A comparative study of the Schmidt-Ruppin and Bryan high titer strains of Rous sarcoma virus
by Clayton Arthur Buck
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in Bacteriology
Montana State University
© Copyright by Clayton Arthur Buck (1965)
Abstract:
The biological properties of the Bryan high titer strain of Rous sarcoma virus, RSV(B) , and the
Schmidt-Ruppin strain, RSV(SR) , were compared. RSV(B) was found to be more pathogenic, in vivo
and in-vitro than RSV(SR). RSV(B) carried neutralizable antigens not found on RSV(SR) and was 3
times more heat stable than RSV(SR). Defectiveness was found to be a property of RSV(B) but not of
RSV(SR). Both RSV(SR) and RSV(B) were produced at the same rate by infected cells; however, there
was always 10 to 100 times more RSV(B) produced than RSV(SR). Puromycin had identical effects on
24 hr. RSV(B) and RSV(SR) production; however, the effects of puromycin on 48 hr. RSV(SR)
production appeared to be different from its effects on 48 hr. RSV(B) production. Both viruses were
inhibited by the addition of actinomycin D to cells within the first 35 hrs. after infection. Possible
explanations of the differences between the 2 viruses were discussed.
r . A-COMPARATIVE-STUDY H3F THE SCHMI D T - RUPP I N AND BRYAN HIGH"Tl TER
STRAINS GF ROUS SARCOMA VIRUS
by
&
; CLAYTON, ARTHUR BUCK
A t h e s i s s u b m i t t e d . t o t h e G r a d u a t e " F a c u l t y in p a r t i a l
f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e d egree
of
DOCTOR OF PHILOSOPHY
in
x
B acteriology
App r o v e d :
H e a d , . Maj o r .'Department
.
" Cha i rman ,/"Ekam i n i ng Comm i t t e e
Dean, G r adu at e D i v i s i o n
MONTANA STATE. COLLEGE
Bozeman,,Montana
June,
I 965
ACKNOWLEDGMENT
T h e a u t h o r was s u p p o r t e d by N a t i o n a l
F e l l o w s h i p number GPM-1 3 , 3 8 2 - Rl f r o m J u l y
I n s t i t u t e s o f Health P redoctoral
I SS'2 t o J u l y
1964 .
. Study and
r e s e a r c h f r o m J a n u a r y t o June 1962 and f r o m September 1964 t o March 1965
we re s u p p o r t e d by P u b l i c H e a l t h S e r v i c e T r a i n i n g Gr ant number 2 - E - 13 1(R1) .
.The a u t h o r w i s h e s t o g r a t e f u l l y ack no wl ed ge t h e a s s i s t a n c e ,
agement and p a t i e n c e o f D r .
in ve stig a tio n .
The a u t h o r
F. .S. . Newman t h r o u g h o u t t h e - co ur se o f t h i s
is
McBee f o r t h e i r s u g g e s t i o n s
encour­
i n d e b t e d t o D r . . J . W. J u t i l a
and Dr . R. H.
in p r e p a r in g the m a n u s c rip t.
The a u t h o r w i s h e s t o t h a n k D r . . P e t e r K. V o g t , o f t h e De part ment o f
Pathology,
U n iv e r s it y o f Colorado/,Denver,
in d e v e l o p i n g t h i s
research p r o j e c t .
D r s . A . . E i s e n s t a r k and A.
f o r h i s h o s p i t a l i t y and h el p
T h e -encouragement and s u p p o r t o f
F . . Borg o f t h e Kansas S t a t e U n i v e r s i t y D e p a r t ­
ment o f B a c t e r i o l o g y t h r o u g h o u t t h e y e a r s o f g r a d u a t e s t u d y was g r e a t l y
appreciated.
typing
A special
the m anuscript.
n o t e o f a p p r e c i a t i o n goes a l s o t o Judy Buck f o r
■i. v
TABLE OF CONTENTS
"LIST OF TABLES .............. ................................................................................. „ ................ .. „ . vi 'i 1
LIST OF FIGURES _____ _.................. ...........................................................................................v r i I
INTRODUCTION TO THE "VIRUS ..................... ...................................... .....................................
. Rous and Hi s V i r u s
................................................................. ...................... ....................
Geneaology o f Rous" Sarcoma V i r u s
Rous Sarcoma V i r u s C l a s s i f i c a t i o n
Assay o f Rous Sarcoma V i r u s
............................ ...............................................
......... ............. ..
....................... ...................................... ..
■ P r o p e r t i e s o f RSV C o n f e r r e d by H e l p e r V i r u s e s
The E f f e c t s o f
......... ........................... ..
I n h i b i t o r s on Rous Sarcoma V i r u s R e p l i c a t i o n
I NTRODUCTI ON TO THE PROBLEM
..............
.............. ..
........................................................................
' P r e p a r a t i o n o f G la s s w a r e ............................ ............. ................
8
lb
10
13
i Prepa r a t ion o f T r i s B u f f e r e d S a l i n e ..................................
20
21
24
..............................................................................
P r e p a r a t i o n and T e s t i n g o f C h i ck Embryo F i b r o b l a s t s
20
20
Prepa r a t ion o f Ti ssue C u l t u r e Med i urn ......................... ..................................
Preparation of T rypsin
6
I'9
MATERIALS AND METHODS ................... ........................................................ ..
Pr e p a r a t i o n o f Wa ter
.1
J
............................ ........................................ .................
- D e f e c t i v e n e s s o f Rous Sarcoma V i r u s
I
...............
25
25
Prepa r a t ion o f Mouse Embryo F i b rob I a s t s
...........................
28
■ P r e p a r a t i o n end Source o f V i r u s S t o c k s
...........................................................
29
Methods o f V i r u s Assay ................................................
3I
I s o l a t i o n o f T r a n s f o r m e d , . N o n - P r o d u c i n g Clones . . . . . . . . . . . . . . . . . . . . .
32
■ Pr e p a r a t i o n o f A n t i - s e r u m
.....................................................................................
34
V
■Preparation o f F lu o re s c e n t"L a b e le d A ntiserum . . . . . . . . . . . . . . . . . . . . . .
34
Fluorescent "Staining o f V iru s
..................................... ..
36
..........................................................
37
Infected C e lls
P r e p a r a t i on o f Pur omyc in S t oc k S o l u t i o n s
P r e p a r a t i o n o f A c t i n o m y c i n D S t ock S o l u t i o n s
■Processing o f C e l l s f o r S c i n t i l l a t i o n
......................
Counting
..................................... ..
RESULTS ........................................................................ ............................. ..
RSV Tumors i n C h i c k en s
. RSV I n f e c t i o n s
Heat
I n a c t i v a t i o n o f RSV(B) and RSV(SR)
and RSV(SR)
•40
...........................
46
................
4$
...............................
53
- RAV I n t e r f e r e n c e wi t h RSV Repl i-c a t i o n
Growth o f RSV(B) and RSV(SR)
40
............................
A n t i g e n i c R e l a t i o n s h i p s Between RSV(B) and RSV(SR)
. D e f e c t i v e n e s s o f RSV(B)
38
40
...................................................................................................
in T is s u e C u lt u re C e lls
38
.............................
in C h i c k F i b r o b l a s t s
.......................... .
57
57
Vi rus Rel eased by . E s t a b l i shed RSV Cel I s ......... .......................................... ..
63
E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f Pur omycin
on. C e l l D i v i s i o n and V i r u s P r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . .
64
K i n e t i c s o f S u p p r e s s i o n o f T w e n t y - f o u r Hour
V i r u s Production by Puromyc in .............................................. .................... ...........
74
K i n e t i c s o f S u p p r e s s i o n o f ' F o r t y - e i g h t Hour
V i rus P r o d u c t i o n by 'Puromyc i n ............................................ ..
77
E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f Puromycin
on F o r t y - e i g h t Hour V i rus P r o d u c t i o n ............................................................
91
" E f f e c t , o f Pur omycin on V i r u s Release by E s t a b l i s h e d Rous C e l l s
P r o t e i n , S y n t h e s i s by RSV(B) and RSV(SR)
.....
I n f e c t e d C e l l s , .....................
E f f e c t o f A c t i n o m y c in. D on V i r u s P r o d u c t i on
SI
99
........................
10,7
D I SCUSS I ON . ...................................................................................................................................
1.09
Vi
SUMMARY •............................................................................................................... .. ..........................
124
LITERATURE CITED ............... ........................................................................................................
126
v ii
LIST OF TABLES
Table
- 1. P r e p a r a t i o n o f Medium 199A IOX .....................
T able
I I . I n d u c t i o n o f N o n - P r od u ce r s
Table
T a b le
.Table
T a ble
22
........................................................
.54
I I I . V i r u s P r o d u c t i o n by Foci O b t a i n e d f r o m Lnduced NP 'Ce11s . .
56
T V . V i rus P r o d u c t i o n by 24 and 48 H r . . C o n t r o l s ...................
81
V. The E f f e c t o f l y t g / m l o f A c t i n o m y c in. D on 48 Hr.
P r o d u c t i o n b y . RSV(B) and RSV(SR) I n f e c t e d Cel I s
VI.
Virus
..........
The E f f e c t o f 0 . IyA g/ml o f A c t i n o m y c in. D on 48 Hr. ■
V i r u s P r o d u c t i o n by. RSV(SR) I n f e c t e d C e l l s ......... ..
--I:-
107
108
vi i i
■LIST OF FIGURES
Figure
I.
. RSV(B) Tumor I1^ Days a f t e r
Figure
2.
RSV(B)
Figure
'3.
RSV(SR)
Figure
-4.
RSV(B)
Figure
- 5
Foci
Foci
.............................................
......................................
41
.42
.......................................................................................................... 42
Focus M a g n i f i e d 30X ........................................................
RSV(SR)
43
Focus M a g n i f i e d 30X .............................................. ......................
• Figure
-6.
E s t a b l i s h e d RSV(SR)
Figure
.7.
E s t a b l i s h e d RSV(B)
' Figure
8.
Figure
9•
.Heat
Infe ctio n
C e lls M agnified
C e lls M agnified
iOOX .............
45
IOOX .................................
I n a c t i v a t i o n o f RlSV(B) and RSV(SR)
45
.........................................
N e u t r a l i z a t i o n o f RSV(B) and RSV(SR) w i t h A n t i -RSV(B)
• Serum ..........
F i g u r e 10.
RAV I n t e r f e r e n c e w i t h RSV(B) and RSV(SR)
F i g u r e 11.
Growth Curves o f RSV(B) and RSV(SR)
Infection
F i g u r e 12.
..........................
:
V i r u s R e l e a s e " b y E s t a b l i s h e d RSV(B) and RSV(SR) C e l l s
F i g u r e 13.
I n h i b i t i o n of. C e l l
43
48
52
.....................59
62
..............
66
Growth by Pur omycin ..............................................
69
F i g u r e 14 . -.Exposure o f I n f e c t e d C e l l s f o r , Extended P e r i o d s o f
Time -to Jy AgZml o f Puromyc i n ....................... ...........................................
71
F i g u r e 15.
F i g u r e -16.
F i g u r e -I 7.
F i g u r e 18.
I n h i b i t i o n o f 24 H r . - V i r u s P r o d u c t i on by I n c r e a s i n g
C o n c e n t r a t i o n s o f Puromyc i n ......... ...................... ....................................
73
E f f e c t o f t h e T i me o f A d d i t i o n o f Pyromycin on 24 Hr.
V i r u s P r o d u c t i o n b y . RSV(B) and RSV(SR) I n f e c t e d C e l l s
.........
76
E f f e c t o f t h e Time o f A d d i t i o n o f Pur omycin on 48 Hr.
V i r u s P r o d u c t i o n b y - RSV(B) and RSV(SR) I n f e c t e d C e l l s
.........
79
E f f e c t o f Ext ended P e r i o d s o f . E x p o s u r e t o P ur omy ci n on
V i r u s P r o d u c t i o n by RSV(SR) I n f e c t e d C e l l s . . ............................ 83
;
F i g u r e 19.
-Ma in t en an c e of' RSV(SR)
T r e a t e d Cel I s
P r o d u c t i o n by PuroMycin
85
iX
F i g u r e 20.
. F i g u r e -21.
F i g u r e -22.
F ig u r e - 2 - 3 .
F i g u r e '24.
F i g u r e 25.
E f f e c t "o f ‘E xt ended P e r i o d s o f E x po su re t o Pur omyci n
o n ' V i r u s P r o d u c t i o n by RSV(B) I n f e c t e d CeiTs .....................88
. M a i n te n an c e -of RSV(B) P r o d u c t i o n by Pur omycin T r e a t e d
C e l l s ......................................................................................................................
90
. E f f e c t o f I n c r e a s i n g C o n c e n t r a t i o n s o f Pur omyci n on
48 Hr. V i r u s P r o d u c t i o n ■...........................................................................
g4
, E f f e c t o f P ur omyc in o n . V i r u s Release by E s t a b l i s h e d
RSV(B) C e l l s ......................................................................................................
96
. E f f e c t o f P u r o m y c i n b n . V i rus R el ease b y , E s t a b l I shed
RSV(SR) C e l l s .........
98
. S t a n d a r d T r i t i u m Curve ..................................... ........................................
F i g u r e 26.
h P^ L eu c in e I n c o r p o r a t i o n
i n t o RSV(B)
F i g u r e 27.
H^-Leucine-Incorporation
i n t o RSV(SR)
I n f e c t e d Cel I s ...........
Infected C e lls
.........
102
104
106
-ABSTRACT
The b i o l o g i c a l p r o p e r t i e s o f t h e Bryan h i g h t i t e r s t r a i n o f Rous
sarcoma v i r u s , RSV(B) , and t h e S c h m i d t - R u p p i n s t r a i n , RSV(SR) , were-com­
pared.
RSV(B) was f o u n d t o b e more p a t h o g e n i c , in v i v o and i n- v i t r o t ha n
RSV(SR).
RSV(B) c a r r i e d n e u t r a l i z a b i e a n t i g e n s n ot f o u n d on RSV(SR) and
was 3 t i m e s more heat, s t a b l e t h an RSV(SR). D e f e c t i v e n e s s was f ou nd t o be
a p r o p e r t y o f RSV(B) b u t n o t o f RSV(SR) . Bot h RSV(SR) and RSV(B) were
-produced a t t h e same - r a t e by i n f e c t e d c e l l s ; however, t h e r e was al way s
-10 t o l OOi't imes more RSV(B) p r odu ce d t ha n. RSV(SR) . Pur omycin had i d e n t i ­
c al e f f e c t s on 24 h r . RSV(B) and RSV(SR) p r o d u c t i o n ; however, t h e e f f e c t s
o f p u r o m y c i n on 48 h r . . RSV(SR) p r o d u c t i o n appear ed t o be d i f f e r e n t f r o m
i t s e f f e c t s on 48 h r . RSV(B) p r o d u c t i o n .
Bot h v i r u s e s were i n h i b i t e d by
t h e a d d i t i o n o f . l e t i nomyc i n,- D t o c e l l s w i t h i n t h e f i r s t 35 h r s . a f t e r
in fe c tio n .
P o s s i b l e e x p l a n a t i o n s o f t h e d i f f e r e n c e s between t h e 2 v i r u s e s
were d i s c u s s e d . '
INTRODUCTION TO THE VIRUS
Rous and Hi s V i r u s
The t umor w h i c h was t o e v e n t u a l I y g i v e r i s e t o v i r u s was r e p o r t e d by
Rous i n i g i 0 .
He d e s i g n a t e d
i t . C h i c k e n Tumor I .
I t a r o s e on t h e - b r e a s t
o f a 15 month o l d Pl ymo ut h Rock hen and wps removed 2 months l a t e r .
o f t h e - t u m o r w e re i n o c u l a t e d
o f t h e same s t o c k .
i n t o the o r i g i n a l
-O rig in a lly
■Plymouth. Rock b i r d s .
hen and i n t o 2 o t h e r hens
t h e t umor grew v e r y s l o w l y . a n d o n l y
In b i r d s o t h e r t h q n h i s
During th e f i r s t
few
t h e g r o w t h s w h i c h r e s u l t e d were e n c a p s u l a t e d and
■only r a r e l y gave - r i s e -to m e t a s t a s e s .
v i r u s from th e o r i g i n a l
in
l a b o r a t o r y s t ock, - ,R ou s ob­
t a in e d t r a n s i e n t growth which eventual I y regressed.
t r a n s f e r s o f t h e t u m or ,
B its
t um or .
No a t t e m p t s were made t o
iso la te
. A t t e m p t s t o grow t umor t r a n s p l a n t
in
pi.geons and g u i n e a p i g s were u n s u c c e s s f u l .
A y e a r l a t e r -2 r e p o r t s appear ed : (Rous,
had s u c c e s s f u l l y
i nduced sarcomas
w h i c h was l a t e r t o be r e f e r r e d
was p r e p a r e d '-(Rous,
191 l a )
I in a warm m o r t a r w i t h
1911a;
1911b')
i n w h i c h Rous
in c h i c k e n s u s i n g a f i l t e r a b l e agent
t o as Rous sarcoma v i r u s .
by g r i n d i n g
-The f i l t r a t e
15. gm o f t r a n s p l a n t e d Chicken 1Tumor
sand and a d d i n g ' R i n g e r ' s s a l t
so lu tio n .
.The de­
b r i s was removed by c e n t r i f u g a t i o n and t h e s u p e r n a t e f i l t e r e d . t h r o u g h a
number '5 B e r k e f e l d f i l t e r .
.The e n t i r e o p e r a t i o n was p e r f o r m e d a t 3§<5 C5
t h e body t e m p e r a t u r e -of a c h i c k e n .
By t h i s
-(Rous,
tim e,
1 911b) .
o f the o r i g i n a l
t h e t umor had shown a d e f i n i t e
Tumors now appeared w i t h i n
30 t o 35 d ay s.
n o d u l e s , wh e re as ,
up t o t h i s
in crease
7 days o f
in v ir u l e n c e
in o c u la tio n
instead
The t umor s a l w a y s . g a v e r i s e t o m e t a s t a t i c
tim e,
m e t a s t a s e s were n ot ed o n l y once (Rous,
2
191 0) .
F in a lly ,
(Rous,
1911b).
ra b bits,
However,
ducks,
In 1913,
t umor s . c o u l d be i nduced
and p i g e o n s s t i l l
spindle c e ll
whether
i n d u ce t umor s
fa ile d
(Rous,
in r a t s ,
.O rig in ally.(R o us,
1910),
g u i ne a p i g s ,
1 91 1bj .
Rous and Murphy d i s c u s s e d some p a t h o l o g i c a l
■tumor had u nd er gon e.
lated
attempts to
in c h i c k e n s o f o t h e r s t r a i n s
changes t h a t t h e
t h e t umor was an encapsu­
sarcoma w h i c h grew r a t h e r s l o w l y .
i nduced by c e l l s o r a ce l I - f r e e f i l t r a t e ,
By '1913,
was a s o f t ,
. n o n - e nc ap s ul a t e d sarcoma w h i c h grew and m e t a s t a s i z e d r a p i d l y .
c e lls
v a r ie d from s p in d le c e l l s
to s h ort,
,gian t c e l l s f r e q u e n t l y being found.
b lu n t,
t h e t umor ,
fria b le ,
The t y p i c a l
o r rounded c e l l s , w i t h
.The t umor was h e m o r r h a g i c and p r o ­
duced :1 a rge q u a n t i t i e s o f a mucinous m a t e r i a l .
..This 1913 f o r m o f t he
t umor resembled t h e -tumors w h i c h were o b t a i n e d
in t h i s
Rous sarcoma v i r u s was i n j e c t e d
l a b o r a t o r y when
i n t o t h e w i n g web o f c h i c k e n s ( M a t e r i a l s
and M e t h o d s ) .
Rous c o n t i n u e d w o r k i n g w i t h h i s f i l t e r a b l e age nt and c h a r a c t e r i z e d
i n a .m an n er w h i c h was n o t c o n s i d e r a b l e
improved upon u n t i l
extensive
v i t r o w o r k w i t h t h e v i r u s was made p o s s i b l e ( Temin and =Rubin,
most e x t e n s i v e g e n e r a l
physico-chemical
p u b l i s h e d by Rous and Murphy i n 1912.
month.
,The a g e n t ,
The
i n 50 p e r c e n t g l y c e r i n f o r
as a g ro un d t umor s u s p e n s i o n ,
ing and t h a w i n g and a l s o h e a t i n g t o 53 C f o r
t h o s e - o f " D o u g h e r t y • ( I 961) .
1958).
They f ou n d t h a t t h e v i r u s c o u l d be
in d r i e d . t i s s u e f o r up t o 7 months o r
15 m i n.
in
c h a r a c t e r i z a t i o n o f t h e v i r u s was
■stored
v i v e h e a t i ng t o '55 C f o r
it
could s u r v i v e
15 mi n, b u t
it
rapid f r e e z ­
could.not sur­
These r e s u l t s c o r r e l a t e c l o s e l y w i t h
- Rous and Murphy (1912)
I
a ls o demonstrated the
- s e n s i t i v i t y o f t h e a g e n t t o s u r f a c e a c t i v e m a t e r i a l s such as b i l e and
3
Saponi n as w e l I as c h l o r o f o r m ,
toluol
Friesen a n d -R u b in -(Ig b l)
that
have,
and a l c o h o l ;
thereby
indicating,
l i p i d s p r o b a b l y p l a y an i m p o r t a n t
as
role
in th e a c t i v i t y o f th e v i r u s .
In i g i I , ' R o u s and Murphy r e p o r t e d t h a t d i s c r e t e t umor s c o u l d be i n ­
duced .on t h e membranes o f d e v e l o p i n g c h i c k embryos w i t h a f i l t r a t e
C h i c k e n . Tumbr I .
It
i s u n f o r t u n a t e t h a t t h e y d i d n o t c o n t i n u e t h i s work
past the p a th o lo g ic a l
c h a r a c t e r i z a t i o n o f t h e tumor s as t h e y were w o r k i n g ,
e v i d e n t l y unknowingly* w i t h
v iru s .
the f i r s t
good means o f q u a n t i t a t i n g
be a b l e t o
Rous and -Murphy (1911)
i n d u ce t umor s
in u t e r o
No f u r t h e r p o s i t i v e
c e rn in g t h i s work,
but
r e p o r t s seem,
i t may w e l l
t h e - c h i c k e n . ( D u r a n - R e y n o l d s,
insinuated th a t
they might
i n mi ce and r a t s w i t h t h e i r t umor ex­
t o have been p u b l i s h e d con­
have been a p r e l u d e t o t h e o r i g i n o f
s t r a i n s o f Rous sarcoma v i r u s whose h o s t
Wi ndl e,
this
T h i s a s p e c t o f Rous sarcoma v i r u s w o r k had t o w a i t 27 y e a r s (Keogh,
1:938) f o r d e v e l o p m e n t .
tra cts.
from
ranges e xt en de d beyond t h a t o f
19^7; Z i l b e r and . Kr y u k o v a j
1957; •Munroe and
1963; A h l s t r b m ejt a_l_. , 1962) .
Rous (1913) was a b l e t o show t h a t b i r d s
and t umor a n t i g e n s .
responded s e p a r a t e l y t o v i r a l
He -f ound t h a t b i r d s w h i c h had managed t o
re je ct t h e ir
t umor s c o u l d n o t be i n f e c t e d w i t h e i t h e r t umor c e l l s o r f i l t r a t e s .
a l s o was a b l e t o o b t a i n b i r d s w h i c h , w e r e r e s i s t a n t t o f i l t r a t e
He
challenge,
/
b u t wo ul d d e v e l o p t umor s
He, t h e r e f o r e ,
i f wh o l e c e l l s w e r e i m p l a n t e d
concluded t h a t
separately or together to
in t h e i r b re a s ts .
t h e v i r u s and c e l l s were a b l e t o a c t e i t h e r
in du ce t u m o r s .
T h i s work was c o n f i r m e d and e x ­
t ended n e a r l y 50 y e a r s l a t e r by Hanafusa e_t aj_. ( 1 9 64 a ) , . Qo ug he r ty and
Morgan ( I 962 ) ,
and Temin ( I 962 ) .
4
• Reu s Vet ,a I . (1919)
attempted t o produce n e u t r a l i z i n g a n t ib o d i e s
a g a i n s t t h e f i l t e r a b l e a g e n t by i n j e c t i n g
ground tumor e x t r a c t s as w e l l
t u m or c e l l s
i n o b t a i n i n g o n l y good a n t i ­
in to
c h i c k e n serum.
ra b b its.
They succeeded
.They f i n a l l y
geese i n t r a v e n o u s l y w i t h
o b t a i n e d n e u t r a l i z i n g a n t i b o d i e s by i n j e c t i n g
t umor s u s p e n s i o n s and b l o o d .
wo u l d s t o p t umor p r o d u c t i o n by t h e f i l t r a t e
mixed p r i o r t o c h a l l e n g i n g t h e c h i c k e n s .
tissu e,
t h e serum and f i l t r a t e w e r e
. A f t e r a d s o rp tio n w i t h chicken
•and Puirdy ( 1933)
c h i c k e n serum.
1961)
n e u t r a l i z e the
Rous and h i s c o l l e a g u e s c o n c l u d e d t h a t a n t i - c h i c k e n sera
wo u l d n o t n e u t r a l i z e t h e i r
( 1 9 56 ;
if
The s e ra o b t a i n e d
t h e g o o s e - a n t i - C h i c k e n Tumor I serum woul d s t i l l
in fe c tin g agent.
as
in fe c tiv e agent.
It
is
in te re stin g
t h a t Gye
r e p o r t e d n e u t r a l i z a t i o n o f t h e Rous a g e n t w i t h a n t i ­
.This
r e p o r t has been s u c c e s s f u l l y
and by F i n k . ( 1 9 6 4 ) .
r e p u d i a t e d by Rubin
, T h e i r e v i d e n c e a g a i n s t Gye and P u r d y ' s
d a t a ( 1933) was based upon t h e f a c t t h a t a n t i - c h i c k e n sera c o u l d p r e v e n t
t u m or d ev e lo p me nt by i n t e r a c t i n g w i t h t h e
i n f e c t e d t i s s u e (Rub i n ,
1956),
and t h e f a c t t h a t v i r u s n e u t r a l i z i n g a n t i b o d i e s p e r s i s t e d even a f t e r e x ­
ten sive adsorption of
r a b b i t a n t i -RSV sera w i t h c h i c k e n t i s s u e ( F i n k ,
196 4) .
Rous and h i s f i l t e r a b l e a g e n t h el pe d l a y t h e f o u n d a t i o n f o r a c o n t r o ­
versy which c o n tin u e s t o plague avia n l e u k o s is
w i t h c h i c k e n t u m o r s , , Rous i s o l a t e d 3 tjj.mors,
research.
,W hile working
Chicken Tumors
I , . V I I , .and
XVI I I , w h i c h we re, ■t r a n s m i s sab I e by a f i l t e r a b l e a ge nt (Rous and Murphy,
1.913b) .
The o b v i o u s q u e s t i o n a r o s e .
. W e r e - t h e s e .3 d i f f e r e n t m a n i f e s t a t i o n s
o f t h e same a g e n t o r s i n g l e m a n i f e s t a t i o n s o f 3 r e l a t e d a g e nt s ?
a g e n t s we re a l i k e
sue, a l l
in t h a t a l l
The 3
c o u l d be s t o r e d w i t h i n d e s i c c a t e d t umor t i s ­
passed t h r o u g h some B e r k e f e l d f i l t e r s
b u t were r e t a i n e d by
5
e x t r e m e l y f i n e ones,
■.Murphy,
we re a l l
1913b) .
and a l l
p r o du c ed m a l i g n a n c i e s
in c h i c k e n s (Rous and
T h e r e f o r e , . Rous and Murphy (1913b)
s ugges ted t h a t t h e y
3 o f I c l a s s , " w h a t e v e r t h a t c l a s s may b e " .
