
resistant cells were tested on line 6 for foci producing
ability but no foci were observed (Table 8).

The response of secondary cells to SR—RSV can be
defined in terms of their patterns of sensitivity or
resistant to the BS—RSV (Table 9). When all the embryos
of the BX are compared, it is apparent that none was
sensitive to BS—RSV and resistant to SR-RSV.

The response of secondary cells of SR-RSV can also
be defined in terms of the patterns of sensitivity or
resistance to HA—RSV (Table 10). Of the embryos sensitive
to HA-RSV, 97%‘were also sensitive to SR—RSV.

When the response of secondary cells to challenge
with BS—RSV is defined in terms of response to HA-RSV,

there is a great difference in host range (Table 11).

25

The X2 test supports the relationship of SR—RSV to both
BS-RSV and HA—RSV but an independence of the BS—RSV and
the HA—RSV. The response of the BX embryo cells to dif-
ferent combinations of the three viruses can statistically

be rated as follows.

 

 

VIRUS PAIR i SIGNIFICANCE
BS—RSV, HA—RSV 0.36 Independence of
infective ability
on same cells.
BS—RSV, SR-RSV 28.6 Dependence of
infective ability
on same cells.
HA-RSV, SR—RSV 14.25 Dependence of

infective ability
on same cells.

26

TABLE l.—-Challenge responses of embryos from several

individual pedigreed backcross dams

 

 

a

 

DAM NUMBER of NUMBER of NUMBER of
NUMBER HA-RSV FOCI BS-RSV FOCI SR-RSV FOCI
144 72 134 141
144 54 130 103
144 0 0 O
144 O .. O
144 O ... O
161 0 0 41
161 O 0 O
161 78 0 0
161 .. O 0
163 0 81 136
163 168 60 169
163 0 O O
163 0 O O
163 60 0 0
163 73 O 116
126 14 300 291
126 O O 0
126 0 O 214
126 28 O 170
126 ... 100 220
132 O 335 293
132 82 310 294
132 204 316 281
132 0 O O
132 O O 0
132 136 O 268
132 ... 11 17
132 ... O 58
132 .. O 19
132 .. 31 94
117 O 263 252
117 14 60 154
117 264 O 271
117 227 O 274
117 24 O 50

 

area, a

a .
All foc1 counts represent 1/10 of the total plate
zero represents a total plate area determination.

27

TABLE 2.--Challenge responses of embryos from several
individual pedigreed backcross damsa

1‘1

 

 

 

DAM NUMBER of NUMBER of NUMBER of
NUMBER HA-RSV FOCI BS—RSV FOCI SR-RSV FOCI
137 O 82 251
137 16 300 132
137 0 O 269
137 O O O
137 O 0 O
137 14 O 200
137 16 O 216
137 109 O O
137 97 O 281
137 ... 195 267
137 ... 0 221
113 0 141 223
113 14 208 284
113 13 142 165
113 0 O 101
113 0 0 86
113 0 O O
116 O 217 257
116 22 300 203
116 202 331 362
116 15 208 202
116 62 O 240
116 52 O 285
116 14 O 307
123 O 314 320
123 O 152 137
123 O 151 175
123 22 54 59
123 O O O
123 O O O
123 14 0 268
123 18 O 242
123 217 O 269
123 33 0 34
123 ... 0 O
a

All foci counts represent 1/10 of the total plate

area, a zero represents a total plate area determination.

28

TABLE 3.—-Challenge responses of embryos from a single
backcross dam (lll)a

 

 

 

NUMBER of NUMBER of NUMBER of
HA-RSV FOCI BS-RSV FOCI SR—RSV FOCI
O 91 44
O 278 264
O 276 248
O 304 321
O 236 ...
O 101 108
O 160 131
O 103 134
O 105 113
O 71 9
13 300 137
18 112 152
15 121 147
67 53 163
O O O
O O 211
O O 0
O O 0
O O O
O O O
O O O
O O O
O O O
18 ... 256
.. 133 63
. O O
.. O O
.. O 110

... 35

 

a .
All foc1 counts represent 1/10 of the total plate
area, a zero represents total plate area determination.

29

TABLE 4.-—Challenge responses of embryos from a single
pen (pen 18) of Line 7 dams

 

 

 

NUMBER Of NUMBER of NUMBER Of
HA-RSV FOCI BS-RSV FOCI SR—RSV FOCI
O 0 O
O O O
O O O
O O O
O O 0
O O O
O O O
O O O
O O O
15 O 116
14 O 121
76 O 217
208 O 145
O O O

 

aAll foci counts represent 1/10 of the total plate
area, a zero represents a total plate area determination.