However,
C h ic ke n Tumor I v i r u s a l w a y s gave r i s e t o a s p i n d l e - c e l l e d
1911a). a n d . C h i c k e n Tumor Vl I v i r u s ,
1.912) and- C h icke n Tumor X V I I I
p e cu lia r
sin ce
sarcoma (Rous,
t o an o s t e o c ho n dr o sa r co m a (Rous e t a l . ,
viru s,
to a sp in d le -c e lle d
i n t r a c o n a l i c ' u l a r p a t t e r n (Rous and Lang,
1913),
sarcoma w i t h a
I t was co nc l ud e d
t h a t t h e s e 3 a g e n t s composed a new group o f e n t i t i e s c a p a b l e o f c a u s i n g
neoplasms o f d i v e r s e c h a r a c t e r s
in c h i c k e n s (Rous and Murphy,
19 1 3 b ) .
' H o w e v e r , . Rous and Murphy p o i n t e d o u t t h e u n c e r t a i n t i e s o f c l a s s i f y i n g
agents s t r i c t l y
the
on t h e b a s i s o f t umor p a t h o l o g y when t h e y showed t h a t ,
d uring the s e le c tio n f o r
r a p i d l y g r o w i n g C h icke n Tumor XVI I I v i r u s ,
the
h i s t o l o g y o f t h e t umor came t o resemble more c l o s e l y t h a t o f Ch ick en Tumor
I.
In 1914,
however,
they demonstrated t h a t chickens which were r e s i s t a n t
t o C h ic ke n Tumor Vl I were n o t
Murphy,
191 4) ;
r e s i s t a n t t o Chi cke n Tumor I ( Rous and
t h e r e b y , o p e n i n g new p a t h s f o r t umor v i r u s c l a s s i f i c a t i o n
and i d e n t i f i c a t i o n
and s u p p o r t i n g t h e i r c o n t e n t i o n t h a t t h e s e were
se para te v i r u s e s .
Rous was n o t q u i c k t o c o n c l u d e t h a t he was d e a l i n g w i t h a v i r u s .
deed,
-firs t
in d i s c u s s i n g t h e f i l t e r a b l e
t e n d e n c y was t o r e g a r d
b u t he a l s o f e l t
that
in t u r n ,
to
1 9 1 1 b ) , he s t a t e s t h a t t h e
i t as a s e l f - p e r p e t u a t i n g p a r a s i t i c o r g an i s m,
i t was p o s s i b l e t h a t t umor c e l l s
s t i m u l a n t which r e s u lt e d
them.,
a g e n t (Rous,
In­
i n n e o p l a s t i c changes
re le a s e the ch em ical.
t i o n o f t h e a g e n t (Rous and Murphy,
s e c r e t e d a chemical
in a f f e c t e d c e l l s c a u s i n g
A f t e r h i s work on c h a r a c t e r i z a ­
1912) , he c o n c l ud e d t h a t
i t was a
6
l i v i n g o r g a n i s m and i n 1913 (Rous and Murphy,
a g e n t as a " c a u s a t i v e v i r u s " .
1 913a) ,
referred
The f a c t t h a t Peyt on Rous,
t o the
l e s s t ha n 15
y e a r s a f t e r B e i j e r i n c k 1s i n t r o d u c t i o n o f t h e c o n c e p t o f f i l t e r a b l e
( B e i j e r i n ck ,
189 9) , was a b l e t o
characte rize
it
p h ysica lly,
iso la te a v iru s ,
recognize
c h e m i c a l l y and a n t i g e n i ca l I y ,
it
viruses
as such,
and s u g g es t t h a t
i t was o n l y I o f a group o f a v i a n t umor v i r u s e s w h i c h m i g h t s e r v e as a
model
f o r t h e cause o f c a n c e r
a b ilitie s
in man, was a t r i b u t e
to h is s c i e n t i f i c
and i n s i g h t .
Geneal ogy o f Rous Sa rcoma V i r u s
The d i v e r s i t y o f c o n d i t i o n s and t h e s e l e c t i o n f o r v a r i o u s c h a r a c ­
te ris tic s
i n t h e many l a b o r a t o r i e s o v e r t h e p a s t 50 y e a r s have r e s u l t e d
several ' ! s t r a i n s "
L i cs.
o f Rous sarcoma v i r u s
(RSV) w i t h d i f f e r i n g
in
c h a r a c t e r ! s-
The f o l l o w i n g g e n e a l o g y o f RSV i s a m o d i f i c a t i o n o f t h a t p u b l i s h e d
by Simons and D o u g h e r t y ( 1 9 6 3 ) :
^
A. C aude
'Y.
(1941)
High t i t e r and
S t a n da r d RSV(B)
W. R. Bryan
V
H. Rubin
P. Rous (1911)
( b e f o r e 1924)
W. E. Gye
W. J .
"~-
I
I
P u r d y - ( 1 9 2 9 ) RSV(29)
(1935)
C. R. Amies
I
I
J . G. C a r r — ——^ Z i l b e r
RSV(Carr-Zilber)
R. J .
(1948)
C. H a r r i s
P. J . Simons------ > RSV(H)
Ch. Oberl ing
(1954)?
Schmic t - R u p p in
NZ
(I960)?
C. G. A h l s t r o m
v
Huebner RSV(SR)
7
The d o t t e d 'I i n e s r e p r e s e n t c o n j e c t u r e ;
approxim ate times o f
the dates
in p a r e n t h e s i s
r e c e i p t o f the v i r u s s to c k s ;
represent
t h e known s t r a i n d e s i g ­
n a t i o n s a r e l i s t e d whe ne ve r p o s s i b l e f o l l o w i n g t h e name o f t h e i n d i v i d u a l
usin g the d e s i g n a t i o n .
-These v a r i o u s s t r a i n s o f RSV have t h e i r own d i s t i n g u i s h i n g c h a r a c ­
te ris tic s .
(RAV)
,RSV(B)
c a rrie s
h i g h c o n c e n t r a t i o n s o f Rous A s s o c i a t e d V i r u s
( R u b i n and V o g t , . 1962)
■culture (se e .b e lo w ).
R$V(SR)
and f o r m s s h a r p ,
d is tin c tiv e foci
is not d e f e c t i v e ,
in t i s s u e
p ro du ce s f o c i w h i c h a r e
• q u i t e d i s t i n c t f r o m t h o s e o f RSV(B)
and w i l l i n o t r e a c t w i t h RSV(B)
tra liz in g
i s a n t i g e n i cal I y d i s t i n c t f r o m RSV(Zg),
s e ra ( s e e b e l o w ) .
produces n o . f o c i
RSV(H)
in t i s s u e c u l t u r e s ,
neu­
and p r o d u c e s tumor s on t h e c h o r i o ­
a l l a n t o i c membrane o f c h i c k s w h i c h a r e d i s t i n g u i s h a b l e f r o m t h o s e o f
RSV(ZSi)
and RSV(B)
i n f e c t mammals,,
w ill
also
( D o u g h e r t y ^ t _aj_., . 1963) .
w ill
i n c l u d i n g p r i m a t e s .(Mun roe and W i n d l e , - 1963) . - RSV(SR)
i n f e c t mammals and cause chromosonal
(Ahl s t rom _et^ a j _ . , . 1962; :N i c h o l s e_t aj _. , 1964) .
(1965)
RSV ( C a r r - Z i l b e r )
have succeeded i n
i n d u c i n g t umor s
b r e ak s
in human c e l l s
■Recent l y ,
R a b o t t i _et a I .
i n t h e b r a i n s o f h am st er s w i t h
RSV(B).
- Rous Sa rcoma V i r u s Cl ass i f I c a t i on
RSV i s a. member o f t h e a v i a n l e u k o s i s group o f v i r u s e s ;( J u n g h e r r et_
a l .,
1 94 1 ).
- Th i s gr oup
e rythrob la stosis,
in c lu d e s the v i r u s e s o f avian lymphomatosis,
m yeloblastosis,
and o s t e o p e t r o s i s , . a s w e l l
)
as v a r i o u s
'8
•sol id t umor v-i r u s e s - s u c h as t h e F u j i n a m i
v i r u s (Beard,
195 7) ^
A ll
the
v i r u s e s o f t h i s group so f a r examined a re • he I i cal : RNA v i ruses. -(Howat son,
1964) . w h i c h ' m u l t i p l y
at
the c e l l
Bea rd ,
in the cytopla sm o f
i n f e c t e d c e l l s and a r e r e l e a s e d
membrane ( Haguenau and Beard,
19 6 3 ) .
S tru c tu ra lly ,
1962’; Vogt and R u b i n,
t h e y resemble t h e m y x o v i r u s e s
19 6 1;
in t h a t they
have an o u t e r e n v e l o p e e n c l o s i n g an i n n e r membrane wh.ich e n c l o s e s t he
n u c l e o i d ( Howatson,
1964;
Beard,
1963; DourmasNon and Simons,
gr oup o f v i r u s e s sh ar e s a common s o l u b l e a n t i g e n w h i c h
p a r t i c l e s and i n n e o p l a s t i c c e l l s
1964) .
1961) ..
is found
in v i r u s
i nduced by t h e v i r u s (Huebner e t a l . ,
These v i r u s e s a l s o sh ar e a number o f n e u t r a l i z i n g a n t i g e n s ,
which are not
This
a ll
r e c i p r o c a l , ! y s u s c e p t i b l e t o t h e same a n t i b o d i e s ( B e a r d ,
Simons and. D o u g h e r t y , 1963;
F r i e s e n and, R u b i n,
of
1957;
I 96 I ) .
Assay o f Rous. Sarcoma- V i r u s
P r o b a b l y t h e most v a l u a b l e c o n t r i b u t i o n
t o t h e s t u d y o f RSV has been
t h e d ev e lo p me nt o f an i n v i t r o assay sys tem (Temin a p d - Rubi n ,
system r e l i e s on t h e f o r m a t i o n o f f o c i
t o t h e amount o f
on a p e t r i
accuracy,
d i s h was shown t o be
in f e c t in g virus.
c e n t o f t h e c e l l s were i n f e c t e d : ( T e m i n and:Rub i n ,
■number o f f o c i
on a p l a t e were k e p t w i t h i n
195 8) ;
u n til
I t o 10 p e r
therefore,
if
the
the l i m i t s o f s t a t i s t i c a l
t h e t i t e r o f a v i r u s s t o c k c o u l d be e xp r es se d
■ u n i t s (FFU)
This
o f t r a n s f o r m e d c e l l s on a m o n ol a y er
o f c h i c k f i b r o b l a s t s . . The number o f f o c i
d ir e c t ly pro po rtio n al
1 958).
in focus formin g
p e r ml o f v i r u s .
D i r e c t m i c r o s c o p i c c o m p ar i s on o f t h e number o f v i r u s e s
•and t h e number o f . f o c i
in a c u l t u r e
o b t a i n e d by f o c u s a ssay showed t h a t t h e r e were a bo u t
9
750 v i r u s p a r t i c l e s p r e s e n t f o r each" focus p reduced -. (Crawford and C r a w f o r d ,
196 1) .
However,
t h e y c a l c u l a t e d t h a t t h e r e . m i g h t a c t u a l l y be 15 p a r t i c l e s
p e r FRU i f one assumed t h a t t h e use o f ' n o n - c o n f I u e n t m o n o l a y e r s l owered
t h e e f f i c i e n c y o f t h e as say system and t h a t ' o h I y 10% o f t h e " c e l l s were i n ­
fected.
Considering the rapid
in a ctiva tio n
(Rubin,
loss o f v i a b i l i t y
o f v i r u s e s due t o , h e a t
1955) , t h e system wo u ld seem t o be r a t h e r e f f i c i e n t .
T h i s system has been p l a g u e d by many d i f f i c u l t i e s .
F irs t,
focus f o r ­
m a t i o n may be supp re sse d by h a v i n g t oo . ma n y c e l l s on t h e p l a t e a t t h e t i m e
of
i n f e c t ion ;(Rub i n,
o r o v e r 10% c a l f
• P r i n c e . (1962)
resulted
1960b ) .
.. Second,
t h e use o f f e t a l
serum s u p p r e ss e s f o c u s f o r m a t i o n
c a lf
(Rubin,
(R IF ) , which
influence of fa c to rs
i n t h e medium,. Rubin ;(1960a)
This d iscovery
l e d t o t h e use o f
in te rfe rin g fac­
c i a t e d vir.uss(RAV).
i n t e r f e r e n c e a ssay f o r a v i a n
19 6 0 a ; ; Rubi n and V og t ,
the d is c o v e r y o f the i n t e r f e r i n g
viru s ca rrie d
1962), w h i c h
. The - f a c t t h a t RAV was f ou n d t o .be p r e s e n t
t o TO p e r c e n t o f t h e c e l l s
and C r i t t e n d e n ejt aj_.
(1963)
chickens g ive r is e to g e n e t i c a l l y
Rous a s s o ­
i n concen­
1963 ) e x p l a i n e d why
i n a p l a t e c o u l d be i n f e c t e d w i t h RSV.
Besides o b t a in in g a r e d u c tio n o f f o c i
i n f e c t e d w i t h . RSV.
in tu r n led t o
i n RSV s t o c k s ,
t r a t i o n s up t o 10 t i m e s t h a t o f RSV',(H a na fu s a e_t
(1964)
f ou n d
i n t e r f e r e d w i t h ' RSV i n f e c t i o n , a n d t h u s s t o pp ed f o c u s f o r ­
■leukosis v ir u s e s (Rubin,
o n l y 'I
Third,
in the suppressio n o f focus f o r m a t i o n .
t h a t t h e embryos f r o m c e r t a i n eggs c a r r i e d a v i r u s , . R o u s
mation.
1 96 0 c ) .
F e t u i n,
found t h a t the omissio n o f t r y p t o s e phosphate b r o t h ; (D ifc o )
•Besides the
tor
serum,
due t o v i r a l
in te rfe re n c e ,R u b in
have shown t h a t c e r t a i n
r e s i s t a n t embryos
stra in s of
w h i c h c a n n o t be
. 10
- D e f e c t I veness o f Ro u s<Sarcema- V i rus
In 1 9 6 2 , Rublh and Vogt
t h e Bryan ihigh' t i t e r
s t r a i n o f RSV.
c a lle d Rous,associated v i r u s
c o n c e n t r a t i o n s t h an RSV.
that foci
r e p o r t e d f i n d i n g "2 v i r u s e s
in ' t h e i r stocks o f
One was RSV i t s e l f and t h e o t h e r t h e y
( RA V) . -RAV was a l wa y s p r e s e n t
i n much h i g h e r
S u b s e q u e n t l y , . Hanafusa e t a I . (1963)
o f RSV t r a n s f o r m e d c e l l s w h i c h were pro du ce d on p l a t e s
w i t h h i g h d i l u t i o n s o f RSV w o u l d n o t p r o du c e v i r u s .
t h e s e n o n - p r o d u c i n g (NP)
c e l l s wo ul d p r o d u c e -RSV o n l y
infected
They a l s o showed t h a t
if
s u p e r!nfected w it h
RAV o r / s o m e o t h e r a v i a n l e u k o s i s v i rus . (Hanafusa e t a j_. , 1963;
Thus,
reported
1964a).
i t was shown t h a t RSV depended upon a h e l p e r v i r u s f o r m a t u r a t i o n .
. They c o n c l u d e d t h a t t h e " ' RSV genome l a c k e d t h e
m a t u r a t i o n - w i t h i n an i n f e c t e d c e l l
:A word o f c a u t i o n s h o u l d
1964a)
data t o
and RSV mu st ,
in je c t e d here.
r e p o r t t h a t RAV was f ou n d i n a l l
t h e r e f o r e , ■be - d e f e c t i v e .
- A l t h o u g h lHanafusa e t a l . . ( 1963 ;
RSV c u l t u r e s t e s t e d ,
t h e r e - w a s no
i n d i c a t e t h a t RSV(H),-RSV ( C a r r - Z i l b e r ) , o r RSV(29) were t e s t e d . .
One s h o u l d n o t c o n c l u d e t h a t a l l
,RSV(B)
in f o r m a tio n necessary f o r
s t r a i n s o f RSV a r e d e f e c t i v e
has been e x t e n s i v e l y - s t u d l e d ,
and,
as w i l l
since o nly
be..shown b e l o w ,
RSV(SR)
has been f o u n d n o t t o b e - d e f e c t i v e .
P r o p e r t i e s o f RSV C o n f e r r e d by H e l p e r Vi r uses
Hanafusa e t a I . ( 1964a) were a b l e t o show t h a t t h e RSV, p roduced by NP
c e l l s as a r e s u l t . o f
helper v ir u s .
. That
induction,
is,
if
c a r r i e d th e n e u t r a l i z i n g a n t ig e n o f the
t h e 'NP c e l l s were, induced by an a v i a n lymphoma\
to s is viru s,
the r e s u l t i n g
l y mp h om a t os i s s e r a .
RSV c o u l d be n e u t r a l i z e d . b y s p e c i f i c a n t i - a v i a n
Therefore,
upon i n f e c t i n g a ce l I , . RSV was shown t o
11
c a s t o f f I t r s p r o t e i n c o a t and exchange i t ' f o r t h a t o f any h e l p e r v i r u s
which mig ht b e " p r e s e n t .
,The h e l p e r t h a t was most handy w a s , o f c o u r s e ,
RAV, b u t by p r o p e r m a n i p u l a t i o n , . any a v i a n l e u k o s i s v i r u s c o u l d serve as
t h e , h e l p e r and t h u s c o n t r i b u t e t h e c o a t o f t h e r e s u l t i n g RSV p a r t i c l e s .
- T h i s was v e r y m u c h'I i k e p h e n o t y p i c m i x i n g . ( N o v i c k and Szi l o r d , . 19 5 11
. S t r e i s i n g e r , . 19 5 6 ) ■ e x c e p t t h a t o n l y I o f t h e v i r u s e s ,
i . e . , ; RAV, c o n t r i b u t e d
the c o a t " p r o t e i n ,
. H a n a f u s a . e t a l , (1964b) ■showed t h a t
" RSV and RAV a t t h e same t i m e ,
l a r t o t h a t o f RAV a l o n e .
lim itin g
step
i nduced NP cel I s pro du ce d b o t h
and t h a t t h e r a t e o f v i r u s
- L a t e r , , R u b i n c o n c l u d e d (1964)
r e l e a s e was s i m i ­
t h a t the ra te
i n , RSV p r o d u c t i o n .w a s t h e c o a t i n g o f t h e v i r u s .
t h a t t h e r a t e a t w h i c h v i r u s was r e l e a s e d f r o m t h e c e l l
This.meant
was d e t e r m i n e d by
■t h e ,heI p e r v-i r u s .
I
■A second p r o p e r t y o f RSV which, has been lShqwn t o ,be a f f e c t e d by t h e
helper
involves v i r a l
in terfe re n ce.
. Rubin (1960a)
f o un d t h a t
in fe ctio n of
c h i c k f i b r o b l a s t s w i t h an a v i a n l e u k o s i s v i r u s p r i o r t o e x p o s u r e o f t h e
same c e l l s
t o iRSV wo u ld p r e v e n t -RSV i n f e c t i o n .
t o be a p r o p e r t y
and Vogt (1965)
RAV 2 ,
i nduced b y ^ RAV ( R u b i n and V o g t , . I 962 ) .
i s o l a t e d 2 s t r a i n s o f RAV.
. H a n a f u s a . ( 1965)
They were - d e s i g n a t e d RAV I and
, The 2 RAV1s : w e r e - f o u n d t o be a n t i g e n i c a l l y d i s t i n c t and d i f f e r e d
th e ir a b ility
ience,
. I n t e r f e r e n c e was shown a l s o
to
in fe c t c e r ta in s tra in s o f chick f ib r o b la s t s .
in
. For co nve n­
t h e RSV genome t h a t was i n s i d e a RAV I c o a t was d e s i g n a t e d RSV(RAV I ) ,
. and t h a t w h i c h . w a s
i n a RAV 2 c o a t ,
RSV(RAV 2 ) .
I f c h i c k - f i b r o b l a s t s were
i n f e c t e d w i t h R A V - I , and s u pe r i n f e c t e d . w i t h RSV(RAV I) , no f o c u s f o r m a t i o n
12
occurred.
If,
h o w e v e r , . t h e RAV I
(RAV 2 ) , f o c u s f o r m a t i o n ,
c e p tib ility
to
i n f e c t e d c e l l s were c h a l l e n g e d w i t h RSV
and t h e r e f o r e
in fectio n ,
occurred.
tio n
therefore,
in to a c e ll
Indeed,
sus­
i n t e r f e r e n c e was f ou nd t o ,be a p r o p e r t y o f t h e v i r u s c o a t s
and h e n c e - a • p r o p e r t y . g o v e r n e d by t h e h e l p e r v i r u s .
seem t o , b e ,
Thus,
a property
i n v o l v i n g v i r u s a d s o r p t i o n and p e n e t r a ­
r a t h e r than a p h y s i o l o g i c a l
t h e . -RSV(B)
producing c e ll
I n t e r f e r e n c e would
c o m p e t i t i o n between 2 v i r u s e s .
supports the growth o f 2 v ir u s e s which
must be -phys.iolo,gi c a l I y c o m p a t i b l e .
, RAV was a l s o f o u n d t o
i n f l u e n c e t h e a b i l i t y o f RSV t o
, c h i c k f i b r o b I a s t s . ( V o g t , ,-1-965;> Ha naf u s a , . J9 6 5 ) .
■The - f i b f o b l a s t s w h i ch were
r e s i s t a n t t o RAV 2 were a l s o . r e s i s t a n t t o RSV(RAV 2 ) ,
.Even t ho ugh p r o p e r t i e s
b u t n o t t o RSV(RAV I ) .
i n v o l v i n g f u n c t i o n s o f t h e v i r u s c o a t have been
shown t o be d e t e r m i n e d by t h e h e l p e r v i r u s ,
its a b ilit y
in fe c t certain
t o t r a n s f o r m norma]
ce lls
t h e o u t s t a n d i n g p r o p e r t y o f RSV,
i n t o t umor c e l l s ,
has been,shown t o
be e x c l u s i v e l y a p r o p e r t y o f t h e RSV genome (Hanaf usa e t a ] . , . 1 9 6 4 a ;
1964; and Temi n, . 1962) .
Thus,
cause t umor f o r m a t i o n
in je cted
et a l .,
1964a),and
if
i nduced
in v i t r o c o u l d
i n t o t h e w i n g webs o f c h i c k e n s .(Hanafuaa
t h e s e t umor s s t i l l
were i n f e c t e d w i t h 'RAV.
non-virus y ie ld in g
non-producer c e l l s
Ru b in ,
c o u l d n o t p r odu ce v i r u s u n l e s s t h e y
T h i s m i g h t e x p l a i n t h e r a t h e r common o c c u r r e n c e o f
t umor s ( S h i m i z u and R u b i n , . 1 9 6 4 ) .
The i n f e c t i o n o f
b i r d s w i t h ' l o w doses o f RSV o r t h e i n f e c t i o n o f b i r d s h a v i n g a n t i - l e u k o s i s
a n tib o d ie s could r e s u lt
f e c t i o n o f RSV c e l l s .
been.found
i n such n o n - p r o d u c i n g tumors by p r e v e n t i n g RAV i n ­
However,
i t - s h o u l d be k e p t
i n t umor e x t r a c t s w h i c h w i l l
in mind t h a t m a t e r i a l
n e u t r a l i z e RSV ( A n d r e w s , .1932;
has
13
- Dougherty . e t a I . ,
i 960 ) .
, T h i s c o u l d a-.l so e x p l a i n why RSV m i g h t n o t be
o b t a i n e d if rom some t u m o r s .
-.The - E f f e c t s - o f
I nhib-i t o r s on Rous Sarcoma Vi rus Repl i c a t i on
S i nc e r i b o n u c l e i c a c i d
■material
o f RSV ( B a t h e r ,
C r a w f o r d , . 196 1) ,
b it
(RNA) was shown t o c o n s t i t u t e t h e g e n e t i c
1 9 5 7 ; ; E p s t e i n and H o l t ,
i t was o n l y l o g i c a l
RSV r e p l i c a t i o n
1 9 5 8 ; ; C r a w f o r d and
t h a t a t t e m p t s wo u ld be made t o
in h i­
u s i n g a n t i m e t a b o l i t e s w h i c h were known t o a f f e c t RNA
'synthesis.
- Gol de and V i g i e r p u b l i s h e d wo rk showing t h a t RSV r e p l i c a t i o n c o u l d be
i n h i b i t e d by 5 - f l u o r u r a c i I
(PU)
( G o l d 4- and V i g i e r , I 9 6 I and 196 3) .
f o u n d t h a t t h e a d d i t i o n o f 20y ^ g / m l
hrs, .a f t e r
in fe c tio n
o f FU t o v i r u s
infecte d c e lls
i n h i b i t e d v i r u s p r o d u c t i o n a t Zk h r s .
They
I t o 10
- They a l s o f o u n d
t h a t t h e a d d i t i o n o f t h e same c o n c e n t r a t i o n s o f FU between I and 20 h r s .
a fte r
in fe c tio n
ever,
that c e lls
su pp r es s ed 48 h r .
v iru s production.
i n f e c t e d w i t h RSV f 0 r 3 days became c o m p l e t e l y r e f r a c t ! I e
t o t h e e f f e c t s o f FU.
. The a c t i v i t y o f FU c o u l d be r e v e r s e d by t h e a d d i t i o n
. of u r a c i l
(200/^g/ml)
e ffe cts.
On t h e b a s i s o f t h e s e e x p e r i m e n t s ,
at
,They d i s c o v e r e d , h o w ­
t o t h e medium.
l e a s t l , and p o s s i b l y ' 2 , . / F U
, T h y m i d i n e wo ul d n o t
' f o r v i r u s p r o d u c t i o n a t Zk h r s .
t h e y c o n c l u d e d t h a t t h e r e was
s e n s i t i v e steps in v o lv e d
, The f i r s t o c c u r r e d between I and 10 h r s .
a fte r
i n RSV i n f e c t i o n .
i n f e c t i o n and was n e c e s s a r y
The second o c c u r r e d between 5 and 20 h r s .
and was n e c e s s a r y f o r v i r u s p r o d u c t i o n a t 48 h r s .
t o 3 days were r e q u i r e d : f o r a l l
r e v e r s e t h es e
th e ir
It
infected c e lls
i s p o s s i b l e t h a t up
to begin producing
■14
V/i!rus and t h a t ' l a t e r a d d i t i o n s o f FU were m e r e l y b l o c k i n g t h e same e a r Iiy
steps
i n t h e c e l l s w h i c h were j u s t b e g i n n i n g t o s y n t h e s i z e v i r u s .
-Following t h i s ,
. a m e t h o p t e r l n (AM),
.(1:963)
w o r k was p u b l i s h e d on t h e u s e o f S c t i nomycin D ( A D ) ,
and m i t o m y c i n C (MG)
t o stop v i r a l
showed t h a t AD a t . c o n c e n t r a t i o n s f r o m 0.1
m u ltip lic a tio n .
from 8 h rs .
Temin
t o 1 0 / c g / m l wo u ld st op RSV
.He -found t h a t t h e a d d i t i o n o f 0.2y%.g/ml o f AD t o c e l l s
b e f o r e t o 2 4 - hrs.. a f t e r
production.
re p lic a tio n .
If,
however, 0 . j y ^ g / m l
in fe ctio n
- w i t h i n 112 t o T6 h r s .
v iru s production
the c e l l s
st op ped RSV
o f t h e a n t i b i o t i c was added t o c e l l s
t h a t had been c a r r y i n g RSV f o r s e v e r a l
,and removed 8 . h r s . l a t e r ,
irre v e rs ib ly
g e n e r a t i o n s - ( e s t a b l i s h e d Rous cel I s ) ,
recovered t h e i r a b i l i t y
a f t e r treatment.
t o produce v i r u s
, T h u s , ,AD wo ul d r e v e r s i b l y
in e s t a b l i s h e d Rous c e l l s .
in h ib it
Temin c o n f i r m e d and e xtended
t h e s e r e s u l t s w i t h e x p e r i m e n t s u s i n g AM - (Tern i n , . 1:964a) .
He f o u n d t h a t 10"" 7
M AM had no e f f e c t on v i r u s p r o d u c t i o n by e s t a b l i s h e d Rous c e l l s , . b u t
. -AD,
i t ,would i n h i b i t v i r u s p r o d u c t i o n
. i ,-e,, I t o 4 h rs .
ever,
re ve rsib le .
irre v e rs ib le .
The e a r l y
if
i t was added soon a f t e r
lik e
in fe c tio n ,
i n h i b i t i o n o f v i r u s p r o d u c t i o n by AM was-, how­
T h i s was i n c o n t r a s t t o t h e e a r l y e f f e c t o f AD w h i c h was
. S i n c e AD a c t s .by b i n d i n g t o . d e o x y r i b o n u c l e i c a c i d .(DMA)
. mol e cu l e s, , t h u s p r e v e n t ing messenger RNA s y n t h e s i s ( G o l d b e r g e t aj_. , 1962) ,
Temin c o n c l u d e d ( 1 9 6 3 ;
■the-cells
1964a;
1964b)
in a p r o v i r u s -state,
.Accordingly,
upon i n f e c t i o n ,
t h a t RSV e x i s t e d and f u n c t l o n e d - w i t h i n
and t h a t t h e p r o v i r u s was composed o f DMA.
t h e RSV genome w h i c h was. RNA, e n t e r e d t h e c e l l
and a c t e d as a t e m p l a t e f o r t h e s y n t h e s i s o f
v iru s
i t s DNA p r o v i r u s .
t h e n d i r e c t e d v i r u s p r o d u c t i o n by t h e c e l l .
The p r o ­
Temin a d d e d - s t r e n g t h t o
15
h i s h y p o t h e s i s by d e m o n s t r a t i n g t h a t t h e r e was a l a r g e r amount o f DNA,: in
■v iru s
infected c e lls
infecte d c e l l s
t h a t wo u l d anneal w i t h v i r a l
( Te mi n,
1:964b).
ence i n t h e amounts o f v i r a l
: and non- i n f e c t e d c e l l s ,
RNA t h a n t h e r e was in non-
A l t h o u g h Temin showed a c o n s i s t e n t d i f f e r ­
RNA w h i c h w o u l d anneal w i t h DNA f r o m i n f e c t e d
t h e d i f f e r e n c e was n o t g r e a t .
f a c t t h a t RSV(B), . w h i c h ' Temin used,
is d e fe c tiv e ,
Also.,
in v i e w o f t h e
t h e h y b r i d i z a t i o n de­
m o n s t r a t e d must have been between RAV RNA and DNA, n o t RSV RNA and DNA.
In v i e w o f t h e s e f a c t s ,
the work w i l l
need c o n f i r m a t i o n , p r e f e r a b l y w i t h a
n o n - d e f e c t i v e s t r a i n o f RSV, u s i n g more s e n s i t i v e t e c h n i q u e s .
V i g i e r and Golde (1:964)
( 1963) .
o b t a i n e d t h e same r e s u l t s w i t h AD as d i d Temin
They a l s o used MC and f o u n d t h a t ,
w i t h i n 24 h r s .
a fte r
in fe c tio n
resulted
as w i t h AD,
in
t h e a d d i t i o n o f MC
irre ve rsib le
suppression o f v i r u s
p r o d u c t i o n . • H o w e v e r , , u n l i k e AD, MC wo u l d n o t s i g n i f i c a n t l y . i n h i b i t v i r u s
production
i f added t o e s t a b l i s h e d Rous c e l l s a t c o n c e n t r a t i o n s t h a t d i d
n o t p r o du c e c e l l
death.
■On t h e b a s i s o f , t h e i r d a t a ,
V i g i e r and Golde pro po se d f o u r models t o
e x p l a i n the e a r l y s e n s i t i v i t y o f the v i r u s t o these a n t ! m e t a b o l i t e s .
F irs t,
an e a r l y enzyme coded by h o s t DNA c o u l d be r e q u i r e d t o u n c o a t t h e v i r a l
RNA.
T h i s model wo u l d f i n d p r e c ed e nc e i n t h e d i s c o v e r y o f such an enzyme
f o r u n c o a t i n g pox v i ruses .(Abe-1 , . 1963) .
Second, a s p e c i a l
enzyme coded by
h o s t DNA m i g h t be r e q u i r e d t o open a se co nd ar y s t r u c t u r e o f RSV RNA.
mode,l was based upon t h e
in h ib itio n of
re p lic a tio n of
a d o u b l e s t r a n d e d RNA v i r u s .(Gomatos and Tamm,
^ t aj _. , . 1962) ; however, .Temin ( 1964b)
1963),
.This
r e o v i r u s 3, w h i c h
is
by AD and MC (Gomatos
showed t h a t RAV RNA and RSV RNA were
16
se n s itiv e to
RNA1s ,
ribonuclease
in d ica tin g
t h a t they are -probably s i n g l e stranded
, T h i r d , . t h e -RNA d ep endent RNA p o l y m e r a s e n e c e s s a r y f o r v i r a l
RNA
■ s y n t h e s i s m i g h t be coded by c e l l u l a r DNA, o r f o u r t h , T e m i n ' s p r o v i r u s model
m i g h t be used t o e x p l a i n t h e r e s u l t s .
Of t h e s e 4 h y p o t h e s e s ,
the f i r s t
c o a t i n g enzyme has been i s o l a t e d .
• s t r a n d e d -RNA t o be- f o u n d w i t h i n
seems most l i k e l y
s i n c e such an un-
.The p o s s i b i l i t y e x i s t s f o r a d ou b l e
t h e c e l I , b u t t o d a t e , -AD has n o t been such
c e s s f u l ' l y combined . w i t h d o u b l e s t r a n d e d RNA ( G o l d b e r g and R e i c h , . 1964) .
■Therefore, . d i r e c t
i n t e r a c t i o n o f A D . w i t h such RNA woul d n o t seem a l i k e l y
explanation f o r v ir a l
in h ib itio n .
Th er e has been no e v i d e n c e p r e s e n t e d
t h a t non i n f e c t e d c e l l s c o n t a i n a n . RNA d ep endent RNA p o l y m e r a s e .
In-fact,
i t s p r e s e n c e has been shown t o be c o m p l e t e l y dependent upon t h e
in fe ctin g
v i r u s ( ( B a l t i m o r e e t a ] _ . , 1 96 3 ).
why t h e - c e l l
wo u ld c a r r y t h e
It
is,.th e re fo re ,
d iffic u lt
t o understand
i n f o r m a t i o n t o code a m o l e c u l e t h a t
n e v e r use as w o u l d be r e q u i r e d by t h e t h i r d model.
i t would
. However, Weiss (1963)
has r e p o r t e d t h a t DNA dependent RNA p o l y m e r a s e can t r a n s c r i b e
inform ation
i n t h e f o r m o f p o l y r i b o n u c l e o t i d e s d i r e c t l y o f f o f RNA m o l e c u l e s .
fore, ' i t
There­
i s p o s s i b l e t h a t RSV RNA c o u l d compete w i t h d e l l u l a r DNA f o r t h e
use o f RNA p o l y m e r a s e and t h e r e b y r e q u i r e - a c e l l u l a r enzyme f o r
re p lic a tio n
T e m i n ' s h y p o t h e s i s and e x p e r i m e n t s have -been based on t he b e l i e f t h a t AD,
,MG, and AM i n t e r f e r e n c e w i t h " RSV r e p l i c a t i o n
v iru s ,
.However, ..AD has been f o u n d . t o
ca tion e a r ly
. 1965) .
i n t he m u l t i p l i c a t i o n
i s u n i q u e s i n c e RSV i s an RNA
in te r fe re w ith
cycle (B arry,
i n f l u e n z a v i r u s rep I i I
e t aj _. , 1962; Whi t e e t a l .
I n f l u e n z a and RSV o b v i o u s l y do n o t behave t h e •same w i t h i n
the c e l l s
17
• T h e r e f o r e , , T e m i n 1s p r o v i rus p o s t u l a t i o n p a r a ! I el ing Tysogeny i s j u s t i f i e d ,
and i f
h i s w o r k can be c o n f i r m e d
in a n o t h e r l a b o r a t o r y ,
t h e r e would be
I i t t I e d o u b t t h a t h i s DNA p cov-i rus s t a t e e x i s t e d .and' must exp I a in t h e s e
data.
B u t-u n til
mo d el s,
t h a t t i m e , , t h e - f i rs.t,
c a n n o t be c o m p l e t e l y e x c l u d e d .
. U n de r any c i r c u m s t a n c e s , . t h e
step
o r f o r t h a t m a t t e r any o f t h e o t h e r
is d i f f i c u l t
to e xp la in .
i r r e v e r s i b i l i t y o f t h e f i r s t AD s e n s i t i v e
I f t h e u n c o a t i n g enzyme model were a d o p t e d ,
one wo u ld have t o assume t h a t t h e v i r u s ,
a c tiv it y w ith in
the c e l l
due t o
being
rather unstable,
in a c tiv a tio n o f v ira l
t e m p e r a t u r e s b e t w e e n , 37 and 4 5 ' C . ( D o u g h e r t y , . l . g b l ) .
v i r qs model we re -a cc e pt e d,
loses
its
n u c le ic acid at
I f : T e m i n 1 s.DNA p r o ­
one must assume -e i t h e r t h a t
t h e n u c l e a s e s of- t h e
ce l I b r e a k down t h e n o n - f u n c t i o n i n g DNA, .which woul d be c o n t r a d i c t o r y t o
re ve rsib le
in h ib itio n occurring
t i m e - ' AD-DNA b i n d i n g
in -.e-stabI i shed'Rous c e l l s ,
i s s t r o n g e r t han, a t a n o t h e r .
Also,
o r t h a t a t one
i n t h i s case, .one
-must e x p l a i n :why MC wo u ld a t t a c k t h e p r o v i r u s DNA a t o n e - t i m e and n ot a t
another.
. The MC d a t a c o u l d be e x p l a i n e d by t h e i n v o l v e m e n t o f d i f f e r e n t
• s i t e s on t h e h o s t genome in e a r l y and l a t e - v i r u s
a hypothesis
i f one f a v o r s
i n v o l v i n g t h e h o s t genome.
T h e - l a t e r AD r e v e r s i b l e
m o r e - d if f ic u l t to e xplain ,
■b u i l t
synthesis
in c o n t r a d i c t i o n
( M C , . A M , a n d PU i n s e n s i t i v e )
step
i s even
as t h e s o l e a d o p t i o n o f T e mi n 1S idea has t h e
stated p re v io u s ly .
However,
i f one-includes
in
iTemin's h y p o th e s is the n e c e s s it y f o r cel IularrDNA i n t e g r i t y f o r v i r u s
r e l e a s e , .a f a i r l y
s e n s i t i v e steps
r e a s o n a b l e e x p l a n a t i o n o f b o t h t h e e a r l y and I at e- AD
is p o s s ib le .
18
■One -very i m p o r t a n t f a c t
sio ns o f the e f f e c t o f
has b e e n ' e m i t t e d
i n h i b i t o r s on RSV.
- h e l p e r v.i r u s , , RAV, . f o r RSV m a t u r a t i o n .
in most p a p e r s a n d . d i s c u s ­
- Th a t
is the n e c e s s it y o f the
S i nc e a l l
o f t h e s e s t u d i e s have
r e t i e d upon t h e f o c u s a ssay f o r m a tu r e RSV p a r t i c l e s ,
■ o m i t t e d any s t u d y o f RAV i n f e c t i o n o r
release.
t h e y have c o m p l e t e l y
,T h e o re tica lly,
one m i g h t
say t h a t t h e e f f e c t o f t h e i n h i b i t o r s on ,RAV i s . being d e t e r m i n e d
i f one
■measures t h e i r e f f e c t on RSV, .as RAV d e t e r m i n e s w h e t h e r o r n o t RSV m a t u r e s .
H o w e v e r , RSV i n f e c t i o n , . u n c o a t i n g ,
form a t ion are
i n d e p e n d e n t o f RAV ( Hanafusa o t a]_., 1 96 4a) .
• or t l y, RAV. e v e n t which
can be measured i n t h e s e - e xp e r i m e n t s
maturation- o f the v i r u s .
of
genomic r e p l i c a t i o n and c e l l u l a r t r a n s -
Work w i l l
T h e r e f o r e , . the
is the f i n a l
have t o be done t o d e t e r m i n e t h e e f f e c t
i n h i b i t o r s on each o f t h e s e v i r u s e s s e p a r a t e l y b e f o r e v i r u s
can be u n d e r s t o o d .
taining v iru s
.In doing t h i s ,
stocks,
as i t
re p lica tion
one . i s f a c e d w i t h t h e p r o b l e m o f ob­
i s easy t o o b t a i n . : R S V - f r e e c u l t u r e s o f RAV
( Hanafusa , e t a I . , . 1 96 3 ), . but t o s tudy. RAV-free-RSV w o u l d r e q u l r e a non­
d e f e c t i v e s t r a i n o f RSV.
Such s t r a i n s ,
however, a r e a v a i l a b l e . ( R e s u l t s ) .
I NTRODUCTI ON TOlTHE PROBLEM
! There e x i s t s among t h e v a r i o u s s t r a i n s o f RSV d i f f e r e n c e s
h g s t range,
serological
In o r d e r t o o b t a i n
re la tion sh ip s,
and d e f e c t i v e n e s s . ( G e n e a l o g y o f RSV) .
some idea o f t h e - e x t e n t o f t h e s e d i f f e r e n c e s ,
o f "RSV, , r e p r e s e n t i n g ^ b i o l o g i c a l
studies.
- The -2 s t r a i n s
s e l e c t e d were t h e Bryan h i g h t i t e r , s t r a i n , .RSV(B),
e x te n s iv e ly studied o f a l l
com parative s tan d ard.
studied,.and
The Bryan s t r a i n
T h e ^ S c h m i d t - R u p p i n s t r a i n has ;been much l e s s e x t e n ­
its a b ilit y
to
i n f e c t mammals and mammalian t i s s u e s ,
therefore,
p r o p e r t i e s o f t h e s e 2 s t r a i n s of, RSV.
structural
and c o m p o s i t i o n a l
s e r o l o g y and s t a b i I i t y
suggest th a t
it
diffe re n ces
t o compare t h e b i o ­
P roperties
In t h e v i r a l
in d ic a tiv e of
p a r t i c l e s . s u c h as
und er t i s s u e c u l t u r e c o n d i t Ions we re s t u d i e d .
p r o p e r t i e s w h i c h wo u l d r e v e a l
diffe re n ces
Also
in t h e f u n c t i o n i n g o f t he v i r a l
genome . w J t hi n t h e c e l l .such as p a t h o g e n i c i t y ,
t o RAV i n t e r f e r e n c e ,
19 6 4 ) ,
p r o p e r t i e s d i f f e r e n t f r o m t h os e o f RSV(B).
T h e - p u r p o s e -o,f t h e . f o i l owing w o r k was,
lo g ica l
has been t h e most
t h e s t r a i n s o f RSV and t h e r e f o r e was used as a
i n c l u d i n g h u m a n , f i b r o b l a s t s ^ B j o r n and. ’B on t e n ,
p os se ss es b i o l o g i c a l
2 strains
e x t r e m e s , , we re chosen f o r c o m p a r a t i v e
and t h e Schmidt- Rupp i n ! S t r a i n , . RSV(SR).
siv e ly
including
defectiveness,
su s c e p tib ility
normal, v i r u s g r o w t h and r e l e a s e f r o m c e l l s ,
and t h e
- e f f e c t o f t h e i n h i b i t o r s p u r o m y c i n d i h y d r o c h l o r i d e and a c t I no my cin. D on
v ira l
rep I i c a t i on ;were - examined.
Although there-were d e f i n i t e
s im ila ritie s
between t h e 2 v i r u s e s , s u f f i c i e n t d i v e r s i t y was f ound t o w a r r a n t o n l y
c a u t i o u s g e n e r a l i z a t i o n s t o o t h e r s t r a i n s o f RSV.
MATERIALS AND: METHODS
Prepa r a t I on ' o f Wa t er
A ll
w a t e r used i n p r e p a r i n g t h e t i s s u e c u l t u r e medium e r s o l u t i o n s
w h i c h ‘were t o come . i n - c o n t a c t w i t h t i s s u e - c e l I s was d o u b l e - d i s t i l l e d ; : t h a t
i s , , w a t e r o b ta in e d from a Barnstead s t i l l
was d i s t i l l e d
a second t i m e in
glass.
• P r e p a r a t i o n ’o f Gl a ss wa r e
I m m e d i a t e l y a f t e r use,
a ll
g l a s s w a r e was submerged i n a w e a k 'Purex
-solution u n t il
i t was t o -be -washed.
fo r
i n a I p e r c e n t s o l u t i o n o f 7X ( L i n b r o - C h e m i c a l : C o v , , New
15 m i n u t e s
H a v e n , , Conn.)
, Fo r c l e a n i n g , . a l l
and a l l o w e d t o s t an d u n t i l
t h e w a t e r was cool
t h e g l a s s w a r e c o u l d be h an d l ed w i t h b a r e hands.
b r us he d v i g o r o u s l y .
- w a t e r . - a n d '3 t i m e s
-A fter
"fo il
it
glassware w a s : b o ile d
. Each-piece-was rin s e d
enough t h a t
T h e . g l a s s w a r e ' was then
10 t i m e s
in r u n n i n g , . w a r m t a p
in d i s t i l l e d w a te r .
had d r i e d ,
and s t e r i l i z e d
a ll
g l a s s w a r e was capped o r c o v e r e d w i t h ‘aluminum
in a . h o t a i r oven..
S y r i n g e s and p i p e t t e s w e r e - t r e a t e d
as o t h e r g l a s s w a r e - e x c e p t t h a t t h e y were c l e a n e d by a u t o c l a v i n g f o r 30 t o
60 m i n . . w h i l e
submerged i n a I p e r c e n t s o l u t i o n o f 7X.
- r i n s e d by hand as. a bov e.
- P i p e t t e s we re r i n s e d o v e r n i g h t
S y r i n g e s were t h e ^
i n -a- p i p e t t e - r i n ­
s e r and t h e n r i n s e d 3 t i m e s . w i t h d i s t i l l e d w a t e r and a l l o w e d t o d r y .
drying,
p i p e t t e s and s y r i n g e s were p l a c e d
b a g s. a nd a u t o c l a v e d .
le a st 6 h rs.
A fte r
in r-D isp o-w ra p-pip e tte -o r syringe
A f t e r a u t o c l a v i n g , . t h e y were d r i e d a t 60 C for- a t
21
P r e p a r a t i o n o f T i s s u e C u l t u r e - M e d i urn
Medium 199 w i t h o u t p u r i n e s o r p y r i m i d i n e s was used e x c l u s i v e l y .
A 10
t i m e s c o n c e n t r a t e d s o l u t i o n o f 199A ( 1 9 9 A ' lOX) was p r e p a r e d a c c o r d i n g t o
t h e p r o c e d u r e - o f Vogt - (1963p) as g i v e n
filte re d
t h r o u g h an 02 S e l a s f i l t e r
t h e -199A I OX appear ed s l i g h t l y
in T a b l e I .
. The s o l u t i o n was then
and s t o r e d a t 4 C.
,P rio r to f i l t r a t i o n ,
t u r b i d due t o t h e low sol ub I H t y
o f some o f
the c o n s t it u e n t s .
A 10 p e r c e n t s o l u t i o n o f s o d i urn b i c a r b o n a t e -was p r e p a r e d by m i x i n g
10 gm o f sodium b i c a r b o n a t e
i n 100 ml o f w a t e r and a u t o c l a v i n g
the m ix tu re
f o r 1 5- m i n .
. Sol u t i on DG ,was p r e p a r e d .in J
I.
s t e p s ( V o g t , , 1963p) :
,The f o l l o w i n g were d i s s o l v e d
i n 700 ml o f w a t e r :
I - C y s t e i n e HCl
A s c o r b ic Acid
G l u t a t h l one
2.
100.0 mg
5 0 . 0 mg
5 0 i 0 mg
100 mg o f v i t a m i n A was added t o 10 ml o f 95 p e r c e n t e t h a n o l .
. A f t e r t h e v-i tami n A was d i ssol ved,
t h e sol u t i o n was mixed wi t h
1.00 ml o f a 5 p e r c e n t s o l u t i o n o f Tween 80 in 95 p e r c e n t e t h a n o l .
3.
The s o l u t i o n s p r e p a r e d
added t o b r i n g
then . f i l t e r e d
the t o t a l
i n s t e p s I and 2 were mixed and w a t e r was
volume t o T 000 m l .
t h r o u g h an 02 S el as f i l t e r
The-DG s o l u t i o n was
and s t o r e d . f r o z e n
ml q u a n t i t i e s .
Medium 199A IX . ( V og t , . 1963a) was p r e p a r e d by m i x i n g :
199A10X
S o l u t i o n r DG
1.0% Sodium p ! c a r b o n a t e
P e n ic iIlin
I 0 0 , ml.
I . ml
- 7 ml
2 x ID'S u n i t s
i n 10
22
'T a b le ' I .
. P r e p a r a t i o n o f 199A I OX
D i s s o l ve . in 200 ml o f b o i I i n g w a t e r :
I -Tyrosine
1-Cystine
0 . 7 2 gm
0 . 3 6 'gm
Add 1 . 5 ml o f IN HCl , 600 ml w a t e r and d i s s o l v e :
144.0 gm
: 7.2 gm
18.0 gm
0 . 3 1 6 gm
' NaCl
- KCl
G l ucose
.Phenol Red
D issolve
i n 100 ml o f w a t e r and add t o t h e above s o l u t i o n
KHjf-P04
NagHPG^
Dissolve
I . 08
I .08
-gm
gm
i n 50 ml o f w a t e r and add t o t h e above s o l u t i o n :
'Mg 564 - 7H2O
3.60
gm
D i ssol ve i n T'50 ml o f w a t e r and add t o t h e above sol u t ion
' 2 . 5 2 gm
CaC 1.2
Weigh i n t o a small
b e a k e r and add t o t h e above s o l u t i o n :
N ia c in , ( N i c o t i n i c Acid)
Ni a c i namide - ( N i c o t i n a m i de)
P y r i d o x i n e HCl
P yr i d o x a l HCl
T h i a m i n e HCl
• R i b o f I a v in
i - Inosi t o I
■
' p-Am i nobenzoJ c a c i d
C h o l i n e 'H1CI
V ita m in E (DL-o<-T a c o p h e ro l)
V i t a m i n K ( M e n a d i o n e . Sodi urn
Bisul fa te )
d-Bio tin
F o l ic A cid
d-Ribose
d-2-deoxyribose
Fe(NO3 ) 3 -SH2O
0.18
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
0.18
0.18
0.18
-9.00
9 .0 0
I .80
mg
mg
mg
mg
mg
mg
0. 45
0. 45
0 . 45
0 . 45
0.18
0.18
.
0.90
0.90
0..90
23
Table
I.
Cent I ruled.
' . D i s s o l v e i n a s o l u t i o n o f 0 . 3 6 ml Tween 80 i n 75 ml o f w a t e r and add : t o
t h e above s o l u t i o n :
. C a lcife ro l
Cholesterol
I ,80 mg
3 .6 0 mg
B r i n g t h e above s o l u t i o n t o a t o t a l volume o f 1800 ml by a d d i n g 6 2 5 • ml o f
w a t e r ; t o t h e f i n a l s o l u t i o n , add t h e f o l l o w i n g w i t h c o n s t a n t s t i r r i n g :
1 - A r g i n i n e . m o n oh y dr o c h l or Tde
■I - H i s t i d i n e HCl
I - L y s i ne HCl
d l - T r yp t o p h a n e
d l -P henyla la nin e
:d l - M e t h i o n i ne
dl - S e r i ne
d l - T h r e o n i ne
d l - L e u c i ne
d l - I s o leucine
d'l - V a l i ne
d l - G l utamic A cid
■d l - A s p a r t i c Ac id
■dl
- A l an i ne
■I - P r o l ine
I - Hyd r oxyp r o l i ne
Glycine
■I - G l u t am i ne
Sodium A c e t a t e
■S t r e p t o m y c i n
' Peni c i 1 1 in
T .26
0.36
I . 26
■0.36
0,90
0.54
:.gm
gm
-gm
gm
gm
gm
0.90 gm
- I .08 gm
2 . 1 6 gm
0 . 7 2 gm
0.90 gm
2 .70 gm
I .08 gm
0.90 gm
0 . 7 2 gm
0 . 1 8 gm
■ 0 . 9 0 gm
1.80 gm
0.90 gm
0 .1 0 gm
2 X -IO^ un.i t s
:
.2 4
'VMye ost at i n
HgO
5 x -IO^ u n i t s
.900 ml
• ' ' M y c o s t a t i n was o b t a i n e d f r o m ’ E. R., Squibb and Sons, .New Y o r k .
<The 1:99A 1TX was f i I t e r e d t h r o u g h an 02 Sel as f i l t e r
and r e f r i g e r a t e d u n t i l
: used.
Complete Medium 1 9 9 " f o r g r o w i n g c e l l s was composed o f t h e - f o l l o w i n g
i n t h e d e s i g n a t e d - p r o p o r t i o n s . ( R u b i n , . i 960 ) :
ingredients
■80:; t r y p t o s e p ho s p h a t e b r o t h " ( B i f c o )
NaHCOg - 2 .
The c a l f
Medium 1 9 9 A ’ 1X -
- - 10 ; c a l f serum - 5 ; 2 .8 p e r c e n t
serum was o b t a i n e d f r o m Hyland L a b o r a t o r i e s , -Los
A n g e l e s , - C a l i f o r m a, and was sh ip pe d f r o m l o t s o f serum t e s t e d b y . D r . - Pv K.
. Vo,gt , U n i v e r s i t y o f ’ C o l o r a d o Medi ca l ' School , . D e n v e r , , f o r maximum cel I
g r o w t h and l e a s t
i n t e r f e r e n c e w i t h f o c u s - , f o rma t io n.
.Medium '199A 2X was p r e p a r e d by do.ub'l ing t h e
ing red ie n ts f o r
199A I X
and a d d i n g 800 ml o f w a t e r .
A ga r o v e r l a y medium was p r e p a r e d by m i x i n g 40 p a r t s
1 . 8 ^ e r c e n t D i f c o ' Bacto a g a r i n w a t e r ,
5 parts c a lf
2 X ,,c a lf
199A2X,.4d p a rts
10 p a r t s t r y p t o s e p ho sp ha t e b r o t h ,
s e r u m , . a n d 2 p a r t s 2 , 8 p e r c e n t sodium b i c a r b o n a t e .
.The -199A
s e r u m , , t r y p t o s e p h o sp ha t e b r o t h and sodium b i c a r b o n a t e were mixed
and p l a c e d
in a 45 C w a t e r bat-h w h i l e t h e m e l t e d a ga r c o o l e d t o 45 C.
t h e t i m e t h e a g a r and 199 were a t 45 C t h e two were m i xe d .
.At
-OnlyyDifco
-Bacto a g a r can be used s u c c e s s f u l l y , . a s o t h e r more ■ p u r e p r e p a r a t i o n s o f
a g a r s up pr e ss f o c u s f o r m a t i o n .