3O

TABLE.5-Challenge responses of embryos from several
pedigreed line 7 damsa

 

 

DAM NUMBER of NUMBER of NUMBER of
NUMBER HA-RSV FOCI BS-RSV FOCI SR—RSV FOCI
315 O O O
315 O O O
315 O O O
315 14 O 29
315 16 0 30
312 O O O
312 22 O 51
312 18 O 14
344 O O O
344 O O O
345 O O O
345 O O O
347 O O O
347 O O O
347 O O O
347 O O O
347 O O O
347 O O O
347 O O O

 

a .
All foc1 counts represent 1/10 of the total plate
area, a zero represents a total plate area determination.

31

TABLE 6.--Challenge responses of embryos from several
pedigreed dams and a single pen of line 6 damsa

 

 

 

DAM NUMBER of NUMBER of NUMBER of
NUMBER HA-RSV FOCI BS-RSV FOCI SR-RSV FOCI
236 44 170 243
251 37 261 248
231 61 144 170
231 40 251 76
231 38 73 72
251 72 202 212
251 68 314 314
251 59 219 192
236 47 137 87
236 29 311 310
PEN
NUMBER
32 22 300 298
32 224 312 346
32 115 321 357
32 166 290 291
32 270 281 248
32 215 308 336
32 117 316 341
32 237 287 263
32 213 314 321
32 167 306 287
32 89 291 264
32 216 314 280
32 188 287 310
32 260 311 318
32 234 267 296
32 163 258 318
32 80 309 328
32 129 310 266
32 212 277 259
32 136 294 302

 

a .
All foc1 counts represent 1/10 of the total plate
area, a zero represents a total plate area determination.

32

TABLE 7.--Test for RIF activity of twelve day old super-
natants from cells resistant to BS-RSV or HA-RSVa

 

 

 

SUPERNATANT from RESISTANT NUMBER of FOCI on
EMBRYO CELLS TO BS—RSV CHALLENGE

18 BS-RSV & HA-RSV 106

18 BS-RSV & HA—RSV 99

123 BS-RSV 89

137 BS—RSV 70

123 HA-RSV 65

111 HA-RSV 117

116 NONE 92

 

a . . . . .
Six days were allowed for RIF induction on senSItive
cells after inoculation of 2 ml per assay plate.

TABLE 8.--Test of supernatant and cell-free extract for
'the presence of virus in the challenged cells which were
resistant to transformation.a

 

 

 

SOURCE of CELLS NUMBER of BS~RSV
and SUPERNATANT or HA-RSV FOCI

 

18
18
123
137
123
111

000000

 

aSix days were allowed for the development of foci
after inoculation of 2 ml per assay plate.

33

TABLE 9.--Frequency of backcross embryos falling in dif—
ferent categories as a result of BS—RSV and SR-RSV challenge

 

 

 

BS-RSV
Sensitive Resistant
Sensitive 41 26
SR-RSV
Resistant 0 26

 

TABLE 10.-—Frequency of backcross embryos falling into
different categories as a result of reponse to HA-RSV
and SR—RSV challenge

HA—RSV
Sensitive Resistant

 

Sensitive 35 25

 

SR-RSV

Resistant 3 22

 

34

TABLE ll.—-Frequency of backcross embryos falling into
different categories as a result of response to BS—RSV
and HA-RSV challenge

 

 

BS-RSV

Sensitive Resistant

 

Sensitive l7 l7

 

HA-RSV
Resistant 19 25

DISCUSSION

The results indicate that separate genes are respon—
sible for the sensitivity or resistance of cells to BS-RSV
and HA—RSV. Homozygous resistance of line 7 chicken embryo
cells to BS-RSV and also the significant degree of resistance
to HA~RSV is in contrast to line 6 chicken embryo cells which
were homozygous susceptible to both viruses.

Quantitative differences in the number of foci in
a given cell culture were within normal variation limits.

The HA-RSV was responsible for the greatest variation in

the number of foci formed. Similar results have been reported
by other investigators (78). Dougherty et a1. (25) indicated
that his HA-RSV strain, in cell culture, produced some foci
which were very diffuse and indistinct thus making the accu—
rate counting of foci difficult. The latter situation may
have been the reason for the variable counts encountered

with this HA-RSV virus.