P r e p a r a tio n 'o f - T r i s B u ffere d Sal ine
T ris
lite rs
s a l i n e was p r e p a r e d by d i s s o l v i n g
o f w ater a n d .a d ju s tin g the s o lu t i o n
the f o l l o w i n g
ingredients
in 3
t o a pH o f 7 . 4 w i t h c o n c e n t r a t e d
25
HCl
( V o g t , . 19 63# :
NaCl
Na^HPQi1.
.'KCl
'Glucose
*Sigma 7-9 o r 21 ' ( t r i s)
PenI c i l I in
; S t r e p t o m y c in
*S i gma Chemi ca l
C o . , , S t . . Lou i s , .Mi sst iur i
T r i s s a l i n e was s t e r i l i z e d
■omitted,
2 4 . 0 gm
0 .3 gm
6 .8 gm
3 .0 'gm
9 - 0 gm
13 x IO^ - u n i t s
0.166 gm
by f i l t r a t i o n
i n ' w h i c h case i t was s t e r i l i z e d
u n l e s s t h e a n t i b i o t i c s were
by a u t o c l a v i n g .
• Prepa r a t i on o f T r y p s i n
T r y p s i n >1;250 was o b t a i n e d f r o m Di f c o L a b o r a t o r i e s , . D e t r o i t , , M i c h i g a n .
A s o l u t ion o f 0.2 5 p e r c e n t . t r y p s i n
, a d d i n g 2 . 5' gm o f t r y p s i n
in t r i s b u f f e r e d s a l i n e was p r e p a r e d by
to I l i t e r o f t r i s
■constantly a t -3 7 C f o r I h r .
t h r o u g h an 02 S el a s f i l t e r
s a l i n e and s t i r r i n g
The s o l u t i o n Jwas t hen f i l t e r
and s t o r e d f r o z e n .
t h u s p r e p a r e d was i n a c t i v a t e d when a l l o w e d t o
f o r 4 t o 8 hours.
.Therefore,
the.m ixture
s te riliz e d
I t was o b s e r v e d t h a t t r y p s i n
remain a t
room t e m p e r a t u r e
c a r e was t a k e n t o f r e e z e t h e t r y p s i n
i m m e d i a t e l y a f t e r use.
• P r e p a r a t i o n and T e s t i nq o f C h i c k - Embryo F i b r o b l a s t s
The eggs used t h r o u g h o u t t h i s
■S t a t e -Col <lege poul t r y f a r m .
r e s e a r c h were o b t a i n e d .from t h e Montana
They were i n cub a te d w i t h t h e ' l a r g e end up a t
9 9 . 9 ' F i n a w a t e r - s a t u r a t e d a t m os p he r e.
P r i m a r y c u l t u r e s o f c h i c k embryo
, f i b r o b l a s t s were p r e p a r e d f o l l o w i n g a m o d i f i c a t i o n o f t h e p r o c e d u r e d es­
c r i b e d by Cunningham ( ] 9 6 3 )
f r o m eggs . i n c u b a t e d .9 t o 11 d ay s .
.The-large
26
end o f t h e egg w a s . s t e r i l i z e d w i t h a I . p e r c e n t s o l u t i o n o f
nol.
iodine
The swabbed end o f t h e egg was t h e n c r a c k e d and t h e s h e l l
from o v e r the a i r
space w i t h
s te rile -fo rce p s.
we re p e e l e d away w i t h a second p a i r o f s t e r i l e
removed by g r a s p i n g
ceps.
was d i s c a r d e d .
removed.
. The u n d e r l y i n g membranes
forceps.
The embryo was
.If
in a s t e r i l e
100 mm p e t r i
d i s h and i t s
t h e embryo was h e m o r r h a g i c o r d ef ormed,
it
.The e v i s c e r a t e d embryo was t he n f o r c e d t h r o u g h a 10 ml
s y r i n g e . i n t o a 12$ nil
E r l e n m e y e r f l a s k c o n t a i n i n g 25 rril o f t r i s
s a l i n e and a T e f l o n c o a t e d s t i r r i n g
s tirre d
removed
i t a round t h e neck w i t h a p a i r o f s t e r i l e c u r ve d f o r ­
The embryo was t h e n p l a c e d
head and v i s c e r a
in .e th a ­
bar.
buffered
The c o n t e n t s o f t h e f l a s k were
r a p i d l y f o r a b o u t 2 m i n u t e s t o f r e e as many b l o o d c e l l s as p o s s i b l e .
A f t e r 2 m i n u t e s t h e chunks o f embryo were a l l o w e d t o s e t t l e o u t o f suspen­
s i o n , . a n d t h e s u p e r n a t e was . dec ant ed. , T w e n t y - f i v e ml o f 0 . 2 5 p e r c e n t
t r y p s i n was t h en added t o t h e f l a s k and t h e c o n t e n t s . d i g e s t e d w h i l e b e i n g
•stirre d
r a p i d l y f o r 20 m i n .
a fte r th is
The l a r g e chunks were a l l o w e d t o s e t t l e o u t
t i m e , . and t h e s u p e r n a t e w a s . d e c a n t e d t h r o u g h a d o u b l e l a y e r o f
s t e r i l e cheese c l o t h
i n t o a c e n t r i f u g e tube c o n t a in in g
. The c e l l s were t h en c e n t r i f u g e d a t 3200 r . p . m .
c e n t r i f u g e f o r 15 m i n.
carded.
10 ml o f medium' 199 »
in an I n t e r n a t i o n a l
.The s u p e r n a t e was removed by -aspi r a t i o n and d i s ­
in 20 ml o f medium
. T h e - c e l I s w e r e suspended by v i g o r o u s p i p e t t i n g
T 99 and f i l t e r e d
this fin a l
ce ll
c lin ic a l
a g a i n t h r o u g h a d o u b l e - l a y e r o f cheese c l o t h .
s u s p e n s i o n was t he n p l a t e d
,Three ml o f
i n t o 100 x 20 mm F al co n p l a s t i c
t i s s u e c u l t u r e d i s h e s c o n t a i n i n g 10 ml o f medium 199»
The c e l I s w e r e i n c u ­
b a t e d a t 37 C i n a n , a t m o s p h e r e o f 5 p e r c e n t CQg in a i r .
Whenever h a n d l i n g
27
c h i c k - e m b r y o s " o r c h i c k f i b r o b l a s t s w h i c h were t o be used t o grew RSV, c a r e
-was t a k e n n o t t o- , mi x o r
in any way cross, c o n t a m i n a t e t h e c e l I s o f one em­
b r y o w i t h ' t h o s e ’o f a n o t h e r because o f p o s s i b l e v i r a ] : ( R u b i n , .igbOa)
tance,
Therefore,
a ll
re sis­
m a t e r i a l s f o r each embryo were -handled s e p a r a t e l y .
. A f t e r t h e ' p r i m a r y c h i c k f i b r o b l a s t c u l t u r e s were i n c u b a t e d f o r 2 d ay s,
. t h e med'i um was removed and 10 ml o f f r e s h med i urn '199 was added t o each
p la te ;
24 h ou rs Ta t e r ,
m a n ne r ,
s e c o n da r y c u l t u r e s w e r e - p r e p a r e d
in the f o l l o w i n g
.The medium was a s p i r a t e d ' o f f t h e - c e l l s . , . a ga i n k e e p i n g t h e m a t e r i ­
a l s u s e d ' f o r t h e c e l l s f r o m one embryo s e p a r a t e f r o m t h o s e u s e d . f o r a n o t h e r .
Four nil o f 0 , 2 5 p e r c e n t t r y p s i n was p l a c e d -.in ,each p e t r i
p l a t e s i n c u b a t e d a t 37. C f o r "]'5 t o .20 m i n .
dish,
and t h e
A f t e r t h i s t i m e , . t he c e l l s we re
- d i s p e r s e d by g e n t l e a s p i r a t i o n t h r o u g h a 5 ml p i p e t t e and t h e s u spe ns io n
pla ced
i n a c e n t r i f u g e t u b e c o n t a i n i n g 8 ml o f medium 199.
p l a t e s o f t h e “same embryo were p o o l e d a t " t h i s p o i n t .
t r i f u g e d a t 3200 r . p . m .
in -an I n t e r n a t i o n a l
.The c e l l s f r o m
.The c e l l s were ce n­
c lin ic a l .ce n trifu g e f o r
15 m i n.
A f t e r t h e s u p e r n a t e was removed t h e c e i l s were suspended i n 20 ml o f medium
199 " and c o u n t e d m i c r o s c o p i c a l l y u s i n g a h e m o c y t o m e t e r ,
. For each embryo a s e t o f secon dar y c u l t u r e s was p r e p a r e d w h i c h co n­
s i s t e d o f (a)
two 60 mm p e t r - i d i s h e s w i t h 5 ml o f medium and I O^ c h i c k . e m ­
bryo f i b r o b la s t s p e r .p la te ;
(b)
two 100 mm p e t r i
and 4 X r l O ^ ' c h i c k - e m b r y o f i b r o b l a s t s p e r p l a t e ;
dishes w i t h
and ( c )
10 ml o f medium
t wo 100 mm p e t r i
•dishes- w i t h i 0 ml o f medium and .6 x I O^ c h i c k embryo f i b r o b l a s t s p e r p l a t e .
. The -60 mm p l a t e s were used f o r t e s t i n g t h e embryo f o r
v ira l
or genetic,
resistance,
e ithe r
and t h e l a r g e r . p l a t e s w e re used e i t h e r f o r e x p e r i m e n t a t i o n
.28
■or f o r ' v i ral
b la st
. T h e - p l a t e s w i t h ' t h e '6 ' x IO^ c h i c k (embryo f i b r o ­
titra tio n .
in itia l
i n o c u l a t i on we re ■ready f o r use o r t r a n s f e r w i t h i n .4 - days;
; h o w e v e r , , t h e - p l a t e s w i t h an i n o cu lu m o f 4 x TO^ c h i c k embryo f i b r o b l a s t s
were not
r e ady f o r use b e f o r e 7 days.
The p r o c e d u r e used . f o r t e s t i n g t h e r e s i s t a n c e of. :embryos t o v i rus
i n f e c t i o n was t h a t o f Rubin : ( i g 6 0 ) .
petri
d i s h e s - f r o m e a ch embryo were i n o c u l a t e d w i t h RSV(B).
each ' s e t was i n o c u l a t e d w i t h
viru s,
Immedi a t e l y a f t e r p l a t i n g , . t h e 2 smalI
One p l a t e f r o m
1000 t o ! 1500 f o c u s f o r m i n g u n i t s -.(FFU) o f
and t h e .remain ing pi ate." f r o m each s e t was i n o c u l a t e d w i t h '100 t o
300 ' FFU o f v i r u s - .
A fte r
i n c u b a t i o n o v e r n i g h t , , t h e - s up er na te was removed
i f r o m each p l a t e - a n d t h e medium r e p l a c e d w i t h 5 ml o f a g a r o v e r l a y medium
( se e a b o v e ) ,
bated,
A f t e r t h e medium had s o l i d i f i e d ,
On day 4 a f t e r
incu­
i n f e c t i o n , . t he p l a t e s w i t h 1000 t o 1500 FFU o f v i r u s
were removed and t h e number o f f o c i
e s t i m a t e d by m i c r o s c o p i c a l l y sc a nn i ng
20' p e r c e n t o f t h e a r e a o f t h e p l a t e .
•on The p l a t e , , t h e - e m b r y o was j u d g e d
ber o f fo c i
t h e p l a t e s we re a g a i n
had t e e n ' r e c o r d e d .
If
.If
t h e r e were l e s s t h a n 1000 foc.i
r e s i s t a n t and d i s c a r d e d a f t e r t h e num­
t h e r e were 1000 o r more f o c i
p l a t e , ,t he embryo was used . f o r - e x p e r i m e n t a t i o n .
..the number o f f o c i , was c o u n t e d ' o n t h e pl a t e
On day 7 a f t e r
on t h e
in fectio n ,
r e c e i v i n g 100 t o 300 FFU o f RSV
and ' t h i s number r e c o r d e d .
-Pr e p a r a t i on• o f Mouse Emb r y o F i b r o b I a s t s
. Mice '15 t O ' 1 8 ' d a y s
A 'I
per cent 's o lu tio n of
'
i n t o p r e g n an cy we re k i l l e d b y c e r v i c a l
iodine
d is lo ca tio n .
in e t h a n o l was t hen p ou re d o v e r t h e dead
: a n I m a l . . .An i n c i s i o n was made a l o n g t h e l e n g t h o f t h e abdomen, , bei ng c a r e f u l
29
net to e n te r the p e r ito n e a l
ca vity.
.The s k i n was t h en p e e l e d away f r o m t h e
abdomen o f t h e mouse, and t h e 'abdominal
s te rile
dish.
scis s o rs .
c a v i t y was opened w i t h a p a i r o f
. The - u t e r u s was removed and p l a c e d
i n a ste.r i I e -pet r i
One end o f each hor n o f t h e u t e r u s was t he n c u t ,
f o r c e d o u t o f t h e - c u t end.
and t h e embryos
Each embryo was decap,i t a t ed and e v i s c e r a t e d .
The p r o c e d u r e used f o r d i s p e r s i o n ,
t r y p s i n i z a t i o n and p l a t i n g o f mouse
embryo f i b r o b l a s t s was t h e same as t h a t d e s c r i b e d p r e v i o u s l y - f o r c h i c k
embryo f i b r o b l a s t s .
P r e p a r a t i o n and. Source o f V i r u s St oc ks
The Bryan h i g h t i t e r
s t r a i n o f Rous sarcoma v i r u s , . RSV( B), ,was ob­
t a i n e d : f r o m : D r . W . .R . Bryan a t t h e N a t i o n a l
,Maryla nd.
via l
,The I yoph.i I i zed s t o c k was d e s i g n a t e d by Bryan as CT933 •
was suspended
web o f each o f
i n I ml o f t r i s
s a l i n e and 0.1 ml
s i x 4 week o l d c h i c k s .
from the wing o f the c h ic k e n s .
to
I n s t i t u t e s o f H e a l t h , Bethesda,
in je cted
-One
in t o the wing
A f t e r . 9 days t h e t u m or s were p e e l e d
The g r o s s p a t h o l o g y o f t h e s e tumors seemed
r es embl e - t h a t d e s c r i b e d by Rous and Murphy ( 1 9 1 3 a ) .
They were s o f t ,
f r i a b l e t u m o r s s u r r o u n d e d and i n f i l t r a t e d w i t h a t r a n s l u c e n t ,
viscous f l u i d
w h i c h showed s p l o t c h e s o f gr een and b r o w n , , p r o b a b l y due t o hemorrhage.
t umor s we re n o t e n c a p s u l a t e d .
The
The w e i g h t o f t h e p o o l e d t u m or s was 4 6 .2 3 gm.
V i r u s was e x t r a c t e d f r o m t h e s e t u m or s u s i n g -a m o d i f i c a t i o n o f t h e
tec h n iq ue o f R u b i n -(1955).
-One p a r t t u m o r c e l l s was mixed w i t h
10 p e r c e n t c a l f serum and l ^ g / m l
o f cold t r i s
s a lin e containing
1u r o n i d a s e .
The m i x t u r e was homogenized
b l en d f e r .
10 p a r t s
in the cold f o r
The homogenate was c e n t r i f u g e d f o r
I mi n .
o f hyai n a Wa r i n g
15 m i n. a t 5000 x g in a
30
re frig e ra te d c e n trifu g e .
and s t o r e d
The s u p e r n a t e c o n t a i n i n g t h e v i r u s was d ecanted
i n s e a l e d g l a s s v i a I s on d r y
embryos we re
ice.
i n o c u l a t e d w i t h 0 . I ml o f t h i s
v iru s p re paration.
•were t r a n s f e r r e d 4 t i m e s a t 2 day i n t e r v a l s .
the c e l l s
Secondary c u l t u r e s o f c h i c k
had become l a r g e and rounded w i t h
These c e l l s
.At: t h e t i m e o f h a r v e s t i n g ,
some g i a n t c e l l s a p p e a r i n g as
d e s c r i b e d by Temin and Ru bi n ( 1 9 5 8 ) .
The v i r u s was h a r v e s t e d a c c o r d i n g t o t h e p r o c e d u r e o f Vogt ( 1 965 ) .
-The c e l l s we re scr ap ed f r o m t h e p e t r i
nates.
d i s h e s and p o o l e d w i t h t h e i r s u p e r -
The p o o l e d c e l l s and s u p e r n a t e s were t he n p l a c e d
and s o n i c a t e d f o r 5 m i n u t e s
o s c illa to r.
q ua n tities
i n an i c e b a t h
i n t h e c o l d u s i n g a Ratheon Model
,The s o n i c a t e s were t h en d i s t r i b u t e d
in 0 . 1 , . 1 , 5 ,
i n t o ampules w h i c h were s e a l e d and p l a c e d on d r y
DFTOl
sonic
o r 10 ml
ice.
The S c h m i d t - R u p p i n s t r a i n o f Rous sarcoma v i r u s , RSV(SR), was s u p p l i e d
by-Dr.
R. J .
Huebner o f t h e N a t i o n a l
c e i v e d was o r i g i n a l l y
. Sweden.
I n s t i t u t e s o f Health.
s e n t t o Huebner by D r .
The sample r e ­
C. G. A h l s t r o m f r o m Lund,
The sample r e c e i v e d her e was A h l s t r o m ' s t umor pool # 1 6 .
sample was d i l u t e d
.fib ro b la s ts .
1:10 and 0.1 ml
The RSV(SR)
on t i s s u e c e l l s .
used t o
in fe c t
This
se co nd ar y p l a t e s o f c h i c k
s t o c k s we re p r e p a r e d e x a c t l y as t h e RSV(B)
Nor RSV(SR)
stockg
s t o c k s were p r e p a r e d f r o m t u m o r s .
Rous a s s o c i a t e d v i r u s was i s o l a t e d f r o m o u r s t o c k o f RSV(B) by t h e
p r o c e d u r e o f Rubin a n d ■V o g t ■(1962 ) .
RSV(B) was d i l u t e d t o 10“ ^ FFU/ml
I ml o f t h i s d i l u t i o n p l a c e d on s ec on d ar y c h i c k embryo f i b r o b l a s t s .
days a f t e r
i n f e c t i o n t h e c e l l s were t r a n s f e r r e d , a n d 4 days l a t e r ,
v i r u s was h a r v e s t e d f o l l o w i n g
and
Four
the
t h e above p r o c e d u r e f o r h a r v e s t i n g RSV f r o m
.
31
tissu e c u ltu re .
By i n t e r f e r e n c e assay ( R u b i n and V og t ,
' o f "t he Rous a s s o c i a t e d VJrus (RAV)
1 96 2 ),
the t i t e r
s t o c k was e s t i m a t e d t o be a bo u t I x I
interference u n its /m l.
- Methods -of V i rus Assay
RSV was assayed a c c o r d i n g t o t h e p r o c e d u r e o f Temi n and. Rubin (1958)
as m o d i f i e d b y , R u b i n ' ( 1 9 6 0 a ) .
, Secondary c u l t u r e s o f t e s t e d c h i c k embryo
f i b r o b l a s t s .were p r e p a r e d as d e s c r i b e d p r e v i o u s l y ( P r e p a r a t i o n and T e s t i n g
o f C h i c k Embryo F i b r o b l a s t s ) , e x c e p t t h a t t h e c e l l s were suspended ,to a
c o n c e n t r a t i o n o f 2 x I O^Vml and 5 ml o f t h i s
petri
d i s h t h a t was t o be used.
s u spe ns io n added t o each 60 mm
I t was f o u n d t h a t o n l y p l a s t i c p e t r i
d i s h e s wo u ld g i v e s a t i s f a c t o r y c e l l
s h e e t s and f o c i
f o r the v iru s .a s s a y .
.The d e s i r e d d i l u t i o n o f v i r u s was t he n added i n a t o t a l
. and t h e pi a t e s a l l owed t o
in cubate overn i g h t .
volume o f 0.1 m l ,
The f o l I ow-i.ng. morn i ng t h e
medium was removed f r o m each p l a t e and 5 ml o f o v e r l a y medium 199 was added
t o each p l a t e .
- Seven days a f t e r
in fe c tio n
t h e number o f f o c i
vwas c o u n t e d u s i n g . a n a u t o m a t i c c o l o n y c o u n t e r .
run i n d u p l i c a t e w i t h t h e f i n a l
p r e p a rin g c e l l s f o r v i r u s assay,
Al I v i r u s
on each p l a t e
t i t r a t i o n s were
t i t e r b e i n g . t h e a v er ag e o f 2 p l a t e s .
i t was f o u n d t o be v e r y
• rap i d l y - s o t h a t t h e c e l l s w e r e n o t o u t o f t h e
When
i m p o r t a n t t o work
i n c u b a t o r more t ha n 2 h r s .
a t a time.
The i n t e r f e r e n c e . m e t h o d o f assay ( R u b i n and V o g t , . 1962 ) was used t o
tite r
RAV.
T h i s , m e t h o d makes use o f t h e f a c t t h a t a c e l l
i n f e c t e d w i t h RAV w i l l
w h ic h has been
n e i t h e r s u p p o r t t h e g r o w t h o f n o r be t r a n s f o r m e d by
RSV ( R u b i n and V o g t , . 1962) .
Secondary c h i c k embryo f i b r o b l a s t ; c u l t u r e s
32
were ‘p r e p a r e d . a s a bo ve "a nd
•to ta l
volume-’o f 0 ,1 ml .
infected w ith
. Fo r each p l a t e
u n i n f e c t e d t o s e r v e as a c o n t r o l .
th e - c e l Is
10 f o l d d i l u t i o n s o f ' RAV i n a
i n f e c t e d w i t h RAV,, I p l a t e remained
A f t e r 4 days a l l
p l a t e s were t r a n s f e r r e d ,
i n one 60 mm p l a t e b e i n g d i v i d e d between 2 new 60 mm p l a t e s .
Four days l a t e r t h e c e l l s were r e - t r a n s f e r r e d and c h a l l e n g e d w i t h . RSV.
• For each d i l u t i o n o f RAV,
t h e r e were 4 p l a t e s , ,2 w h i c h were
RAV and 2 w h i c h s er ve d as t h e u n i n f e c t e d c o n t r o l s .
infected w ith
. Each o f t h e 4 p l a t e s
was t he n c h a l l e n g e d , w i t h ' 1 00 t o 300 FFU o f RSV.
A fte r overnight
o v e r l a y medium was a d d e d .
p l a t e s were examined f o r
fo c i.
Seven days l a t e r , t h e
The p l a t e c o n t a i n i n g t h e h i g h e s t d i l u t i o n o f RAV w h i c h
number o f f o c i
end p o i n t .
incubation,
reduced t h e
by a t l e a s t ^O p e r c e n t o f t h e c o n t r o l was c o n s i d e r e d t h e
,The t i t e r o f RAV was t h e n e x p r e s s e d as t h e - r e c i p r o c a l
of th is
d ilu tio n .
. I s o l a t i o n o f ' T r a n s f o r m e d , N o n - P r o d u c i n g - Clones
Transform ed,
n o n - p r o d u c i n g (NP)
f o r m e d . f ib r o b l a s t s t h a t did not
l e c t e d by i n f e c t i n g
RSV(B)" p e r p l a t e .
c l o n e s were f o c i w h i c h c o n t a i n e d t r a n s ­
r e l e a s e any v i r p s .
..These - c l o n e s . w e r e • se­
s e con dar y c h i c k embryo c e l l s w i t h
10 o r f e w e r FFU o f
. The c e l l s were o v e r l a y e d w i t h medium 199 c o n t a i n i n g a
1:50 d i l u t i o n o f a n t i -RSV(B)
serum.
, On day 7 a f t e r i n f e c t i o n , , w e l l
f o c i were p i c k e d f o l l o w i n g
t h e p r o c e d u r e o f V o g t ■(1964^1.
p ar e d f o r . p i c k i n g t h e ; f o c i
by p u l l i n g
f i n e -p o ints.
the t i p
iso la ted
Pip ettes..were p r e ­
of c a p i l l a r y p ip e tte s out to
. The f o e i w e r e p i c k e d , , w h i l e b e i n g o bse rv ed u nd e r a d i s s e c t i n g
microscope,
by t e a r i n g a c i r c l e
in t h e l a y e r o f c e l l s a ro un d t h e f o c u s w i t h
a -p ip e tte .
The f o c u s .was t h e n , s u c k e d
i n t o t h e p i p e t t e and t r a n s f e r r e d t o a
33
i n medium 199 .
■ p l a t e - c o n t a i n i n g ’ IO^ - s e c d nd a ry mouse -embryo f i b r o b l a s t s
A n t i - R S V serum-was t h en added t o t h e medium t o a - d i l u t i o n
o f 1:50.
Si nce
j t h e mouse-'-f i b r o b l a s t s w i l l ■n o t s u p p o r t t h e g r o w t h o f ‘ RAV, , t h e use o f them
as f e e d e r c e l l s
h el p e d
r educe t h e p o s s i b i l i t y o f
w i t h RAV o r RSV ' ( T em i n , , 19 6 2 ) „
ferred,
in fectin g
, A f t e r 7 t o 10 days,
t h e NP ce l I s
t h e s e - f o e i •w e r e • t r a n s ­
I t was p o s s i b l e t o m a i n t a i n t h e s e c e l l s f o r o n l y 3 o r 4 t r a n s f e r s
- (20^30 da:ys) „ - A f t e r t h i s
t i m e , '.the - ce l I s were d i f f i c u l t
cause o f t h e l a r g e number o f g i a n t c e l l s p r e s e n t .
. T h e s e - c e l l s were t e s t e d
- f o r Yfirus p r o d u c t i o n , a f t e r t g o t r a n s f e r s by i n o c u l a t i n g
of chick f ib r o b la s t s w i t h !
t o t r a n s f e r be­
se c on d ar y c u l t u r e s
ml o f t h e - sup.e m a t e - f r o m ' t h e -c l o ne s.
By f o l ­
l o w i n g t h i s p r o c e d u r e , .85 p e r c e n t o f t h e c l o n e s p i c k e d p r o v e d t o be nonproducers.
An a l t e r n a t i v e method f o r p i c k i n g , . t e s t i n g , , a n d
v e l o p e d . w h i c h had s e v e r a l
sary;
s e c o n d , .g f o c i
advantages.
F irs t,,a
f e e d e r l a y e r was n o t n ec e s ­
c o u l d be h an dl ed on I p e t r i
0 , 5 ml o f medium was r e q u i r e d p e r f o c u s .
Small
dish;
mm p l a s t i c
d i a m e t e r o f 5 mm.
i n e t h y l e n e d i o x i d e , ,5 o f t h e s e c y l i n d e r s w e r e p l a c e d
tissu e c u ltu re p e tri
dish.
T hr ee- ml
A fte r
in a 00
, Each c y l i n d e r was
h a l f f u l l . w i t h medium 199 u s i n g a c a p i l l a r y p i p e t t e .
t h e n p i c k e d and I '■f o c u s p i aced i n s i d e -each c y l i n d e r .
th e n 'fille d
than
o f '2 p e r c e n t a g a r i n t r i s
b u f f e r e d s a l i n e was t h e n p i p e t t e d a ro un d t h e c y l i n d e r s .
t h en , f i l l e d
and t h i r d , . l e s s
p l a s t i c c y l i n d e r s were
- p r e p a r e d w h i c h w e r e -6 ,mm h i g h and had an i n t e r n a l
s te riliz a tio n
i n d u c i n g f o c i was de­
'Foci were
- T h e ■c y I i n d e r s were
t h e r e s t o f t h e way w i t h medium and i n c u b a t e d a t 37 . C in an
. a tmo sp h er e o f 5 p e r c e n t COg i n . a i r .
Successful
g r o w t h was o b t a i n e d w i t h
34
'90 p e r c e n t o f t h e - f o c i
the c y l i n d e r
if
picked.
, F o ci c o u i d be m a i n t a i n e d f o r 2 weeks w i t h i n
t h e medium was changed e v e r y '3 o r 4 d ay s .
-be - t e s t e d and i n d u c e d . w i t h i n .the c y l i n d e r .
foci
They c o u l d a l s o
No v i r u s d i f f u s e d between t h e
d u r i n g t h i s p e r i o d o f time-.
■P r e p a r a t i o n .of A n t i serum
A n t i s e r u m a g a i n s t .RSV was p r e p a r e d a c c o r d i n g t o t h e p r o c e d u r e o f Rubin
(1960a) w i t h ' t h e e x c e p t i o n t h a t 10, 000 FFU we re used as t h e
lum,
,The v i r u s was p l a c e d
inocu­
i n t h e - w i n g web o f 4 t o 6 week o l d c h i c k s .
mors were - e v i d e n t by 5 - t o 7 days a f t e r
in je ctio n .
r e j e c t e d o r showed marked d e g e n e r a t i o n ,
. T u­
A f t e r t h e tumors were
t h e b i r d s we re i n j e c t e d w i t h 1 0 ^
' FFU i n t r a v e n o u s l y and b l e d t o d e a th 3 t o 4 weeks l a t e r .
p a r e a ht i - R S V( S R)
in itia l
. A ttempts to p r e ­
serum in t h i s manner f a i l e d .
• E x c e l l e n t a n t i - R S V . sera may a l s o ^be p r e p a r e d by i n j e c t i n g 0.1 ml o f
■7
' R A V , , o r a b o u t 10'
in te rfe re n c e u n its ,
intravenously
i n t o week o l d c h i c k s
and b l e e d i n g them 4 t o 6 w e e k s - l a t e r ( V o g t , . 19 6 3 ) .
Prepa r a t i on o f • F l u o r e s c e n t L a b e l ed A n t i serum
The p r o c e d u r e o f Vogt cand Rubin ' ( I g B l )
of flu oresce n t
labeled a ntib o dy.
was f o i l o w e d . f o r t h e p r e p a r a t i o n
-Only a n t i s e r a w h i c h would
n e u t r a l i z e 90
p e r c e n t o f t h e v i r u s a t a 1:100 o r g r e a t e r , d i l u t i o n we re -used.
s a l i n e was p l a c e d
i n a 125 ml f l a s k a l o n g w i t h a T e f l o n - c o a t e d s t i r r i n g
The s a l i n e was p l a c e d
f q r 5 mi n.
10 m i n.
.Ten-ml
of
bar.
i n an i c e b a t h o v e r a m a g n e t i c s t i r r e r and s t i r r e d
. Ten ml o f a n t i s e r u m was t h en added and t h e m i x t u r e s t i r r e d f o r
A fte r this
tim e,
10 ml o f s a t u r a t e d (NHit) 2 ^ 4 ' was s l o w l y added
35
.The m i x t u r e o f s a l i n e , , s e r u m anci'.'(NH4 ) 2 SO4 was s t i r r e d f o r 30
d r op wi s e.
min,
.The p r e c i p i t a t e d gamma g l o b u l i n was t h e n removed by c e n t r i f u g a t i o n
' a t '5000 x . g ' f o r 10 m i n .
i n a- r e f r i g e r a t e d c e n t r i f u g e .
r esuspended i n h a l f s a t u r a t e d (NH4 ) a n d
was s u b s e q u e n t l y d i s s o l v e d
- b u f f e r , , pH 8 . 5 ,
The s edi men t was
re -ce n trifug e d.
T h i s s edi men t
i n 10 ml o f 0 . 0 5 M sodium c a r b o n a t e - b i c a r b o n a t e
and d i a l y z e d 24 h r s . a g a i n s t 4 changes o,f 100 volumes o f
the c a r b o n a t e - b i carbonate b u f f e r .
per cent ( C a l i f o r n i a
Biochemical
F l u o r e s c e i n ! s o t h i o c y a n a t e on c e l i t e
C o r p o r a t i o n , ,Los Ang el es)
10
was t hen added
t o - t h e d i a l y z e d g a m m a : g l p b u l i n t o a c o n c e n t r a t i o n o f 25 mg f l u o r e s c e i n i s o t h i c y a n a t e p e r 10 ml o f gamma g l o b u l i n ' ( R i n d e r k n e c h t ,
1,962).
The m i x ­
t u r e was ^shakdn..a t
room t e m p e r a t u r e f o r 10 m i n. and t h e e x c es s dye removed
by c e n t r i f u g a t i o n .
. The l a b e l ed a n t i serum was t he n d i a l y z e d
in the c o l d
a g a i n s t 100 volumes o f pH 6 . 5 , 0 . 0 2 M p h o s p h a t e b u f f e r e d s a l i n e - u n t i l
'.flu orescence could :be -d e tecte d
in the d i a l y z i n g f l u i d ,
no
,Ten ml o f d i a l y z e d
f I u o r e s c e n t a n t i serum was t h e n a dsorbed t w i c e - f o r I h ou r p e r i o d s a t - room
t e m p e r a t u r e w i t h ,250 mg o f
chick f ib r o b la s t s .
l i v e r powder and once w i t h
O
10° normal t e s t e d
The l a b e l e d a n t i s e r u m was s t o r e d f r o z e n
i n t he d a r k
u n t i I ■used.
• L i v e r powder was p'repar ed by g r i n d i n g 25 t o 30 gm o f b e e f l i v e r w i t h
an equal
volume o f s a l i n e
i n a Wa ri ng b l e n d e r a t 4 C.
-The c o n t e n t s o f t h e
b l e n d e r we re t h e n p ou r ed i n t o , a 2 l i t e r b e a k e r and 4 volumes o f a c et o ne
w e re added,
u n til
-The p r e c i p i t a t e was t h e n c e n t r i f u g e d and washed w i t h s a l i n e
ho h e m og l o bi n was v i s i b l e
was suspended i n , a n , e q u a l
in t h e s u p e r n a t e .
The washed p r e c i p i t a t e
volume o f s a l i n e , a n d 4 v o l u m e s - o f a c e t o n e were
36
' a d de d .
A f t e r t h e suspended m a t e r i a l ; h a d s e t t l e d ,
c a r d e d , a n d 4 .more volumes e f acetone were added.
was t h e n h a r v e s t e d u s i n g a Buchner f u n n e l .
w ith
several
stored
• F l u o r e s c e n t S t a i n i n g o f V i rus
V irus
This acetone-suspensio n
The p r e c i p i t a t e was washed
I i t e r s o f a c e t o n e - a n d . d r ied a t 3 7 ' C.
in a r e f r i g e r a t o r u n t i l
t h e s u p e r n a t e was d i s ­
The l i v e r powder was
used.
I n f e c t e d -Cel I s
i n f e c t e d ce l I s .were s t a i ned w i t h f I u o r e s c e r i t ■I abel ed . a n t i b o d y
a c c o r d i n g t o t h e p r o c e d u r e o f Vogt and Rubin ( 1 9 6 1 ) . . Secondary c u l t u r e s o f
c h i c k f i b r o b l a s t s were p r e p a r e d and i n f e c t e d as p r e v i o u s l y d e s c r i b e d e x c e p t
t h a t two 18 x 18 mm, , number '0 c o v e r s l i p s were p l a c e d
60 mm p e t r - i
dish.
-The - c o v e r s l i p s were p r e p a r e d by w i p i n g
t i s s u e p a p e r and s t e r i l i z i n g
tio n ;
i »e . , w a s h i n g , o n l y
f a c e - o f the c o v e r s h ip s .
From l
them g e n t l y w i t h
Any f u r t h e r m a n i p u l a ­
t o a dh e re t o t h e s u r ­
in fe c tio n ,
A fte r this,
s ta in in g ja r s containing t r i s
saline
t h e y we re r e mo ve d, f ro m t h e j a r s and t h e
They were t h en p l a c e d
.The chamber c o n s i s t e d o f a - l a r g e p e t r i
in a w a te r s a tu ra te d
d i s h w i t h a w a t e r soaked
p i e c e ' o f / f i l t e r p a p e r i n t h e b o t t o m and a b a r o f s t a p l e s
r e s t i n g so t h a t
t h e c o v e r s l i p s c o u l d be p l a c e d o n : t h e t i p s o f t h e s t a p l e s .
flu oresce n t
the c o v e r s l ips
d i s h e s and t h e medium d r a i n e d f r o m t h e i r s u r ­
s a l i n e d r a i n e d f r o m them.
chamber.
of ce lls
t o 4 days a f t e r
They were t h e n p l a c e d i n small
and soaked i f o r 5 m i n.
tris
them i n a h o t a i r oven.
reduced t h e a b i l i t y
we,re removed f r o m t h e p e t r I
face.
in t h e b o t t o m o f each
One drop o f
l a b e l e d a n t i s e r u m was d e l i v e r e d t o each ^cover sl ip f r o m a c a p i l ­
la ry p ip e tte .
The chamber c o n t a i n i n g t h e c o v e r s l ip s was r o t a t e d so t h a t
t h e serum c o a t e d t h e e n t i r e s u r f a c e - o f t h e - c o v e r s l i p s .
,The chamber and
37
c o v e r s ! i p s -were - i ncub'ated a t 37, C ' f o r ' I O m i n .
f r o m t h e -chamber and,washed f o r two >5 m i n .
The - c o v e r s ! i p s were removed
in tervals
in f r e s h t r i s
sa line.
.The ' c o v e r s ! i p s were t h e n mounted on m i c r o s c o p e s l i d e s no g r e a t e r t han I ; mm
th ick,
usin g a mounting f l u i d
•b u f f e r . (pH • 7*0)
c o n s i s t i n g o f 5 p e r c e n t 0 . I : M p ho sp ha t e
i n :gl y c e r o l . - T h e ' s l i d e s were examined f o r f l u o r e s c e n t
s t a i n i n g und er a - ^ e i t z --.SM model m i c r o s c o p e e q u i p p e d . w i t h a d a r k - f i e l d co n­
d e n s e r and an u l t r a - v i o l e t
lig h t
source.
s c h e i be f i l t e r were used between t h e l i g h t
The BG12 f i l t e r p l u s t h e S t r e u source-and th e condenser.
o ra ng e O G l , . 2 . 5 mm b a r r i e r f i l t e r s :were used in t he eye p i e c e s .
t r i n o c u l a r head,
.The
With the
t h e r e was n o t enough l i g h t a l l o w e d t h r o u g h t h e mo n oc u la r
arm f o r p h o t o g r a p h s t o be t a k e n u s i n g . t h i s mri cto scope.
,The f l u o r e s c e n t
l a b e l e d serum was t e s t e d f o r s p e c i f i c i t y
(I)
by
s t a i n i n g - n o b - i n f e c t e d c h i c k f i b r o b l a s t s , ( 2 ) by b l o c k i n g t h e s u r f a c e a n t i ­
gen on I n f e c t e d ce l I s by a 10 mi n. p r e - t r e a t m e n t o f t h e c e l l s w i t h s p e c i f i c
n o n -flu o re s c e n t antiserum,
w i t h ; I a b e l e d normal
and ( 3 ) by a t t e m p t i n g - to s t a i n
infected c e lls
c h i c k e n :serum.
P r e p a r a t i o n o f P u r o m y c i n 1S t oc k S o l u t ions
Pur bmycin d i h y d r o c h l o r i d e was o b t a i n e d f r o m t h e N u t r i t i o n a l : B i o c h e m i c a l s C o r p o r a t i o n , , C l e v e l a n d , , O hi o .
s o l v i n g t h e puromyc i n
. S t o c k s o l u t i o n s were p r e p a r e d by d i s ­
i n c o m p l e t e 199 ' t o - a c o n c e n t r a t i o n o f
IpbzA g Z m l .
,These s o l u t i o n s were s t o r e d f r o z e n and were thawed j u s t b e f o r e use and
refrozen
im m edia tely.
38
P r e p a r a t i gn o f A c t i nomyc i ru:D S t oc k 'Sol u t i ons
A c t i nomyci n 'D was a g i f t
West P o i n t , .Pa.
ethanol
d ilu te d
A c t i n o m y c i n ^ D was d i s s o l v e d
in double d i s t i l l e d
and Gol d ^ ,
o f M e r c k , . Shar p : and Dome Research ' L a b o r a t o r i e s ,
1 96 4 ) .
i n 50 p e r c e n t
water to a conce n tratio n o f
. T h e - a c t i n om y c i n ; D was s t o r e d
re d is tille d
100/K.g/ml ' ( V i g i e r
in t h e d a r k a t -20 C and
t o t h e d e s i r e d c o n c e n t r a t i o n j u s t p r i o r t o use.
• P r oces s i n g o f C e l l s f o r S c i n t i l I a t i on Count i rig
A f t e r the
i n c o r p o r a t i o n o f H^-Leucine,
moved f r o m s u s p e n s i o n by c e n t r i f u g a t i o n .
c e l l s were t r y p s i n i zed and r e ­
.The r e s u l t i n g p e l l e t o f c e l l s was
suspended i n t r i s - s a l i n e and r e c e n t r i f u g e d .
T h i s p e l l e t o f wahsed cel I s
was suspended i n 'I ml o f c o l d t r i s - s a l i n e , a n d
to th is
suspension,.]
c o l d IM t r i c h l o r o a c e t i c a c i d (TCA) was added.
The c e l l s
ml o f
i m m e d i a t e l y f or med
a w h i t e p r e c i p i t a t e w h i c h was washed 3 t i m e s w i t h '2 ml o f c o l d IM TCA.
Between e ac h w a s h i n g , . t h e p r e c i p i t a t e d c e l l s were c e n t r i f u g e d t o m i n i m i z e
•lo ss o f c e l l u l a r m a t e r i a l .
A f t e r the t h i r d
TCA w a s h i n g , 4 ml o f a s o l u t i o n
o f - a b s o l u t e e t h a n o l : e t h e r , ,3:1 , was added t o t h e p r e c i p i t a t e . . . F o l l o w i n g
c e n t r ’i f u g a t i o n, , e t h e r - was added •t,o t h e p r e c ip i t a t e .
p r e c i p i t a t e ' f r o m e t h e r by c e n t r i f u g a t i o n ,
56 'C f o r d r y i n g .
via l
ml o f
i n 0 . 5 ml o f IN NaOH i n , a
. O n e - t e n t h ml was t h e n p l a c e d
c o n t a i n i n g 20 ml o f s c i n t i l l a t i o n
I N NaOH-in :20 ml o f s c i n t i l l a t i o n
counting.
sepa r a t i n g t h e
t h e p r e c i p i t a t e was p l a c e d a t
The d r i e d m a t e r i a l was d i g e s t e d
b o i l i n g w a t e r b a t h f o r 30 m i n .
A fte r
flu id .
A control
in a c o u n t i n g
c o n s i s t i n g o f 0.1
f l u i d . w a s used f o r b ackground
39
The s c i n t i l l a t i o n
f l u i d : w a s .designed f o r t h e s u s p e ns i o n o f aqueous
s o l u t i o n s and c o n s i s t e d - o f :
I , 4 - D i oxa ne
T o lu en e
A b s o l u t e E t ha nol
2 , 5 - 0 i p h e n y l o x a z o l e (PPQ)
N a p h t h a l ene
-360 ml
-360 ml
216 ■ml
5 gm
80 gm
' RESULTS
. RSV1Tumors
iri Chi c k e n s
V i r u s was i n j e c t e d
cribed
( M a t e r i a l s and M e t h o d s ) .
- days a f t e r
w ith in
i n t o t h e wi n g web o f c h i c k e n s as p r e v i o u s l y d es­
, F i g u r e I shows an i n t a c t . RSV(B)
i n j e c t i o n o f the v i r u s .
t h e t u m or was d i s c o l o r e d .
t umor 14
, The t umor measured : 9 .5 by 5 cm.
Tissue
The t umor was s o f t arid f r i a b l e as d i s ­
cussed p r e v i o u s l y ( M a t e r i a l s and M e t h o d s ) .
The g e n e r a l
c o n d i t i o n o f the
c h i c k e n was v e r y p o o r .
The b i r d e x h i b i t e d c o n s i d e r a b l e w e i g h t l o s s and
somnolescence as w e l l
as l o s s o f comb c o l o r as d e s c r i b e d by Rous ( 1 9 1 1 b ) .
Upon p o s t mo.rt.um e x a m i n a t i o n ,
tained
t h e l i v e r e x h i b i t e d d i s c o l o r a t i o n and co n­
la rg e y e llo w b lo tch e s c h a r a c t e r i s t i c o f v iru s
i n v o l v e m e n t (Rous,
. 19 M b ) .
I n c c o n t r a s t t o t h i s marked p a t h o l o g y ,
d e v e l o p e d t umor s much more s l o w l y .
b i r d s c h a l l e n g e d w i t h RSV(SR)
Fo ur weeks was r e q u i r e d . f o r tumors t o
reach a s i z e o f 2 x 3 c m , , and many remained t h e s i z e o f a hazel
t umor s r e g r e s s e d more - f r e q u e n t l y t h a n d i d RSV(B)
if
they d id not
regress,
i nduced t u m o r s ;
nut.
These
however, ,
t h e b i r d e v e n t u a l l y became i l l , , e x h i b i t e d w e i g h t
I o s s , and d l e d .
■RSV I n f e c t i o n s
in Tissu e C u lt u r e C e lls
F i g u r e s 2 and 3 , show t y p i c a l
RSV(B)
and RSV(SR) f o c i
re sp ective ly.
The c e l l s w e re i n f e c t e d . a n d o v e r I a y ed as p r e v i o u s l y d e s c r i b e d ( M a t e r i a l s
and M e t h o d s ) .
.A fte r
i n c u b a t i o n f o r 7 d ay s ,
t h e y were s t a i n e d f o r p h o t o ­
g r a p h i n g by p l a c i n g 3 o r 4 d r o p s o f an 0.1 5 p e r c e n t aqueous s o l u t i o n o f
neutral
red d i r e c t l y on t h e o v e r l a y a g a r .
. A f t e r 2 t o 2^ h r s . , ,enough o f
Figure I .
RSV(B) Tumor 14 Days a f t e r
t h e s t a i n had been t a k e n up by t h e c e l l s
foci
from the su rro u nd in g monolayer.
u s u a l l y not s t a i n e d .
t o a llo w c l e a r d i s t i n c t i o n o f the
Fo r r o u t i n e c o u n t i n g ,
As may be seen i n F i g u r e 2,
d i s t i n c t p i l e s o f c e l l s on t h e m o n o l a y e r ; whereas,
d iffu s e centers o f
Infe ctio n.
RSV(B)
t h e f o c i were
f o c i were s m a l l ,
RSV(SR)
f o c i were l a r g e ,
i n f e c t i o n w h i c h were n o t a lwa ys e a s i l y d i s t i n g u i s h e d
from the monolayer ( F i g u r e 3 ) .
F i g u r e 4 i s a p i c t u r e o f an RSV(B)
clump o f
f o c u s m a g n i f i e d 30 t i m e s .
rounded c e l l s w h i c h have l o s t c o n t a c t
F r e q u e n t l y t h e s e clumps o f c e l l s woul d p u l l
la ye r,
in h ib itio n
(Vogt,
Note t h e
1963).
away f r o m t h e r e s t o f t h e mono-
as t h i s one has done, and e v e n t u a l l y f l o a t away u nd e r t h e o v e r l a y
medium l e a v i n g a p l a q u e .
is p o s s ib le to d is t in g u is h
N o t i c e a l s o t h a t around t h e edge o f t h e f o c u s
the deeply s t a in e d ,
large,
rou nd ed,
f e c t e d c e l l s from the u n in f e c te d c e l l s o f the monolayer.
viru s
in­
In c o n t r a s t ,
it
42
F i g u r e 3.
RSV(SR)
F o ci.
43
F i g u r e 5-
RSV(SR)
Focus M a g n i f i e d 30X.
4A
■F i g u r e 5 -shows an RSV(SR)
f o c u s u nd e r t h e same c o n d i t i o n s .
I t was i m p o s s i ­
b l e t o d i s t i n g u i s h the c e i l s o f the focus from those o f the monolayer.
fact,
i t was i m p o s s i B i e - t o - c o u n t ' RSV(SR) ’ f o c i
because o f t h e l a r g e , d i f f u s e
■f o c i we re f r e q u e n t l y
. F i g u r e 6 , however,
n a tu re o f the f o c i .
the c e l l s
, C e l l s w i t h i n RSV(SR)
i n RSV(B) f o c i ,
shows t h a t RSV(SR) c e l l s d i d e v e n t u a l l y become
rounded and t en ded t o l o s e c o n t a c t
in h ib itio n .
c e l l s 20 days a f t e r
i n \ RSV(SR) c u l t u r e s .
, F i g u r e 6 i s a 100 x mag ni ­
in fe c tio n .
t e r i s t i c m o r p ho l og y o f e s t a b l i s h e d RSV(SR)
e v er developed
a c c u r a t e ! y usin g a microscope
l a r g e r t h an t h o s e o f t h e - m o n o l a y e r , b u t were seldom
rounded o r s t a c k e d l i k e
f i c a t i o n o f RSV(SR)
c e lls .
Here i s seen t h e c h a r a c ­
. Very f ew g i a n t c e l l s
These RSV(SR) c e l l s c o u l d be . m a i n t a i n e d
t h r o u g h ’ 16 - t r a n s f e r s o r a b o u t 90 days
in t i s s u e c u l t u r e .
. F i g u r e 7 shows.a p i c t u r e o f 20 day o l d e s t a b l i s h e d RSV(B)
100 t i m e s .
. Note t h e l a r g e d e g e n e r a t i n g g i a n t c e l l
photograph.
C h a r a c t e r i s t i c a l l y , RSV(B) c e l l s f i r s t
s m a ll,g ra n u la r,
o f the -giant c e l l
RSV(SR)
i n F i g u r e 7.
c e l l s magnified
n ea r t h e c e n t e r o f t h e
had t h e appear ance o f
These c e l l s a l s o resembled t h e e s t a b l i s h e d
H o w e v e r , a f t e r 2 o r 3 t r a n s f e r s , . a l a r g e number
o f g i a n t c e l l s were -f oun d i a; RSV(B)
c u ltu re s .
g i a n t c e l l s became.; t h e - d o m i n a n t c e l l
not d i v i d e , c e l l
, In ! c o n t r a s t ,
rounded c e l l s , s o m e o f w h i c h may be seen ar oun d t h e edges
c e l l s o f Figure 6 .
transfer,
In
By t h e -se ve nt h t o - te nt h
t y p e . , „ and because t h e y d i d
t r a n s f e r was no l o n g e r p o s s i b l e .
c o u l d be m a i n t a i n e d
in c u l t u r e o n l y 50 t o 60 days.
T h e r e f o r e , RSV(B) c e l l s
45
Figure 6 .
E s t a b l i s h e d RSV(SR)
F i g u r e 7-
E s t a b l i s h e d RSV(B)
C e lls Magnified
100X.
C e l l s M a g n i f i e d IOOX.
46
■Heat - I n a c t i v a t i on o f RSV(B) and RSV(SR)
T h e " p u r p o s e - o f t h e s e e x p e r i m e n t s was t o d e t e r m i n e t h e s t a b i l i t y of
each v i r u s t o t i s s u e c u l t u r e c o n d i t i o n s .
warmed, c o m p l e t e medium 199 and p l a c e d
The v i r u s e s w e r e d i l u t e d
in a t i s s u e c u l t u r e
i n c u b a t o r a t 37
C i n a w a t e r s a t u r a t e d a tmo sp h er e o f 5 p e r c e n t COg in a i r .
t i m e s samples were t a k e n and s t o r e d on d r y
v i r u s p a r t i c l e s c o u l d be d e t e r m i n e d .
ice u n t i l
in p r e ­
.At v a r i o u s
t h e number o f s u r v i v i n g
Z er o t i m e was t h e t i m e o f a d d i t i o n o f
t h e v i r u s t o medium 199 «
,F ig u re 8 gives
the r e s u l t s from these experim ents.
• s t a b l e t o t i s s u e -cul t u r e c o nd i t i o n s t h a n . RSV(SR).
RSV(B)
. RSV(B) was more
The s h o u l d e r ' on t h e
c u r v e between' 0 and 4 h r s . was p r o b a b l y due t o v i r u s a g g r e g a t i o n
( Ha naf usa ejt. _a]_., .1964b) .
Heat
i n a c t i v a t io n constants f o r the 2 viru se s
c o u l d be e s t i m a t e d u s i n g an e q u a t i o n d e r i v e d by Rubin ( 1 9 5 5 ) :
Vt
V0
t
K
=
=
=
=
number o f v i r u s p a r t i c l e s s u r v i v i n g a t t i m e t
number o f v i r u s p a r t i c l e s a t t i m e 0
t i m e in m i n u t e s a t 37 C
heat i n a c t i v a t i o n c o n s ta n t
S u b s t i t u t i n g the a p p r o p r i a t e data from F ig u re 8 i n t o t h i s e q u a tio n ,
h ea t
in a c tiv a tio n
min.™' .
c o n s t a n t f o r RSV(SR) (I<sr ) was f ou nd t o be 8 . 8 5 x ICT^
The h e a t i n a c t i v a t i o n c o n s t a n t f o r RSV(B) (Kg)
was 2 . 9 6 x :10™3 m i n . ™ ' .
RSV(B)
curve
the
between 4 and 12 hrs..
, The a s s u m p t i o n was made t h a t t h i s p o r t i o n o f t h e
represented the
in a c tiv a tio n of
s i n g l e RSV(B) p a r t i c l e s .
.The
Kg o f RSV(B) between 4 and 12 h r s . was f o u n d t o be n e a r l y 3 t i m e s g r e a t e r
t h a n t h e Kg between 0 and 4 h r s . ,
in d ic a tin g th a t,
in itia lly ,
t i on o f c l u s t e r s o f 2 t o 3 v i r u s p a r t i c l e s was b e i n g measured.
t h e • i n a c t i va*-
47
. Heat
I p a c t i v a t i on o f RSV(B) and RSV( SR) .
Open c i r c l e s
r e p r e s e n t d a t a f o r RSV(SR); c l o s e d c i r c l e s
s e n t d a t a f o r RSV(B). , Each p o i n t
■ ex p e r i m e n t s .
is the average o f 2
re pre
48
F ig u r e 8 .
Heat
I n a c t i v a t i o n o f RSV(B) and RSV(SR)
RSV(B)
RSV(SR)
Hours a t 37 C
By s e t t i n g V0 equal
t o 2 and Vt equal
to I,
the h a l f - l i f e
v i rus c o u l d be - c a l c u l a t e d us I ncj t h e above e q u a t i o n .
,The h a l f - 1 i f e -of
RSV(B) was f o u n d t o ,be 234 m i n . and t h a t o f RSV(SR), , 7 8 . 4 m i n .
und er t h e s e e x p e r i m e n t a l
cond itio n s,
o f each
Therefore,
RSV(B) was n e a r l y 3 t i m e s as s t a b l e
as RSV(SR) .
A n t i g e n i c Rel a t i o n s h i p ; Between RSV(B)
and' RSV(SR)
A' s t u d y . w a s made o f t h e a n t i g e n i c
RSV(SR)
of
u s i n g serum n e u t r a l i z a t i o n
infected c e lls .
r e l a t i o n s h i p between RSV(B) and
t e s t s and f l u o r e s c e n t a n t i b o d y s t a i n i n g
Since th e lo ss o f
i n f e c t i v i t y o f a / v i r u s due t o t r e a t ­
ment w i t h a n t i s e r u m p r o b a b l y r e s u l t s f r o m t h e i n t e r a c t i o n o f a n t i b o d i e s
w i t h t h o s e a n t i g e n s on t h e v i r u s
and s u bs eq ue nt r e l e a s e - o f v i r a l
t h e serum n e u t r a l i z a t i o n
r e s p o n s ib le f o r a d s o r p t io n t o the cel I
n u c le ic acid
t e s t wo u ld
(Rubin,
1957; G r a n o f f , 1965 ) ,
i n v o l v e a l i m i t e d number o f a n t i g e n s .