35

36

Many viruses have the prOperty of inducing the forma-
tion of an antiviral substance in vivo and also in Vitro.
The RSV and leukosis viruses apparently have this ability
(7, 71). If interferon were responsible for the resistance
tested in the present study, there would have been no selec—
tive resistance to one virus and not the others. Evidence
for the genetic nature of the resistance is manifested by
the fact that the resistance to BS-RSV of progeny of line
7 females can be changed to sensitivity by mating with a
sensitive male (21). The line 7 embryos, which are at times
resistant to both HA-RSV and BS-RSV, are free of subgroup A
or B viruses (78).

Extracts from representative resistant, challenged
cells, failed to produce any foci when inoculated on sensi—
tive cells, thus indicating that the virus did not multiply
in these cells. Evidence has previously been presented
which suggests that resistance of cell cultures to BS—RSV
extends to viral synthesis as well (52).

The host range of the BS-RSV and the HA-RSV places
them in different subgroups and X2 analysis supports this
interpretation. This means that the genetic control of

infection is different for each of these viruses. When

37

SR-RSV and BS-RSV are compared, their host range is essen—
tially the same but it is also true that the host range of
SR—RSV and HA—RSV are fundamentally the same. The X2
analysis suggests that BS—RSV and HA-RSV are related to
SR-RSV. One possible eXplaination is that the SR—RSV used
is a mixture of two or more viruses, one being similar to
the HA-RSV while the other acts similar to the BS-RSV. The
possibility of a mixture of viruses in the SR-RSV strain
is supported by the recent isolation of two SR RSV strains
on the basis of host range. One was designated SR-RSV—l
and belonged to the A subgroup like BS—RSV; the other was
designated SR-RSV-2 and belonged to the B subgroup like
HA—RSV. The A and B subgroups are characterized by two
other criteria, (1) their antigenic character, because
there is cross neutralization within the group and (2) the
interference pattern i.e. foci inhibition, occurs only
when the helper and the RSV belong to the same group.

Four cell phenotypes have been identified on the

basis of host range (79).

C/O = cells resistant to neither A nor B subgroup
C/A = cells resistant to A subgroup viruses
C/B = cells resistant to B subgroup viruses

C/AB = cells resistant to both subgroup viruses

38

Embryo cell responses to challenge may be used to
tentatively genotype as well as phenotype lines 6 and 7.
The following is a theoretical model where small a repre-
sents the gene controlling the A subgroup and small b
represents the gene controlling B subgroup. The super-
scripts s and r represent susceptibility and resistance

respectively.

LINE

GENOTYPE

39

PHENOTYPE

 

C/O

C/A

C/A

C/AB

POSSIBLE GAMETES

 

The cell culture data from the total backcrosses

(Table 11), when presented in the following manner, reveal

a new cell phenotype unlike either of the original parent

produces.
BS-RSV
Sensitive
Sensitive
Resistant

Resistant

HA—RSV
Sensitive

Resistant

Resistant

Resistant

 

 

OBSERVATIONS PHENOTYPE
l7 C/O
l9 C/B
l7 C/A
25 C/AB

Recombination apparently has taken place in the

6 X 7 genetic material to lead to the formation of a C/B

cell phenotype.

40

The following is a representative recombination in
the 6 X 7 genetic material;

 

 

 

 

 

Line 6 Line 7
Possible gametes asbS arbr, arbs
Possible 6 X 7 individuals (Fl) asbS asbS
arbr arbS
Possible (F1) gametes asbs, asbr, arbr, arbs
(Fl) x line 7 could lead to a C/B asbr
cell type; (first backcross) arbr

 

When the first backcross (BX-l) is mated to line 7
the progeny are referred to as the second backcross (BX—2).
Gametes of the first and second backcross are the same ex-
cept that there may be some C/B chickens in the matings of
the second backcross. This would increase the number of
C/B cells of the BX-2 over the BX-l.

The fact that crossover occured indicates that there
are two separate loci involved, one controlling presence or
absence of A subgroup attachment sites and one controlling
the presence or absence of B subgroup sites. The crossover

further suggests that these loci are not closely linked.

41

The cell types which were expressed here are in agreement
with those described recently. (78) However, the pedigreed
matings used in this study enabled us to show how these

particular cells came about.

10.

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