The f l u o r e s c e n t a n t i b o d y s t a i n i n g w o u l d n o t be l i m i t e d t o t h e s e few a n t i g e n s ,
and t h u s s h o u l d have a b e t t e r c h a n c e - o f d e t e c t i n g w h a t e v e r common a n t i g e n s
m i g h t e x i s t on t h e -2 v i r u s e s .
antigens
. However,
if
i n common, and t h e s e f ew a r e n o t
t h e v i r u s e s have o n l y a few
involved w it h
in fe c tiv ity ,th e y
wo u l d n o t be d e t e c t e d by t h e s e e x p e r i m e n t s because t h e amount o f l a b e l e d
a n t i s e r u m w h i c h c o u l d be adso rb ed t o a c l u s t e r o f viruses woul d be t oo
■ l i m i t e d t o be d e t e c t e d by f l u o r e s c e n t m i c r o s c o p y .
. Serum n e u t r a l i z a t i o n
F r i e s e n :and Rubi n ■(I 9 6 I ) .
as p r e v i o u s l y d e s c r i b e d
t e s t s were p e r f o r m e d f o l l o w i n g t h e method o f
A n t i s e r u m h ea ted t o 56 C f o r 30 m i n . and p r e p a r e d
( M a t e r i a l s and M e t h o d s ) , was d i l u t e d
and warmed t o 3 7 . C. , A known q u a n t i t y o f v i r u s was p l a c e d
i n medium 199
in the d i l u t e d
■5:6
a n t i s e r u m a n d - i n c u b a t e d - f o r 40 m i n.
.A fte r this
tim e ,th e
amount o f a c t i v e
v i r u s was d e t e r m i n e d by f o c u s a s s a y ' ( M a t e r i a l s and . Me t h o d s ) .
consisted o f v iru s d ilu te d
The c o n t r o l
in 199 and i n c u b a t e d f o r 40 m i n . a t 3 7 ;C.
The r e s u l t s o f such an e x p e r i m e n t a r e shown in F i g u r e 9 , .where t h e
f r a c t i o n o f n o n - n e u t r a l i zed v i r u s
serum,
is p l o t t e d a g a in s t the d i l u t i o n s o f a n t i ­
. T h e r e was o b v i o u s l y no n e u t r a l i z a t i o n o f RSV(SR)
, ev en a t - d i l u t i o n s w h i c h
T h e r e f o r e , , t h e ' RSV(B)
by t h i s
i n a c t i v a t e 99 p e r c e n t o f t h e RSV(B)
antig e ns which were responsible f o r
serum,.
present.
i n f e c t i o n .were
n o t t h e same as t h o s e p r e s e n t on .RSV(SR).
RSV(B)
and' RSV(SR)
la b e le d anti-RSV(B)
serum ( M a t e r i a l s and M e t h o d s ) .
f l u o r e s c e n c e - o n 'RSV(B)
■showing no a n t i g e n i c
i n f e c t e d c e l l s w e re t he n s t a i n e d w i t h f l u o r e s c e n t
Serum w h i c h gave, good
c e l l s gave no f I u o r e s c e n c e -on RSV(SR) c e l l s ,
again
r e l a t i o n s h i p between t h e 2 v i r u s e s .
Vpgt (1964b)' has shown t h a t RSV(SR) may be n e u t r a l i z e d by a n t i s e r u m
a g a i n s t a s t r a i n o f : RSV(B) o t h e r t h an t h a t used in t h i s
. Huebner e t a I , , ( 1964). have f o u n d a s o l u b l e a n t i g e n
la b ora to ry.
in RSV(SR)-InBuced tumor s
f r o m b o t h h am st er s and c h i c k e n s w h i c h s t i m u l a t e s complement f i x i n g
b o d i e s t h a t c r o s s r e a c t w i t h RSV(B)
:seems t o be a g r ou p s p e c i f i c
,avian
t u m or and v i r a l
soluble antigen,
However, ■
antigens.
a n ti­
. This a n tig e n
a s . i t has been f o u n d in a l l
Ieukosis viru s e s te s te d .
U n f o r t u n a t e l y , , d u e t o e i t h e r t h e p o o r a n t i g e n i c i t y o f . RSV(SR) o r t h e
l i m i t e d amount o f a n t i g e n a v a i l a b l e ,
n e u tra liz in g antibodies
i t was n o t p o s s i b l e t o t e s t f o r RSV(B)
i n a n t i -RSV(SR)
d e s i r a b l e s i n c e Andrews .(1933)
serum.
. Such^a t e s t wo u ld be h i g h l y
has shown t h a t v i r u s e s e x i s t w i t h i n
the
51
N '
F i g u r e S_.
N e u t r a l i z a t i g n o f RSV(B) arid RSV(SR) w i t h ' A n t i -RSV(B)
Open c i r c l e s
r e p r e s e n t t h e s u r v i v i n g f r a c t i o n o f RSV(SR), and
the clo sed c i r c l e s
H alf f i l l e d
are
Serum.
r e p r e s e n t t h e s u r v i v i n g f r a c t i o n o f RSV(B).
c i r c l e s a r e p o i n t s w h e r e -RSV(B) and RSV(SR) v a l u e s
id e n tic a l.
52
F ig u r e 9 .
N e u t r a l i z a t i o n o f RSV(B) and RSV(SR) w i t h A n ti-RSV (B )
Serum
RSV(SR)
Fractio
Z 0.60
RSV(B)
1:200
1:800
Serum D i l u t i o n
1:100
53:
avian
l e u k o s i s ,group w h i c h w i l l
i n d u c e -RSV n e u t r a l i z i n g a n t i b o d i e s ,
but
w h i c h a r e n o t t h e m s e l v e s n e u t r a l i z e d by a n t i - R S V serum.
'■D e f e c t ! v e n e s s o f RSV(B)
and RSV(SR)
Hanafusa e t a l . (1963)
showed t h a t RSV(B) was d e f e c t i v e .
S in ce many
o f t h e p r o p e r t i e s o f RSV(B) were shown t o be d e t e r m i n e d by t h e h e l p e r v i r u s
■( Ha naf us a e t ;aj_., 1964;
Rub i n ,
1964; V o g t ,
19 65 ; Hanafusa,
ments were c a r r i e d o u t t o t e s t f o r d e f e c t i v e n e s s
t o be d e f e c t i v e , i t
1 965),
in. RSV(SR).
expe ri­
. F o r RSV(SR)
s h o u l d depend upon t h e p r e s en c e o f a h e l p e r v i r u s ,
RAV, f o r m a t u r a t i o n ,
and t h e r e f o r e f o r m n o n - p r o d u c i n g (NP)
fo c i.
i.e „,
.Experi­
ments we re d e s i g n e d t o t e s t f o r each o f t h e s e p r o p e r t i e s .
If
t h e h e l p e r v i r u s w h i c h m i g h t be c a r r i e d by RSV(SR) were l i k e RAV,
. i t wo u ld p r odu ce i no m o r p h o l o g i c a l
infected c e lls ;
v i r u s would
hence,
u tiliz e
a l t e r a t i o n s o r . c y t o p a t h o g e n i c e f f e c t on
t h e most s e n s i t i v e t e s t f o r t h e p r e s e n c e o f a h e l p e r
its a b ilit y
to
in du ce v i r u s p r o d u c t i o n
i n NP c l o n e s .
• The i n f e e t i dn o f even I NP c e l l w i t h t h e h e I p e r v i rus w o u l d l e ad t o t h e
- r e p l i c a t i o n and m a t u r a t i o n o f b o t h t h e h e l p e r v i r u s and t h e d e f e c t i v e v i r u s .
A f t e r several
cycles o f
re p lica tio n ,
h e l p e r v i r u s e s wo u l d r e s u l t
and d e f e c t i v e v i r u s e s .
t h e p r e s e n c e - o f a v e r y small
number o f
i n t h e p r o d u c t i o n o f a l a r g e number o f h e l p e r
■T h e - p r e s e n c e -of a h e l p e r v i r u s w o u l d t h en b e . e v i ­
denced by t h e p r e s e n c e o f f o c u s - p r o d u c i n g ,
m a tu r e RSV i n t h e s u p e r n a t e o f
c el I s -which p r e v i o u s l y gave r i s e t o no RSV.
I
. An e x p e r i m e n t was . designed t o t e s t f o r t h e p re s en c e o f a h e l p e r v i r u s
i n yRSV( SR) by i n d u c t i o n .
c e l l s as d e s c r i b e d
. S ev e r a l
f o c i were p i c k e d f r o m RSV(B)
in M a t e r i a l s and.Methods.
infected
A f t e r 2 t r a n s f e r s , ,0. 5 ml o f
54
■t h e ' s u p e r n a t e -from t he-NP c e l I s were t e s t e d - f o r t h e - p r e s e n c e - o f 1RSV(B) and
■t h e r e f o r e , . , i n d i r e c t i y . f o r t h e p r e s e n c e - o f RAV.
w ere ^se le cte d .fo r
duction,
E i g h t o f t h e s e -NP c l o n e s
i n d u c t i o n as sh own ■i r r T a b l e - I I .
Immediately b efo re
in­
t h e s u p e r n a t e - f r o m each o f " t h e s e c l o n e s was a g a i n t e s t e d , : f o r 'RSV(B) ,
and "as e v i d e n c e d by t h e - s u p e r n a t e c o n t r o l 1( T a b l e
. In ic a s e - t h e - RSV(SR)
h e l p e r v i r u s m i g h t be p r e s e n t
n o t e x c e e d i n g t h a t o f RSV(SR)
h
' IO - FFU o f RSV(SR)
I I ) , ,no v i rus was d e t e c t e d .
( T a b l e -I I ) .
its e lf,
in c o n c e n tr a t io n s
6 o f t h e -c l o n e s w e r e i n f e c t e d . w i t h
S e v e n t y - t w o h ou rs a f t e r
in fectio n ,
the
s u p e r n a t e - w a s removed and t e s t e d f o r t h e p r e s e n c e o f v i rus by f o c u s f o r m a ­
tio n .
Qf t h e 6 c l o n e s
.T a b le 'll.
, I n d u c t i o n i o f . Non-Producers.
■N o n- P ro du c er
■Number
I
:2
•
'3
■4
-5
6
7
8
,
-RSV(SR) I O ' 6
' Control
(a)
,(b)
:( c )
,(d)
i n d u c e d , .6 p r odu ce d v i r u s .
• S u pe r n at e
" Cont r o l
Oa
■0
0
0
0
0
'NT
0
0
-RSV(SR)
IO4 FFli
+b
-NT
+
+
+
+
.NT
+
'
.RSV(SR)
10"° d i l u t i o n
NTc
0
NT
NT
0
o•
0
NT
RAV
10'^ d i l u t i o n
'NT
+
■NT
NT
NT
NT
+
NT
0 = no v i r u s f o u n d : i n 0 . 5 m l ■o f t h e m e d i urn
+ = v i rus •found i n 0 . 5 ml o f t h e medium
NT = n o t •t e s t e d .
0 . 5 ml o f a 1 0 " ° d i l u t i o n o f RSV(SR) was t e s t e d : f o r t h e p re s en c e -of
RSV(SR)
The p o s s i b i l i t y a l s o e x i s t e d t h a t a n y ; RSV(SR)
be p r e s e n t
in v e ry hig h c o n c e n t r a t io n s .
h e l p e r m i g h t , , l i k e RAV,
, T h e r e f o r e , 4 c l o n e s were i n f e c t e d
5-5
w i t h a -IO ^ d i l u t i o n
f o c i when-tested J l
o f RSV( SR)
hrs.
c l o n e s we re i n d u c i b l e ,
II).
la te r.
(T a b le ■(!)„
None o f t h e s e c l o n e s pro du ce d
As a p o s i t i v e c o n t r o l
2 we re i n f e c t e d w i t h a 10~3 d i l u t i o n
Bot h o f t h e s e c l o n e s - p r o d u c e d v i r u s a f t e r
The r e s u l t s
t o show t h a t t h e s e
in Table
I I indicate.! t h a t
o f RAV ( T a b l e
ind uctio n .
i f RSV(SR) was d e f e c t i v e ,
h e l p e r v i r u s was p r e s e n t a t c o n c e n t r a t i o n s c l o s e t o t h a t o f RSV(SR)
,They a l s o
i n d i c a t e t h a t RSV(SR) m i g h t e i t h e r m u l t i p l y
o r s e r v e as a h e l p e r and t h u s
i n RSV(B)
in du ce v i r u s p r o d u c t i o n
f o r RSV(B), t h e s u p e r n a t e f r o m some-of t h e
d ilu te d
we re t he n p i c k e d
. RSV(B)
b le
i n low d i l u t i o n s
c o u l d s e r v e as a h e l p e r
on t e s t p l a t e s .
II)
was
,The f o c i
i n t o w e l l s as d e s c r i b e d e a r l i e r ( M a t e r i a l s a n d •M e t h o d s ) .
f o c i were p i c k e d f r o m a r e a s a d j a c e n t t o RSV(SR)
in o rd e r t o
NP c e l l s ,
i nduced c l o n e s ( T a b l e
so as t o g i v e v a r i o u s numbers o f f o c i
its e lf.
i n RSV(B) NP c e l l s .
In o r d e r t o a n a l y z e f o r . t k e p r e s e n c e o f a h e l p e r v i r u s
o f RSV(SR) , and t o . d et e rm i ne w h e t h e r o r n o t RSV(SR)
the
f o c i whenever p o s s i ­
i n c r e a s e t h e chance o f c o n t a m i n a t i n g t h e RSV(B) f o c u s w i t h
h e l p e r v i r u s f r o m RSV(SR).
Table
t h e RSV(B)
I I I shows t h a t b o t h RSV(B)
non-producers
f r o m RSV(SR)
and RSV(SR) were p r od uc e d by some o f
i nduced w i t h RSV(SR);
in duce d c l o n e s f a i l e d
to
that a ll
release v ir u s ;
b u t I RSV(B) f o c u s
that a ll
p r odu ce d v i r u s even when p i c k e d f r o m uncrowded p l a t e s ;
and t h a t al l: RSV(B)
f o c i w h i c h a r o s e as a r e s u l t o f RAV i n d u c t i o n y i e l d e d v i r u s .
l e ad t o t h e f o l l o w i n g c o n c l u s i o n s .
l e u k o s i s gr oup o f v i r u s e s
RSV(B).
S e c o n d , . RSV(SR)
(Rubin,
F irs t,
These r e s u l t s
l i k e o t h e r members o f t h e a v i a n
1 9 6 4 ) , ,RSV(SR)
is not d e f e c t iv e .
RSV(SR) f o c i
can s e r v e as a h e l p e r f o r
-The second c o n c l u s i o n
is
5&
■ Table - I I I .
• Focus
.'Number
I
2
'3
4
5
.6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
'23
24
(a)
(b)
Ce)
:(d)
. V i r u s P r o d u c t i o n by Foci
■ Focus
M o r p h o l oqy
Ba
B
.3
■B
B
■B
"B
B
:B
B
B
B,
-SRb
SR
.SR
SR
.SR
SR
SR
SR
■B
'B
-B
-B
O b t a i n e d f r o m Induced NP C lo nes.
NPd
Number
I
I
6
6
■6
6
6
6
6
. 6
I,
I
4
3
I
I
.6
'5
5
5
2
2
2
2
Number and
Types o f Foci
On P l a t e f r o m
Which Focus
Was P i cke d
. Type o f
Vi rus
Produced
by Focus
6 SR
22SR
Tl B
62B
•53
'SB
8 SR 164 b
-.8SR 1:64b
8 SR T64B
8 SR T64 b
8SR-164 b
8 SR 164 b
2 2SR 62B
.22 SR 62 B
64 SR
32SR
22SR 62B
6 SR TlB
■8 SR•I 24 b
27SR
27SR
•27SR
333
33B
'3 3 B
33B
0=
0
0
0
0
0
0
0
0
■o
SR & B
0
SR
SR
SR
-SR
SR
SR
..SR
SR
B
B
B
B
: B - Focus m o r p ho l og y c o r r e s p o n d i n g t o t h a t o f RSV(B)
' SR- = Focus m o rp h ol o g y c o r r e s p o n d i n g t o t h a t o f RSV(SR)
0 = -No v,i ru.s p roduced
NP Number r e f e r s t o t h e n o n - p r o d u c e r number in T a b l e I l o f t h e
in duce d c l o n e whose - s u p e r n a t e was used as a s o ur c e o f ' v i r u s .
57
" S t r e n g t h e n e d by t h e o b s e r v a t i o n s t h a t no f o c i w i t h t h e m o rp h o l o g y o f .'RSV(SR)
w e r e - f o u n d -t o be n o n - p r o d u c e r s ,
and t h a t a l l
RSV(B)
foci
d e r i v e d f r o m RAV-
i nduced c l o n e s c o n t i n u e d t o p r o du c e v i r u s .
. RAV I n t e r f e r e n c e w i t h ' RSV. R e p l i c a t i o n
S i n c e d e f e c t i v e n e s s was n o t f o u n d t o be a p r o p e r t y o f RSV(SR), ,an
e x p e r i m e n t was d e s i g n e d t o t e s t t h e s u s c e p t i b i l i t y o f RSV(SR)
-ference,
Secondary c h i c k . f i b r o b l a s t s we re i n f e c t e d w i t h 2 - f o l d
d i l u t i o n s o f RAV.
tio n ,
t o RAV i n t e r -
- The i n f e c t e d c e l l s were - t r a n s f e r r e d 4 days a f t e r
and ^a ga in 4 days l a t e r .
a known amount o f RSV(B)
transferred
serial
At t h i s
and RSV(SR).
t i m e t h e c e l l s were c h a l l e n g e d w i t h
Control
u n i n f e c t e d c u l t u r e s were
t h e same as RAV i n f e c t e d c e l l s and c h a l l e n g e d w i t h
q u a n t i t i e s o f RSV(B)
t h e same
and RSV(SR).
The r e s u l t s o f t h i s e x p e r i m e n t a r e shown i n . F i g u r e 10.
d i l u t i o n o f RAV w h i c h
infec­
,The h i g h e s t
i nduced i n t e r f e r e n c e t o -RSV(B) was . 10™^.
Therefore,
, t h e t i t e r o f ' RAV used i n t h i s e x p e r i m e n t was IO^ i n f e c t i o u s u n i t s / m l .
t h ou gh t h e r e seemed t o be some i n t e r f e r e n c e w i t h RSV(SR)
:o f 0 . 5 x I O " 8 , t h i s was o f d o u b t f u l
c l u d e d t h a t RgV(B)
a nd 1RSV(SR)
sig n ific a n c e .
a t a RAV d i l u t i o n
.Therefore,
showed s i m i l a r degrees o f
Al -
i t was c o n­
su s c e p tib ility
to
RAV i n t e r f e r e n c e .
Growth o f - RSV(B)
and RSVCSR)
i n .;Chi c k F l b r o b l a s t s
E i g h t e e n t o <24 h r . c u l t u r e s c o n t a i n i n g 10& s e con dar y c h i c k f i b r o b l a s t s
we re exposed t o 10& FFU o f RSV(B)
o f I ml f o r I h r . , a t 37 C.
o r I O^ FFU o f RSV(SR)
A fte r th is
time,
in .a to ta l
volume
each p l a t e was washed once
58
F i g u r e 10.
RAV I n t e r f e r e n c e w i t h RSV(B)
and RSV(SR)
in fe c tio n .
..
Open c i r c l e s
r e p r e s e n t t h e d a t a f o r RSV(B), and X 1s r e p r e s e n t
t h e d a t a f o r RSV(SR).
Each p o i n t
is the average o f 2 p l a t e s .
The r e s u l t s a r e p l o t t e d as FFU o f RSV on RAV i n f e c t e d c e l l s
d i v i d e d by FFU o f RSV on t h e c o n t r o l
p l o t t e d as < ^ 0 . 1
p le te ly
RSV(SR) .
c e lls .
Those p o i n t s
FFU RAV
a re t h e r e s u l t s f r o m c e l l s comFFU C o n t r o l
r e s i s t a n t t o i n f e c t i o n w i t h 4 ,0 0 0 FFU o f RSV(B) o r
59
F ig u r e 10.
RAV I n t e r f e r e n c e w i t h RSV(B)
RAV C e l l s
n t r o l Ce)
RSV(B)
RSV(SR)
0 .0 1 ---------------------- L _
Contro l 0 . 5 x 1 0 ' °
D i l u t i o n o f RAV
and RSV(SR)
In fe c tio n
w i t h 2 ml o f warm medium 199•
p l a t e and t h e - c u l t u r e s
■of 5 p e r c e n t
sampling.
F i v e ml o f warm 199 was t h e n added t o each
i n c u b a t e d a t 37 C in a w a t e r s a t u r a t e d atmosphere
CQ2 in a i r .
P l a t e s were removed a t t h e d e s i g n a t e d t i m e s f o r
, The ■s u p e r n a t e s f r o m d u p l i c a t e p l a t e s were p o o l e d and s t o r e d
' s e a l e d ampules on d r y
ice u n t i l
t h e y we re t i t e r e d .
in
The number o f c e l l s on
each p l a t e was d e t e r m i n e d arid t h e a v e r a g e o f d u p l i c a t e samples was used t o
c a l c u l a t e focus form ing u n i t s / c e l I (F F U /c e lI ) .
- F i g u r e -Tl
viru s
g i v e s t h e r e s u l t s o f such an e x p e r i m e n t .
r e l e a s e between 12 and 24 h r s . a f t e r
The r a t e o f
i n f e c t i o n can be c a l c u l a t e d
• f r om t h e f o l l o w i n g e q u a t i on:
Iog V t 2 -
Vtj
log V t ] '
_ K
= F F U /c e lI a t time t j
■V t 2 = F F U / c e l I a t t i m e ^ t 2
‘ *■
t
= Time i n h r s .
.K
Rate c o n s t a n t f o r
release
By s u b s t i t u t i n g v a l u e s f r o m F i g u r e I I
' f o r RSV(B)
in cre ase
in to t h i s equation,
in v i r u s
the valu e o f K
i n f e c t e d c e l l s was c a l c u l a t e d t o be I . 08 x I 0 ” ^ FFU/cel I / h r .
and K f o r RSV(SR)
i n f e c t e d c e l l s was 1 . 1 8 x 10""^ FFU/cel 1 / h r .
v i r u s p r o d u c t i o n by b o t h c u l t u r e s
between 12 and 24 h r s . a f t e r
i n c r e a s e d a t a p p r o x i m a t e l y t h e same r a t e
in fe c tio n .
A t 24 h r s . , t h e
p r o d u c t i o n d e c r ea s ed : f o r b o t h s e t s o f c e l l s and f i n a l l y
a t 36 h r s . a f t e r
in fe c tio n .
Therefore,
RSV(B)
rate o f v iru s
reached a maximum
i n f e c t e d c e l l s m a i n t a i n e d t h i s maximum
r a t e o f v i r u s p r o d u c t i o n f o r 48 h r s . ; however, . RSV(SR)' p r o d u c t i o n d ecr ea se d
6,1
{;
‘ F i qure -I I .
Growth Curves o f RSV(B)
The open c i r c l e s
c irc le s
and =RSV(SR).
r e p r e s e n t d a t a f o r RSV(SR), and t h e c l o s e d
r e p r e s e n t d a t a f o r RSV(B) .
For c o m p a r a t i v e pur po se s
t h e c u r v e s we re drawn on t h e same g r a p h .
The r i g h t m a r g i n
c o n t a i n s t h e o r d i n a t e v a l u e s f o r RSV(SR) , and t h e l e f t m a r g i n
t h o s e f o r RSV(B).
Note t h e v a l u e s a t any p o i n t d i f f e r by I
to 2 Iogs.
• \
. /-
62
F ig u r e 11.
Growth Curves o f RSV(B) and RSV(SR)
-ORSV(SR)
- * RSV(B)
Sample Time in Mrs. A f t e r
In fe c tio n
63
- b e t w e e n <36 and 4 8 - h r s . a f t e r
in fe c tio n .
Indeed, as w i l l
be seen bel ow,
RSV(SR)
v i r u s p r o d u c t i o n g e n e r a l l y d e c r ea s ed n e a r l y a l o g between 24 and
4 8'hrs.
a fte r
in fe c tio n ,
whereas RSV(B)
p r o d u c t i o n was m a i n t a i n e d a t a
f o r up t o .9 6 ' h r s .
maximum l e v e l
Not ekbhe d i f f e r e n c e
i n t h e amount o f t o t a l
a n d -RSV(SR) a t any g i v e n t i m e .
. g r e a t e r t ha n t h a t o f RSV(SR).
Total
RSV(B)
v i r u s p r od uc e d b y . RSV(B)
FFU was a l w a y s I t o 2 l o g s
T h i s d i s c r e p a n c y was a l s o t r u e o f v i r u s
re­
lease from
e s t a b l i s h e d RSV(B) and
RSV(SR) c e l I s . ( s e e b e l o w ) . Ther e wo u ld
seam t o be
s o m et h i ng l i m i t i n g t h e
amount and n o t t h e r a t e o f RSV(SR)
p r o d u c t i o n t h a t k e e p s . i t b el ow t h a t o f RSV(B).
Vi rus Rel ease b y ; Est a b ! I shed RSV Cel I s
E s t a b l i s h e d - RSV(B) o r RSV(SR)
s ec on d ar y c h i c k - f i b r o b l a s t s w i t h
A fte r
d ay s .
IO^ FFU o f e i t h e r RSV(B)
i n c u b a t i o n f o r 4 t o 5 days ,
order to .m a in ta in established
.The
transfer.
the
RSV
IO^
o r RSV(SR) .
i n f e c t e d c e l l s were t r a n s f e r r e d .
In
c e l l s , t r a n s f e r s were made e v e r y 5 t o 7
medium was u s u a l l y changed on t h e t h i r d o r f o u r t h day a f t e r
. E s t a b l i s h e d RSV c e l l s were n e v e r used f o r e x p e r i m e n t a l •p ur po s es
p r i o r t o the t h i r d
transfer.
To measur e v i r u s
r e l e a s e by t h e s e c e l l s , d u p l i c a t e p l a t e s were p r e ­
p ar e d f o r each samp I e , t i m e .
mm p e t r l
c e l l s we re o b t a i n e d by i n f e c t i n g
dish.
I n i t i a l l y , .I
. A f t e r 18 t o 24 h r s .
of
ce l I s were p i aced in each 60
incubation,
t h e medium was removed
f r o m t h e p l a t e s and each washed once w i t h 2 ml o f warm '199-
A f t e r washing,
,5 ml o f warm medium 199 was p l a c e d i n t h e p l a t e s , . and t h e y we re i n c u b a t e d
i n a w a t e r s a t u r a t e d a tmosp her e o f 5 p e r c e n t COg in a i r a t 37 C ( z e r o
64
'.tilme)"'
A t : specified
■incubator,
i n t e r v a l s ■sample p l a t e s w e r e ■removed f r o m t h e
supernates from d u p l i c a t e p l a t e s pooled,
per p la te determined.
'dry
tim e
ice u n t i l
. P oo l ed s u p e r n a t e s were s t o r e d
RSV(B)
c e l l s displayed a rapid
c e l I s - appear ed t o
8 and 16 h r s .
increase,
in s e a l e d ampules on
t h e i r v i r u s c o n t e n t c o u l d be d e t e r m i n e d by f o c u s a ssay.
■The r e s u l t s o f such an e x p e r i m e n t a r e g i v e n
and RSV(SR)
and t h e number o f c e l l s
in F i g u r e 12.
Both RSV(B),
r e l e a s e o f v i r u s between 0 and 8 I h r s .
r e a c h ' t h e i r maximum ;v i rus r e l e a s e p o i n t between
, The amount o f v i r u s
r e l e a s e d by RSV(SR)
c e l l s continued to
b u t a t a -much s l o w e r r a t e , between 8 and 16 h r s .
. Maximum" RSV(SR)
1r e I ease was o b t a i n e d by 16 h r s .
. Bot h RSV(B) 1and- RSV(SR)
viru s
r e l e a s e decr ea se d a f t e r
RSV(SR) d e c r e a s i n g a t a g r e a t e r r a t e t h a n RSV(B).
a r a p i d d ec r e a s e
in v i r u s p r o d u c t io n a f t e r
c h a r a c t e r i s t i c o f RSV(SR)
infected c e lls ,
a l wa y s d e cr e as e d a t a much s l o w e r r a t e .
.A s w ill
16 h r s . . w i t h
be seen l a t p r ,
r e a c h i n g a maximum l e v e l was
w h e r e a s -RSV(B)
v ir u s production
The I l og o r g r e a t e r d i f f e r e n c e
i n t h e amount o f RSV p r odu ce d was a l s o c h a r a c t e r i s t i c .
, F o r any g i v e n t i m e ,
t h e r o was a l w a y s a t I e a s t 10 t i m e s more RSV(B) p roduced p e r c e l l
t han t h e r e
-was RSV(fSR) . 1
, E f f e c t o f I n c r e a s i n g -C o n c e n t r a t i on o f Pur omyc in o n Cel I ! D i v i s i o n and V i r u s
Production
When s t u d y i n g t h e e f f e c t s o f a m e t a b o l i c
(PU)
on a v i r u s
t h e c el I ,
it
re p lica tin g
system w h i c h
i n h i b i t o r such as p ur omy ci n
requires constant v i a b i l i t y of
i s necessary to-determin e the c o n c e n tr a t io n o f the
•.which w,i I I p r o du c e t h e g r e a t e s t
in h ib itio n of virus
in h ib ito r
r e p l i c a t i o n w i t h the
65
1
F i g u r e 12.
V i rus Rel ease by E s t a b l I shed- RSV(B)
Open c i r c l e s
and RSV(SR)
r e p r e s e n t t h e d a t a f o r RSV(B)
the clo sed c i r c l e s
t h a t f o r RSV(SR) .
viru s
The l e f t
c o n t a i n s t h e s c a l e f o r t h e RSV(B) o r d i n a t e ,
Cel I s .
release,
hand m a r g i n
and t h e r i g h t
hand m a r g i n c o n t a i n s t h e s c a l e f o r t h e RSV(SR) o r d i n a t e .
Each p o i n t
i s t h e a v er a ge o f 2 p l a t e s .
X
and
66
F ig u r e 12.
V i r u s Release by E s t a b l i s h e d RSV(B)
and RSV(SR)
C e lls
RSV(SR)
Sample Time in Mrs. A f t e r Washing
6(7
l e a s t damage t o t h e c e l l s .
In o r d e r t o do t h i s ,
se co nd ar y c u l t u r e s o f
c h i c k f i b r o b l a s t s were p r e p a r e d ( M a t e r i a l s and M e t h o d s ) .
was i n f e c t e d w i t h
IO^ FFU o f RSV( B)
One s e t o f p l a t e s
i n I ml o f medium 199 f o r I h r .
p l a t e s w e r e t hen washed once w i t h 2 ml o f medium 199 ( z e r o t i m e ) .
These
A fte r
t h i s , .2 ml o f 199 c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f PU were added t o
each p l a t e .
Fo r each c o n c e n t r a t i o n o f PU, 2 p l a t e s we re p r e p a r e d .
v i r u s t i t e r s were d e t e r m i n e d 24 h r s .
a fte r
i n f e c t i o n by p o o l i n g t h e s u p e r -
n a t e s o f p l a t e s c o n t a i n i n g t h e same c o n c e n t r a t i o n s o f 'PU.
infecte d co n tro l
A s e r i e s o f non-
p l a t e s were t r e a t e d e x a c t l y as above.,
F i g u r e 13 phows t h a t
to
The
i n f e c t e d and n o n - i n f e c t e d c e l l s
i n c r e a s i n g c o n c e n t r a t i o n s o f PU.
o c c u r r e d when 2 / t g / m l
respond s i m i l a r l y
Maximum i n h i b i t i o n o f c e l l
o f PU w e r e i n c o r p o r a t e d
i n t o t h e medium.
d iv is io n
At co nce n­
t r a t i o n s o f PU g r e a t e r t h an l y ^ g / m l , d e g e n e r a t i v e changes such as v a c u o l i ­
z a t i o n and r o ug h e n i n g o f t h e c e l l
s u r f a c e were n o t e d .
o f PU g r e a t e r t h an I t o 2ytg/m1 were l e f t
t h a n 24 h r s . ,
t h e c e l l s d i e d and e i t h e r
I f concentrations
in c o n ta c t w i t h
the c e l l s
longer
slo ughed,from the p l a t e s o r did
not survive t r y p s i n i z a tio n .
■In a n t i c i p a t i o n o f e x te n d e d e x p o s u r e s o f
infected c e l l s
■counts we re p e r f o r m e d on p l a t e s exposed t o ^lzA g Z m l
, 96 h r s . a f t e r
considerably
in fe c tio n .
retarded,
o f puromycin f o r 0 t o
,,As may be seen i n F i g u r e 14, c e l l
and no d e c r e a s e
in c e l l
t o PU,.cel I
d i v i s i o n was
numbers was n o te d u n t i l
96
hrs.
• F i g u r e 15 i l l u s t r a t e s
the e f f e c t o f
on v i r u s p r o d u c t i o n 24 h r s . a f t e r
i n c r e a s i n g c o n c e n t r a t i o n s o f PU
L rifecti on.
,One/tg/ml
o f PU i n h i b i t e d
68
}
rrv
.
I n h i b i t i o n o f Ce-I I Growth by Puromyci n .
Open c i r c l e s
represent
represent u ninfe c te d c e ll
infected c e ll
counts.
counts.
.Each p o i n t
b e r o f c e l l s on d u p l i c a t e p l a t e s .
Open t r i a n g l e s
i s t h e a v er ag e num­
69
I n h i b i t i o n o f C e ll Growth by Puromycin
u m b e r /P la t
F ig u r e 13.
A
I n f e c t e d Cel I s
n i n f e c t e d Cel I
C o n c e n t r a t i o n o f Puromycin ^<g/ml
&8
A
f
*
■F i g u r e - 14 .
E x p o s u r e - o f I n f e c t e d Cel I s f o r Ext ended P e r i o d s o f Time t o
o f "P u r o m y c i n .
Open c i r c l e s
IzAgZml
represent c e l l
o f puromycin.
counts from p la te s tre a te d w it h
Cl osed c i r c l e s
f r o m c e l l s n o t exposed t o p u r o m y c i n .
represent c e ll
Each p o i n t
ij
counts
I
re presents
t h e a v e r a g e number o f c e l l s on d u p l i c a t e p l a t e s .
X
.
.:
71
F ig u r e 14.
Exposure o f I n f e c t e d C e l l s f o r Extended Pe rio d s o f Time
t o Iy * g / m l o f Puromycin
U n tr e a t e d C e l l s
PU-t r e a t e d C e l l s
Time in Mrs. A f t e r
In fe c tio n
72
!;!
/
I
F i g u r e 15.
I n h T b i t i on o f 24 Hr. V i rus P r o d u c t i o n by I n c r e a s i n g C o n c e n t r a ­
t i o n s o f Puromycin .
Open c i r c l e s
r e p r e s e n t t h e amount o f v i r u s p r odu ce d by RSV(B)
i n f e c t e d c e l l s 24 h r s . a f t e r
i n f e c t i o n when v a r i o u s c o n c e n t r a ­
t i o n s o f PU we re added a t t h e t i m e o f
i s t h e a v er a g e o f 2 e x p e r i m e n t a l
in fe c tio n .
p la te s.
Each p o i n t
\
73
F ig u r e 15.
I n h i b i t i o n o f 2 k Hr. V i r u s P ro d u c tio n by I n c r e a s i n g C o n c e n tra tio n s of Puromycin
C o n c e n t ra t io n o f PuromycinygZm i
v iru g p roduction b y .^8-per cent.
Maximum i n h i b i t i o n was o b t a i n e d w i t h 4
■and ' S / ^ g / m l .
• One/c-g/ml
o f PU was s e l e c t e d f o r use i n v i r u s
as t h i s c o n c e n t r a t i o n
reduced v i r u s - p r o d u c t i o n by n e a r l y 2 l o g s ( F i g u r e 15)
and a I I owed -cel I s u r v i v a l
:w h e r e a s , , c e l l
i n h i b i t i o n experiments
f o r e xt en de d p e r i o d s o f e x p o su r e ( F i g u r e 1 4) ;
damage was n o t e d w i t h c o n c e n t r a t i o n s o f 2 y ^ g/ ml
o r more o f PU,
K i n e t i c s o f t h e . S u p p r e s s i o n o f T w e n t y - f o u r Hour V i rus P r o d u c t i on by Pur omyc i n .
C e l l s were i n f e c t e d , t r e a t e d w i t h PU and sampled as d e s c r i b e d
previous section w ith
times,
t h e e x c e p t ion t h a t J / ^ g / m l
o f PU was used a t a l l
and t h a t p l a t e s were i n f e c t e d w i t h 2 x IO^ FFU/ml o f RSV(SR)
t h i s was t h e maximum amount o f v i r u s o b t a i n a b l e .
of w ashing.the in fecte d c e l l s .
PU was added a t t h e d e s i g n a t e d t i m e s , and
infected c e lls
c o n t r o l s a t the tim e o f sampling
might occur d uring t i t r a t i o n
, F i g u r e 16 i l l u s t r a t e s
' RSV(B) and RSV(SR)
have s to pp ed c e l l
since
. Zero t i m e was t he t i m e
remained ,in c o n t a c t w i t h t h e c e l l s u n t i I .'..the t i m e o f s a m p l i n g .
consisted o f v ir u s
in the
n o t exposed t o PU.
Controls
PU was added t o t h e
in o r d e r t o o m i t any d i s c r e p a n c i e s w h i c h
o f t h e samples due t o t h e p r e s e n c e o f PU.
t h e e f f e c t o f t h e t i m e o f a d d i t i o n o f PU on
i n f e c t e d c e l l s 24 h r s .
a fte r
in fe c tio n .
d i v i s i o n a t t h e t i m e i t was a dd e d .
,PU appeared t o
T h e r e f o r e , ■even
though, s a m p l e s . w e r e n o t c o u n t e d f o r 4 t o 20 h r s . a f t e r e x p o s u r e t o PU, t h e
re su lting
increase
in c e l l s per p l a t e
r e se mb les t h a t o f a normal
c u r v e w i t h a g e n e r a t i o n t i m e o f 20 t o 24 h r s .
a l a g - p e r i o d as t h e y had been in c u l t u r e
began.
g r owt h
The c e l l s d i d n o t e x p e r i e n c e
18 t o 24 h r s . b e f o r e t h e e x p e r i m e n t
75
Il
;,v:
■Figure 16 .
E f f e c t o f t h e Time -o f Add1 1 i on o f Puromycin on 2k Hr. V i r u s
P r o d u c t i on by RSV(B) and RSV(SR) I n f e c t e d Cel I s .
Open c i r c l e s
r e p r e s e n t d a t a f o r RSV(B); c l o s e d c i r c l e s
s e n t d a t a f o r RSV(SR).
m e nt s .
.The e x p e r i m e n t a l
so e v i d e n t h e r e .
lin e
Each p o i n t
i s an a v e r a g e o f 4 e x p e r i ­
f l u c t u a t i o n f o u n d a t 48 h r s . was n o t
The l o w e r g r ou p o f X1s c o n n e c t e d by .a sol id
represents a ty p ic a l
curve of c e il
c e i l s d u r i n g an e x p e r i m e n t .
times.
repre­
One^Ag/ml
c o u n t s o f PU t r e a t e d
o f PU was used*- a t a l l
76
F ig u r e 16.
E f f e c t o f the Time o f A d d i t i o n of Puromycin on 24 Hr. V i r u s Prod uc tion
____________________ by RSV(B) and RSV(SR) I n f e c t e d C e l l s ______________
'er o f C e ll s / P I a t
RSV(SR)
•RSV(B)
T i m e P u r o m y c i n A dd ed
in
M rs.
A fte r
In fe c tio n
.
Twenty-four hr.
b o t h RSV(B)
.
ri
v i r u s p r o d u c t i o n was i n h i b i t e d
and RSV(SR)
in the'same m an ne r-fo r
i n f e c t e d c e l I s ( F i g u r e -16) . ' .The -data wef:e p l o t t e d
as - f o c u s f o r m i n g u n i t s p e r c e l l :■( F FU / ce l I ) , , p e r c e n t o f t h e c o n t r o l
o r d e r t o compare t h e d eg r ee o f
w ith 'd iffe rin g
i n h i b i t i o n o f v i r u s p r o d u c t i o n among p l a t e s
numbers o f c e l l s .
, F i g u r e 16 ■shows t h a t t h e r e was a t l e a s t
• one "PU s e n s i t i v e - e v e n t o c c u r r i n g between 0 and 12 h r s . a f t e r
„ Th i s
in
in fectio n .
i s n o t s u r p r i s i n g as t h e l a t e n t p e r i o d o f b o t h v i r u s e s was abo ut 12
hrs,
, Th er e Was a l s o a - PU s e n s i t i v e e v e n t w h i c h o c c u r r e d between I'2 and 24
hrs,
a fte r
in fe c tio n ,
; K i n e t i c s o f t h e Suppr e s s i on o f F o r t y - e i g h t Hour V i rus P r o d u c t i o n by
Puromyc i n
'
The e f f e c t o f
i n h i b i t o r s on 24 h r .
been shown t o be - q u i t e - d i f f e r e n t
1964; Temin,
1963, . 1 9 6 4 a ) .
and 48 h r .
( G o l de and V i g i e r , . 1963;
Therefore,
viru s
time,
The 2 v i r u s e s behaved d i f f e r e n t l y a t t h i s
RSV(B) a f t e r w h i c h t i m e v i r u s
if
F ig u re I 7 g iv e s the re­
, Some i n h i b i t i o n was n ot ed between O and 4 h r s .
RSV(SR)
v iru s
a fte r
in fe ctio n w ith
r e l e a s e i n c r e a s e d t o a maximum a t 20 h r s .
r e l e a s e was l i m i t e d b y 'PU i f
t h e amount o f v i r u s
V i g i e r and G o l de;
i n f e c t e d c e l l s were exposed
t o 'PU as d e s c r i b e d above and sampled a t 48 h r s .
s u l t s o f these experim ents.
v i r u s p r o d u c t i o n has
i t was added b e f o r e 16 h r s . , as
r e l e a s e d was p r o p o r t i o n a l
t o t h e amount o f p r e c u r s o r
p r o t e i n p r odu ce d up t o t h e t i m e RU was a d d e d , b u t t h e r e was no l ag t o
cate the n e c e s s ity o f
w i t h RSV(B).
in itia tin g
A su rp risin g
stim ulate v iru s
in d i­
t h e s y n t h e s i s o f t h e p r o t e i n as t h e r e was
r e s u l t was t h a t t h e p r e s en c e o f PU seemed t o
r e l e a s e f r o m RSV(SR)
c e lls .
-Note t h a t a f t e r 4 h r s .
the
78
•'
’
'
!:
-
F i g u r e 11 .
E f f e c t o f t h e Time o f A d d i t i o n ' o f Pur omycin on 48 Hr. V i rus
P r o d u c t i o n by RSV(B) arid RSV(SR) I n f e c t e d Cel I s.
F ille d
c irc le s
Open c i r c l e s
r e p r e s e n t t h e d a t a f o r RSV(SR)
r e p r e s e n t t h e d a t a f o r RSV(B)
- Because o f t h e v a r i a b i l i t y
the r e s u lt s of
closed c i r c l e
open c i r c l e
A typical
n o t e d f ro m e x p e r i m e n t t o e x p e r i m e n t ,
r e p r e s e n t s t h e a v er a ge o f 3 e x p e r i m e n t s .
Each
Each
re p re s e n ts th e average o f 5 expe rim en ts.
c u r v e showing c e l l
counts from I o f the experiments
\
the f i g u r e .
The r i g h t hand m a r g i n
g i v e s t h e u n i t s o f measure,
t h e t i me s o f s a m p l i n g a r e i d e n t i c a l
those o f th e v i r u s
used t h r o u g h o u t t h e e x p e r i m e n t s .
a fte r
infected c e lls .
i n de p e n d e n t e x p e r i m e n t s we re a v e r a g e d ;
i s shown a t t h e b o t t o m o f
w ith
infected c e l l s .
in fe c tio n .
PU was
Al I t i m e s a r e g i v e n
in h r s .
79
E f f e c t o f the Time o f A d d i t i o n of* Puromycin on 48 Hr. V i rus P ro d u c tio n
by RSV(B) and RSV(SR) I n f e c t e d C e l l s
-#
RSV(SR)
RSV(B)
T i m e P u r o m y c i n A dde d
in
M rs. A f t e r
In fe c tio n
ier o f Cel I s / P l a t
C ells
80
amount ■'o f v i r u s
re-1 eased by RSV(SR)
r e l e a s e d by t h e c o n t r o l
o f PU t r e a t e d RSV(B)
c e lls .
c e lls
i n f e c t e d c e l l s was 1 .6 t o 6 t i m e s t h a t
T h i s was i n marked c o n t r a s t t o t h e - b e h a v i o r
in w h i c h t h e amount o f v i r u s
b u t n e v e r exceeded t h a t o f t h e c o n t r o l s .
■RSV(B)
r e l e a s e d approached
F o rty -e ig h t hr.
c e l l s app ear ed t o be 8 hrs-. b e h i n d t h a t o f RSV(SR)
e x a m p l e , ,RSV(B)
r e l e a s e d i d n o t a pp ro ac h l i n e a r i t y
u n til
viru s
r e l e a s e by
c e lls .
For
8 hrs. a f t e r
f e c t i o n , whereas r e l e a s e was l i n e a r f r o m t i m e 0 w i t h RSV(SR).
Also,
in­
v iru s
r e l e a s e appro ache d a s t a t i o n a r y phase bet ween 12 and 16 h r s . w i t h RSV(SR)
c e lls ,
but not u n t i l
The i n c r e a s e
explained
between 20 and 24 h r s . w i t h . RSV(B)
c e lls .
in v i r u s p r o d u c t i o n by PU t r e a t e d RSV(SR)
i n 2 ways.
c e l l s c o u l d be
. E i t h e r t h e a d d i t i o n o f PU s t i m u l a t e d a massi ve r e l e a s e
o f p re-formed v i r u s p a r t i c l e s ,
o r t h e c o n c e n t r a t i o n o f PU u se d. . (] y k. g/ ml ) was
s u f f i c i e n t t o u p s e t a m e t a b o l i c b a l a n c e between t h e c e l l
such a way t h a t w ha t p r o t e i n
and t h e v i r u s
in
s y n t h e s i s was t a k i n g p l a c e wo u l d c o n t r i b u t e
m ainly to v ir u s p ro d u c tio n .
In T a b l e
IV a r e l i s t e d
t h e amounts o f v i r u s
released p er c e l l
■ c o n t r o l s o f 4 s e p a r a t e e x p e r i m e n t s o f t h e k i n d d e s c r i b e d a bo ve .
lease p er c e l l
i n RSV(B)
s t a y e d n e a r l y t h e same o r
infected c e lls .
In c o n t r a s t ,
t o a low a t 48 h r s .
V irus re­
ro se s l i g h t l y bet ween 24 and 48 h r s .
v iru s production
c e l l s c o n s i s t e n t l y d r o p p e d , n e a r l y 90 p e r c e n t
h i g h a t 24 h r s .
from th e
in. RSV(SR)
in some-experiments,
infecte d
from a
T h e r e f o r e , ■PU seemed t o be s t a b i l i z i n g
v i r u s p r o d u c t i o n by RSV( SR) c e l l s by p r e v e n t i n g t h e d e c r e a s e w h i c h n o r m a l l y
o c c u r r e d between 24 and 48 h r s .
a fte r
in fectio n .,
84
Table
IV.
V i r u s P r o d u c t i on by 24 and448 Hr.
RSV(B)
24 h r . . V i r u s
48 h r ; V i r u s
' Pr o d u c t i on
P r o d u c t I on
Experiment
Number
I
■2
3
4
(a)
C o n trols
3.0
7 .0
2.0
5.2
X--IOaJ t a)
x .IO"2
x 10“
x 10"1
2.0
‘ 1,9
2.4
1.2
x
x
x
x
■ RSV(SR)
48 h r . V i r u i
24 h r . V i r u s
■P r o d u c t ion
■P r o d u c t ion
1.1 x I O ^
I .2 x 1 0 "2
10")
10"
1 0" !
:10 U
5.2 x
1. 7 x
1.6.x
I .1 x
: 9. 8 X 10";
9 .3 X 10 ^
10
10" ;
10" ;
- I O "2
V i r u s p r o d u c t i o n as F F U / c e l I „
If
t h e a d d i t i o n o f PU t o RSV(SR)
c e lls
s t a b iliz e d v ir u s production
■r a t h e r t h a n in duce d a sudden r e l e a s e o f v i r u s p a r t i c l e s f r o m t h e - c e l I ,
s t a b i l i z a t i o n m i g h t l a s t o v e r a lo ng p e r i o d o f t i m e .
ment was c a r r i e d o u t . a s b e f o r e ,
4 8,
72, and 96 h r s .
the
. T h e r e f o r e , . an e x p e r i ­
e x c e p t t h a t v i r u s samples .were t aken a t '24,
The r e s u l t s o f t h i s . e x p e r i m e n t a r e shown in F i g u r e s 18
t h r o u g h 2 1.
In F i g u r e -18, 48 and 96 h r .
several
This
fo ld
v i r u s p r o d u c t i o n by RSV(SR)
c e l l s was
h i g h e r t h a n t h a t o f t h e c o n t r o l w h i c h was s e t a t
i n d i c a t e s t h a t "PU s t a b i l i z e d v i r u s p r o d u c t i o n a t c l o s ^ t o
level
r a t h e r t h an
t h e 72 h r .
i nduced a sudden r e l e a s e - f r o m t h e c e l l s .
100 p e r c e n t .
i t s maximum
Note a l s o t h a t
c u r v e d i d n o t show s i g n i f i c a n t l y more v i r u s p r o d u c t i o n by PU
treated c e lls
t ha n by c o n t r o l
c e lls .
In a n t i c i p a t i o n o f a 48 h r .
dro p.in
v i r u s p r o d u c t i o n by t h e c o n t r o l , . t h e medium was changed on a l l . : R-SV(SR)
c u l t u r e s a t 48 h r s .
trol .
-control
in an a t t e m p t t o
The p r o c e d u r e was s u c c e s s f u l ,
nearly
,This
i n c r e a s e v i r u s p r o d u c t i o n by t h e c o n ­
f o r by 72 h r s .
reached t h a t o f t he 1PU t r e a t e d
i s .more c l e a r l y
released from c e l l s
illu s tra te d
tre ate d .a t
virus
p r o d u c t i o n by t h e
samples.
i n F i g u r e 19.
Here,
the v ir u s
t h e - s t a t e d t i m e w i t h PU was p l o t t e d a g a i n s t
82
F ig u re 18.
E f f e c t o f E x t e n d e d . P e r i o d s ' ' o f E xposure t o Puromyc i n on V i r u s
P r o d u c t i on by RSV( SR) I n f e e t e d Cel I s .
X 1s r e p r e s e n t 48 h r ,
s amp les ; open sq ua re s r e p r e s e n t 72 h r .
sampl es; and open t r i a n g l e s
convenience,
t h e sample t i m e
represent
96 h r . samples.
For
is l i s t e d o p p o s it e the l a s t
p o i n t oh t h e c u r v e r e p r e s e n t i n g t h e c o r r e s p o n d i n g group o f
samples.
Each symbol
i s t h e a v er a ge o f 2 p l a t e s
t h e p o o l e d samples o f 2 e x p e r i m e n t a l
was used t h r o u g h o u t .
in fe c tio n .
representing
p la te s.
Al I t i m e s a r e g i v e n
in h rs . a f t e r
PU
83
F ig u r e 18.
E f f e c t o f Extended P e rio d s o f Exposure to Puromycin on V i r u s P r od uc tion
by RSV(SR) I n f e c t e d C e l l s
96 Hr.
72 Hr.
T im e P u r o m y c i n A dde d
in
H rs.
A fte r
In fe c tio n
84
|!l
''''
i
|:,i
I
' F i g u r e 1 9.
.Mai n t e n a n c e o f RSV(SR)
Open c i r c l e s
P r o d u c t i o n by Pur omyci n T r e a t e d Cel I s .
r e p r e s e n t samples t r e a t e d w i t h PU a t z e r o t i m e ;
X 1s r e p r e s e n t samples exposed t o PU a t 8 h r s .
open t r i a n g l e s
PU a t 12,
r e p r e s e n t an a v er a ge o f a l l
16, 20, and 24 ' h r s . a f t e r
c o n n e c t e d by a d o t t e d
posed t o PU.
lin e
One^t g/ ml
are in h rs . a f t e r
in fe c tio n ;
p l a t e s exposed t o
in fe c tio n .
represent c o n tro l
o f PU was used a t a l l
in fe c tio n .
a fte r
F ille d
s q u a re s
samples n o t e x ­
times.
Al I t i m e s
%
85
M ainten ance o f RSV(SR)
F ig u r e 1 9 .
P r od uc tion by Puromyc in T r e a te d C e l l s
1 2 -2 4 Mrs.
M Control
8 Mrs.
O Mrs.
S am p le T i m e
in
Mrs.
A fte r
In fe c tio n
%
the/sample time
i n , o r d e r f o ' o b t a i n ■some • i dea o f t he - a mo u nt o f
inhibition
i
and t h e s t a b ! T i t y o f v i r u s
r e l e a s e - w h e n -PU was added t o t h e - i n f e c t e d cel Is..
- , I f 'PU was J d d e d : at 0 ' t i m e to. RSV(SR)
infected c e lls ,
vbrus
r e l e a s e was
c o n s t a n t l y s u p p r e s s e d , a b o u t '2 l o g s ;b e l o w t h a t o f maximum v i r u s
•■(12-2^ h r .
curve)
w i t h no n e t - g a i n
in v i r u s p r o d u c t i o n o c c u r r i n g o v e r t h e
-96 h r . , p e r i o d , - . I f "PU was added a t 8 h r s . , v i r u s
s ta n tly
supp re sse d b el o w t h a t o f c e l l s
• showed a s i i g h t
was n ot e d
hrs.
release o c c u r rin g
of viru s
in t h e s e samples.
showed^t he e x p e c t e d d e c r e a s e i n v i r u s
- However,
Stabi I i t y
release
samples .when,PU was added between I Z and 2 4 ' h r s . w i t h t h e
-maximum amount o f v i r u s
cu ltures
r e l e a s e was a g a i n -con­
t r e a t e d w i t h 'PU a t 12- 24 h r s . , b u t
i n c r e a s e o v e r t h e 4 day - p e r i d d .
in -a ll
rele ase
c h a n g i n g t h e medium a t 48 h r s .
The c o n t r o l
r e l e a s e bet ween 24 and 48
seemed t o p r o v i d e . m a t e r i a l
f o r a new mound o f v m r u s . s y n t h e s i s , . f o r by 72 h r s . . t h e c o n t r o l
■sheddi ng-as -much-viiirus as t h e -PU t r e a t e d c e l l s .
c e l l s , were
However, 48 h r s . a f t e r
t h e m e d i u m change (96 h r . . s am p le ) , , v i r u s p r o d u c t i o n by t h e c o n t r o l
ce lls
hpd once - a g a i n ■d e c r e a s e d .
. F i g u r e s .20 a r i d '21 g i v e t h e same d a t a f o r RSV(B)
irifected c e l l s .
In
' F i g u r e '20 i t may be seen t h a t 48 and 72 h r . . v i r u s p r o d u c t i o n behaved. as
e x p e c t e d f r o m F i g u r e -17.
■However, . a t -9.6 , h r s . , v i r u s p r o d u c t i o n exceeded
t h a t ■o f t h e - c o n t r o l : b y -5 times., muph -as i t
had done p r e v i o u s l y wi t h RSV(SR)
■ .
■ cells,
. An e x p l a n a t - i o n f o r t h i s may be d e r i v e d f r o m F i g u r e 21 .
the -viru s
r e l e a s e d by c e l l s exposed To-sPU a t v a r i o u s t i m e s wa,s p l o t t e d
a g a i n s t t h e t i m e - a t w h i c h samples w e r e t a k e n .
rise
.Here-again
in the-amount o f v i r u s
-. The - c o n t r o l
c e lls
r e l e a s e d between .24 and 48 h r s .
showed:a
and a drop
8:7
I;
Is
1
F i q u r e 20.
. E f f e c t o f E xt ended P e r i o d s q f Exposure t o -Puromycin on V i r u s
P r o d u c t i o n by; RSV(B) I n f e c t e d " : Cei I s ; -
Open t r i a n g l e s
r e p r e s e n t t h e d a t a f r o m '72 h r .
p r e s e n t t h e d a t a f r o m 48 h r .
s e n t t h e d a t a f r o m $6 h r .
samples; X 1S r e
samples; and open squares r e p r e ­
samp les.
The sample t i m e s a r e w r i t
t e n o p p o s i t e t h e l a s t p o i n t on t he c u r v e r e p r e s e n t i n g t h e
- c o r r e s p o n d i n g group o f s a mp l e s .
a ll
tim es.
A ll
,OneytgZml
times are in h rs . a f t e r
o f PU was used a t
%
in fe c tio n .
88
Figure 20.
E f f e c t o f Extended Periods o f Exposure to Puromycin on
Virus Production by RSV(B) Infe c te d Cells
72 Hr.
T im e P u r o m y c i n Added
in Mrs. A f t e r
In fe c tio n
89
?
/
F i g u r e 21:
. Ma I n t e n a n c y o f RSV(B)
Open c i r c l e s
fille d
P r o d u c t I on by Pur omyc! r r ' I r e a t e d C e l l s .
r e p r e s e n t samples t r e a t e d w i t h PU a t z e r o t i m e ;
c irc le s
r e p r e s e n t samples t r e a t e d w i t h PU a t 4 h r s „ ;
dpen s q ua re s r e p r e s e n t samples t r e a t e d w i t h PU a t 8 h r s . ;
open t r i a n g l e s
X1s r e p r e s e n t
r e p r e s e n t samples exposed t o PU a t 12 h r s . ;
16 h r . e x p o s u r e s t o PU; f i l l e d
samples g i v e n PU a t 20 h r s . ;
g i v e n PU a t 24 h r s . ; f i l l e d
represent c o n tro l
Al I t i m e s a r e
data.
fille d
r e p r e s e n t samples
sq ua re s c o n n e c t e d by a d o t t e d l i n e
OnezAgZmI
in h rs . a f t e r
tria n g le s
squares r e p r e s e n t
o f PU was used t h r o u g h o u t .
in fectio n .
90
F ig u r e 2 1 . M ainten ance o f RSV(B)
Pro d u c tio n by Puromycin T re a te d C e l l s
Control
16 Mrs.
12 Mrs.
8 Hrs.
4 Hrs.
Samp I e T im e
in
H rs.
A fte r
In fe c tio n
between ? 2 ' a n d g6 Mrs.
. T h i s d r o p :was matched -by an i n c r e a s e
■I ease -by 20 and 24 h r .
treated c e ils
so t h a t by g6 h r s .
c e l l s were p r o d u c i n g more -v,i.,inus t han t h e c o n t r o l .
a ll
tim e
in te rv a ls ,
v iru s
release
in d ica tio n
.The - e x c e p t i o n s
i n f e c t i o n . ' These
r e l e a s e bet ween 24 and 48 h r s .
samples t h e n ro se -to maximum v i r u s
g i v e n iPU a t 0 h r s .
t h e PU t r e a t e d
i n c r e a s e d between 24 and 48 h r s . .and t h e n
.were c e l I s t r e a t e d w.i t h 'PU a t 0 , 20, and 24 h r s . a f t e r
hr,
. The -20 and 24
r e l e a s e . b y 72 h r s . w h i l e t h e samples
continued to d e c lin e
in v i r u s
t h a t s o me th in g n e c e s s a r y f o r 48 h r .
p r e s s e d between O a n d 4 h r s .
re-
Note a l s o t h a t f o r n e a r l y
e i t h e r remained s t e a d y o r d ecr e as e d as d i d t h e c o n t r o l .
showed a d e c r e a s e i n v i r u s
rn v i r u s
-Maximum v i r u s
re le ase g i v i n g f u r t h e r
v i r u s p r o d u c t i o n was sup­
r e l e a s e by PU t r e a t e d c e l l s
n e v e r s i g n i f i c a n t l y exceeded t h e maximum o f t h e c o n t r o l
RSV(SR)• i n f e c t e d c e l l s even t hough v i r u s
c e lls
i i i RSV(B)■o r
r e l e a s e may have been m a i n t a i n e d
over longer periods o f time.
- E ffe ct o f
I n c r e a s i nq Concent r a t ions o f P ur omy ci n on 48 H r . Vi rus - Product i on,
The r e s u l t s
i n t h e p r e v i o u s s e c t i o n sugg est ed t h a t a c o n c e n t r a t i o n o f
- o f PU a d m i n i s t e r e d t o RSV(SR)
.a fte r
i n f e c t e d c e l l s between 16 and 24 h r s .
i n f e c t i o n was a b l e t o - s t a b i l i z e v i r u s p r o d u c t i o n . a t n ea r maximum - l e ­
v e l s ..for e x t e n d e d p e r i o d s o f t i m e due, . p e r h a p s ,
- g l i n g -which ■i n h i b i t e d c e l l u l a r p r o t e i n
a ffe ctin g v ira l
p rotein
synthesis.
If
t o- some p h y s i o l o g i c a l
syntehsis w! thout appreciably
such was t h e ca s e,
t r a t i o n s -of PU m i g h t be -expect ed t o s u pp r e s s 48 h r ,
of
i n c r e a s e d s.uppresss.ion o f p r o t e i n
. c e l l s were p r e p a r e d . a n d
ju g -
synthesis.
in fe c te d .a s described
h i g h e r concen­
v i r u s p r o d u c t i o n 'because
.To t e s t t h i s
hypothesis,
in t h e p r e v i o u s s e c t i o n . .
,At
92
16 h r s . a f t e r
i n f e c t i o n v a r i o u s c o n c e n t r a t i o n s o f "PU w e re added t o d u p l i ­
cate p la te s .
C ontrols consisted o f
. At" '48 h r s .
i n f e c t e d c e l l s n o t t r e a t e d w i t h "PU.
t h e s u p e m a t e s were h a r v e s t e d arid t h e number o f c e l I s p e r p l a t e
- d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d . •
C o n c e n t r a t i o n s o f PU g r e a t e r t h a n lyM-g/ml
b i I i zed 48 h r .
v iru s production,
tio n
c o n c e n t r a t i o n s o f PU had an i n h i b i ­
p r o d u c t i o n a t 48 h r s . ,
a l t h o u g h t h e amount o f
i n c r e a s e d es t h e -PU c o n c e n t r a t i o n was i n c r e a s e d .
and r o ug h e n i n g o f t h e edges was n ot e d
E x t e n s i v e damage and k i l l i n g
r a t h e r t han s t a -
as may be seen i n F i g u r e 2 2. ' As wo ul d be
p r e d i c t e d . ' f r o m ' F i g u r e s .1 7 and 20, a l l
t o r y . e f f e c t on RSV(B)
in h ibite d
in c e l l s
was n o t e d on c e l l s
in h ib i­
Again v a c ti o li z a t i o n
t r e a t e d w i t h 2/«.g/ml
o f PU.
t r e a t e d w i t h more t han
2y«.;g/ml o f ■PU'.
. E f f e c t o f P u r o m y c i n on V i r u s Rel ease: b y , E s t a b l i shed R o u s : C e l I s
E s t a b l i s h e d Rous c e l l s w e r e - p r e p a r e d as d e s c r i b e d p r e v i o u s l y .
.Two s e t s
o f c e l l s we re -used
per v i r u s .
the o th e r s e t , t h e
co ntro ls.
p le time.
At zero
t i m e , .each p l a t e was washed once w i t h 2 ml o f warm me­
dium 199«
.To each
volume o f 2 m l .
-One s e t composed t h e e x p e r i m e n t a l group and
D u p l i c a t e p l a t e s were p r e p a r e d f o r each sam­
experimental
To each c o n t r o l
taken a t the tim es design a te d
p l a t e was added .lyu-g/ml
pi a t e /was added 2 ml o f
i n , F i g u r e s 23 and 24.
,Cell
o f "PU in a t o t a l
199 -
Samples were
c o u n t s were made
- and : t h e -number o f FFU o f RSV in t h e s u p e r n a t e s was d e t e r m i n e d .
F i g u r e 2’3 shows t h e r e s u l t s o f t h i s . e x p e r i m e n t w i t h " RSV(B) c e l l s .
•contro l
c u l t u r e s d i s p l a y e d normal
viru s
,The
r e l e a s e o v e r t h e p e r i o d o f 24 h r s .
• Note - t h a t maximum v i r u s p r o d u c t i o n was a c h i e v e d a b o u t ■12 t o 14 h r s . a f t e r
■ ' F i g u r e 22.
E f f e c t o f I i n c re asi ng Concent r a t i ons o f Puromyc in on 48 Hr.
V i rus P r o d u c t i o n .
Open c i r c l e s
c irc le s
r e p r e s e n t t h e d a t a f o r RSV(B)
t h a t f o r RSV(SR).
and t h e c l o s e d
The b r ok en p o r t i o n s o f t h e gr aph
r e p r e s e n t p o i n t s o f d i s c o n t i n u i t y due t o changes i n t he
o rd in a te scale.
. Each p o i n t
i s t he a v e r a g e o f 2 p l a t e s .
94
F ig u r e 2 2 .
E f f e c t o f I n c r e a s i n g C o n c e n tra tio n s o f Puromycin
on 48 H r. V i r u s P ro d u c tio n
RSV(SR)
RSV(B)
Puromycin Concentration iny.g/m l
,
F i g u r e 23.
E f f e c t o f Pur omycin on V i r u s R e l e a s e ' b y E s t a b l i s h e d RSV(Ei)
Open c i r c l e s
represent c o n tro l
dat a and t h e c l o s e d c i r c l e s
r e p r e s e n t d a t a f r o m PU t r e a t e d c e l l s .
given
in h r s . a f t e r
in itia l
,The sample t i m e
w a s h i ng o f t h e c e l l s .
is
Each p o i n t
i s t h e a v e r a g e o f 2 p l a t e s and r e p r e s e n t s t h e p oo l e d s u p e r n a t e s f r o m d u p l i c a t e s a mp les.
C ells
x
96
F ig u r e 2 3 .
E f f e c t o f Puromycin on V i r u s Release by E s t a b l i s h e d RSV(B)
C e lls
C o n tro l
P U -T re a te d C e l l s
Sample Time in Mrs.
97
i;
?!
I
E f f e c t o f P ur om yc In on' V i r u s R e ! ease by E s t a b l i shed RSV(SR)
CeT I s .
Open c i r c l e s
represent c o n tro l
d a t a , and t h e c l o s e d c i r c l e s
r e p r e s e n t d a t a f r o m PU t r e a t e d c e l l s .
in itia l
w a sh i ng o f
,The sample t i m e
given
in h rs . a f t e r
point
i s t h e a v e r a g e o f 2 p l a t e s and r e p r e s e n t s t h e p o o l e d
. stipe m a t e s f r o m dupl i c a t e samples.
t he c e l l s .
is
, Each
98
F ig u r e 2 4 .
E f f e c t o f Puromycin on V i r u s Release by E s t a b l i s h e d RSV(SR) C e l l s
Sample Time in Mrs.
washing.
.In itia l
o f t-fie c o n t r o l
viru s
r e l e a s e w i t h PU t r e a t e d c e l l s was s l o w e r t han t h a t
c e l l s , . w i t h maximum, r e l e a s e b e i n g a c h i e v e d by 6 h r s . a f t e r
w a s h i n g , ,6 t:o 8 '.hrs. b e f o r e t h a t o f t h e c o n t r o l
•control
ce lls .
ce l I s b o t h showed d ecr e as e d r a t e s o f v i r u s
maximum r e l e a s e p o i n t ;
PU t r e a t e d c e l l s
however,
t h an w i t h
Experimental
r e l e a s e f o l l o w i n g t he
t h e - r a t e o f d ecr ea se was g r e a t e r w i t h
the c o n t r o l s .
the
Note t h a t t h e maximum amount o f
v i r u s p r odu ce d b y 'PU t r e a t e d c u l t u r e s was 7.5 FFU p e r IO c e l l s ,
that of
and
t h e c o n t r o l s was 40 FFU p e r 10 c e l l s ,
whereas
5 t i m e s t h a t o f t h e PU
t r e a t e d pfates... •
F i g u r e - 2 ' 4 g i v e s t h e r e s u l t s o f t h e same t y p e o f e x p e r i m e n t w i t h RSV(Sji)
c e lls .
-c e lls .
I t w o u l d a p p e a r t h a t PU had no e f f e c t on v i r u s
Note t h a t a maximum v i r u s
r e l e a s e by t h e se
r e l e a s e o f 3 . 5 FFU p e r 10 c e l l s was
reached between 6 ’and 8 h r s . a f t e r w a s h i n g .
. P r o t e i n . S y n t h e s ! s b y ; RSV(B) and RSV(SR)
I n f e c t e d . Cel I s
Secondary c h i c k . f i b rob l a s t s were i n f e c t e d w i t h IO^ FFU o f RSV(B) o r
IO^ FFU o f RSV(SR)
in a t o t a l
volume o f
I ml.
A fte r I h r.,
each p l a t e ' w a s
washed once w i t h 2 ml o f warm 199 , and 1 . 8 ml o f w a r m '199 was added t o e a c h
p l a t e -(zero tim e ).
D u p l i c a t e p l a t e s were p r e p a r e d . f o r each sample - t i m e .
• C o n t r o l s c o n s i s t e d o f u n i n f e c t e d se co nd ar y c h i c k f i b r o b l a s t s p r e p a r e d and
treated
lik e
the v i r u s
infecte d c e l l s .
A t t h e t i me s d e s i g n a t e d
in F i g u r e s
26 and 2 7 , . I . Oy-C o f t r i t i a t e d - l e u c i n e was added t o t h e a p p r o p r i a t e
and c o n t r o l
p la te s.
- Four hrs..
infected
l a t e r t h e medium was removed and t h e number
o f c e l l s p e r sample was d e t e r m i n e d .
cribed p re v io u s ly f o r s c i n t i l l a t i o n
The c e l l s were t he n t r e a t e d as des­
counting.
. Samples we re c o u n t ed -on a
100
•P ac ka rd T r i - C a r b
a c tiv ity )
s c in tilla tio n
counter„
D L - I e u c i n e -(5400 mC/mM s p e c i f i e
l a b e l e d on t h e f o u r t h and f i f t h
-E n g la n d -N u c le a r .C o r p . , . Boston,
Mass.
. To ch ec k t h e c o u n t e r f o r p r o p o r t i o n a l
s e ria l
d ilu tio n s of tr it ia t e d
ml p l a c e d
carbons- :was o b t a i n e d f r o m New
counting o f t r i t i u m ,
t h y m i d i n e were p r e p a r e d
i n . 20 ml o f s c i n t i l l a t i o n
flu id
i n IN NaOH and 0.1
f o r counting.
t h a t t h e number o f c o u n t s p e r m i n u t e we re p r o p o r t i o n a l
la bel
present
i n t h e sa mp le.
2-fold
F i g u r e . 25 shows
t o t h e amount o f
The e f f i c i e n c y -of c o u n t i n g was between 2 .5
and 3 . 0 p e r c e n t .
The r e s u l t s o f an e x p e r i m e n t w i t h RSV(B)
in f e c t e d c e l l s are given
F i g u r e 2 6. ' Th er e w e r e -2 c y c l e s o f p r o t e i n s y n t h e s i s .
in
The f i r s t began a t
z e r o t i m e and was c o m p l e t e d by 8 h r s . , r e a c h i n g a maximum between 2 and 4
hrs. a f t e r
in fe c tio n .
tinued u n t il
d iffic u lt
The second began 8 h r s .
20 h r s . a f t e r
in fe c tio n .
t h e r e were 2 c y c l e s o f p r o t e i n
and t e r m i n a t e d a t 12 h r s . '
synthesis.
The i n c r e a s e d l e u c i n e
Comparison o f t h e s e 2 f i g u r e s
c e lls ,
r e q u i r e s an e a r l y
probably
enzymes.
The f i r s t
Again,
began a t z e r o t i m e
and c o n t i n u e d
i n F i g u r e 26
infected c e l l s .
shows t h a t b o t h RSV(B)
increase
is
synthesis.
I n c o r p o r a t i o n noted
in RSV(SR)
it
in co rpo ra tio n
infecte d c e l l s .
The second began a t 12 h r s .
between 20 and 24 h r s . was n o t f o u n d
synthesis
in le u c in e
r e p r e s e n t s a new c y c l e o f p r o t e i n
F i g u r e 27 shows t h e same-data f o r RSV(SR)
t h r o u g h 24 h r s .
i n f e c t i o n and co n­
On t h e b a s i s o f t h e s e d a t a ,
t o determine whether or not the r i s e
between 20 and 24 h r s .
a fte r
and RSV(SR)
i n p r o t e i n - s y n t h e s i s by t h e i n f e c t e d
in d ic a t iv e o f the form a tion of e a r ly v i r a l
Al:so, a second c y c l e ; - o f p r o t e i n
synthetic
s y n t h e s i s was r e q u i r e d ,
this
I
IO O a
b e in g
to
th e
p e rh a p s v i r a l
tim e
of
firs t
a n tig e n
or
co a t p ro te in ,
s ig n ific a n t v iru s
as
re le a s e
its
s y n th e s is
(F ig u re
1 1 ).
c o rre s p o n d e d
F i g u r e 2 5. . . S t a n d a r d T r i t i urn.C u r v e .
Each p o i n t
i s t h e a v e r a g e o f 3 one m i n u t e c o u n t s o f t h e s ample.
The b a c kg r o un d c o u n t a v e r a g e d 37 CPM a t t h e s t a r t and t h e
■ fin is h o f c o u n tin g the set o f
s a m p l es .
f o r b a c kg r o un d b e f o r e b e i n g p l o t t e d .
Each p o i n t was c o r r e c t e d
\
102
F ig u r e 2 5 .
Standard T r i t i u m Curve
103
:i!
4
■ F i g u r e 26. ' P r o t e i n - Synt he s I s by RSV(B)
I n f e c t e d Cel I s .
The X.'s c o n n e c t e d by a d o t t e d l i n e
poration
in to p ro te in s of control
c o n n e c t e d by a s o l i d
lin e
i n t o p r o t e i n s o f RSV(B)
r e p r e s e n t ami no a c i d
c e lls .
r e p r e s e n t amino a c i d
infected c e lls .
p o o l e d samples o f d u p l i c a t e p l a t e s .
f o r 3 one m i n u t e
graph.
The f i l l e d
in co r­
c irc le s
incorporation
Each p o i n t
represents
Each sample was c o un t ed
i n t e r v a l s and t h e a v er a ge p l o t t e d on t h e
The a v e r a g e b a c k g r o u n d c o un t was 39 CPM a t t h e s t a r t
and f i n i s h o f c o u n t i n g .
For p ur po se s o f s t a n d a r d i z a t i o n ,
the
a v e r a g e sample c o u n t s we re c o r r e c t e d t o t h e number o f c e l l s
p e r p l a t e assuming each c e l l
t h e amino a c i d .
i n c o r p o r a t e d equal
q uantities of
104
F ig u r e 2 6 .
H^-L eucine
Inc o rp ora tio n
i n t o RSV(B)
Infected C e lls
RSV(B)
X Control
Time Hj - L e u c i n e Added in H rs. A f t e r
In fe c tio n
Figure 27 .
P r o t e i n S y n t h e s i s by RSV(SR)
The X1s c o n n e c t e d by a d o t t e d
poration
I n f e c t e d Cei I s .
lin e
in to p ro te in o f control
c o n n e c t e d by a s o l i d
lin e
i n t o p r o t e i n o f RSV(SR)
r e p r e s e n t amino a c i d
c e lls .
The f i l l e d
r e p r e s e n t amino a c i d
in fecte d c e lls .
p o o l e d samples o f d u p l i c a t e p l a t e s .
in co r­
c irc le s
incorporation
Each p o i n t r e p r e s e n t s
, Each sample was c o u n t e d
f o r 3 one m i n u t e i n t e r v a l s and t h e a v er a ge p l o t t e d on t h e
graph.
The a v er a g e b a c k g r o u n d c o un t was 39 CPM a t t h e s t a r t
and f i n i s h
o f counting.
F o r p u r po s es o f s t a n d a r d c o m p ar i s o n ,
t h e a v e r a g e c o u n t s p e r sample were c o r r e c t e d t o t h e c e l l
p e r p l a t e assumi ng equal
ce l I .
amino a c i d
incorporation
count
i n t o each
106
F ig u r e 2 7 .
H^-L eucin e I n c o r p o r a t i o n
i n t o RSV(SR)
Infected C e lls
RSV(SR)
Contro l
Time Hj - L e u c i n e Added in H rs. A f t e r
In fe c tio n
107
^ E f f e c t o f A c t i n om yc i n D i on V i rus -Pr o d u c t i on
The e x p e r i m e n t s u s i n g - A c t i nomycin D(AD) were v e r y - p r e l i mi n a r y .
" e x p e r i m e n t s we re p e r f o r m e d - m a i n l y t o see i f
and Gol d ^ (1964)
and-.Temin >( 1963)
, The
t h e - r e s u l t s o b t a i n e d by V i g i e r
c o u l d be - r ep eat ed u s i n g RSV(SFt), . A
m a j o r d i f f e r e n c e between t h e t i s s u e c u l t u r e systems o f t h e s e a u t h o r s and
t h a t :us.ed. i n t h i s
l a b o r a t o r y was t h a t V i g i e r ,
Gol de and Tenii n used E a g l e ' s
medium.
Secondary c u l t u r e s o f c h i c k ' f i b r o b l a s t s were p r e p a r e d and i n f e c t e d as
in t h e PU e x p e r i m e n t s .
At v a r io u s times - a f t e r
the c u l t u r e s f o r the s t e t e d tim e
in te rv a ls .
The c e l l s were washed once
- w i t h -2 ml o f warm medium 199 and i n c u b a t e d
sampled a t 48 h r s , a f t e r
T a b l e V,
The E f f e c t o f IyAgZml o f A c t i n o m y c i n - D on 48 H r . V i r u s
P r o d u c t i o n by RSV(B) and RSV(SR) I n f e c t e d ; C e ! I s .
O - 5
5 -10
10 - 15
15 - 20
20 - -30
30 - 35
-Control
FFU/celI
■ RSV(B)
FFU/celI
RSV( SR)
'•% C o n t r o l
RSV(B)
I .IxIO "4
6.3x10-5
--(a)
0.02
0.01
0.02
1.70
0.69
9.00
100.00
‘1.Ix T O "4
9.2x'.10"j
3.7x10-3
5.0x10-2
5 .4 x 1 0 - 1
No RSV(SR) was d e t e c t e d
t o 30 h r s . a f t e r
-"i
-
9.3x10-5
I .55x107
3 .7 0 xl0 -4
2.10x10-3
I Ag/m l
i n f e c t i on i n h i b i t e d 48 h r .
i n d i c a t e s t h a t 48 h r .
was n o t a p p r e c i a b l y
-
%,Control
RSV(SR)
—
— —
4.38
7.30
•17.40
100.00
i n c e l l s t r e a t e d w i t h AD a t t h e s e t i m e s .
. From, Tabl e V i t may -be •seen t h a t
T a b l e Vl
i n 2 ml o f 199 a t 37 C u n t i l
in fe c tio n .
. I n t e r v a l o f ’ E xposure t o
AD i n H r s . . A f t e r I n f e c t i o n
(a.)
i n f e c t i o n r AD was added t o
o f AD added any t i m e f r o m 0
v iru s production.
v i r u s p r o d u c t i o n by RSV(SR)
i n h i b i t e d by e x p o s u r e t o 0 . 1 / k g / m l
However,
infecte d c e lls
o f AD.
. T h i s was
i n c o n t r a s t t o t h e - r es ul t s of. Temin »(1.96,3) and GoTde and V i g i e r " (1964) who
' o b t a i n e d 90 p e r c e n t o r g r e a t e r
irre v e rs ib le
in h ib i t i o n o f v iru s production
w i t h "0, 2 and 0 . 1X*-g/ml o f AD r e s p e c t i v e l y .
. T a b l e aVT.
The E f f e c t o f 0 . I zA g Z m V o f :;Act i n o m y c in. D on 48 H r . Vi rus
P r o d u c t ion by RSV(SR) ' I n f e c t e d C e l l s .
. I n t e r v a l o f Exposure
t o AD i n Mrs. a f t e r
I n f e c t ion
■ FFUZcel I
0 - 8
8 - 1.6
■i:6 - 24
24 - 32
'32 - 40
40 - 48
. - Control
1.86 x
• 1 . 7 6 -x
1.16 x
2.10 x
6.82 x
3.20 x
I . 96 x
These r e s u l t s
o r RSV(SR)
hr., v i r u s p r o d u c t i o n ,
on 48 h r .
.
95
90
59
'107
34
163
100
i n d i c a t e - t h a t t h e a d d i t i o n o f lytug/ml o f AD t o RSV(B)
infected c e lls f o r short
infected c e lls
10“ 2
TO"?
IO"2
IO"2
10"?
TO"2
IO"2
■
' % o f -Cont ro)
in te rv a ls a fte r
in fe c tio n
but t h a t the a d d i t i o n o f 0 . T^gZml
f o r 8 hr.
in te rv a ls
v iru s production.
sh ortly a fte r
i n h i b i t 48
o f AD t o RSV(SR)
i n f e c t i o n had no e f f e c t
- DI SCUSSION
I n j e c t ion o f
l a r g e doses o f e i t h e r RSV(B)
o r RSV(SR)
i n t o the-wing
web o f c h i c k e n s p r odu ce d e s s e n t i a l l y t h e same r e s u l t s w i t h t h e e x c e p t i o n
t h a t RSV(B)
i n f e c t e d b i r d s d e v e l o p e d t u m or s more r a p i d l y and d i e d sooner
t han. RSV(SR)
t h a t RSV(B)
infected b ir d s .
. T h i s c o u l d p er ha ps be e x p l a i n e d by t h e f a c t
i n f e c t e d c e l l s c o n s i s t e n t l y p r odu ce d more v i r u s t h a n RSV(SR)
infected c e lls
(Figures
c e l I u l a r p r o l i f e r a t i on,
Il
and 12) .
By ra p. id v i r u s
o n l y o n e - h u n d r e d t h as much v i r u s as RSV(B).
as p a r a d o x i c a l
by i n f e c t e d b i r d s .
as
RSV(B) wo u ld be e x p e c t e d t o overcome t h e h o s t ' s
d e f e n s e Mechanisms more r a p i d l y t h a n . RSV(SR) w h i c h
e s t i n g -as w e l l
r e l e a s e as w e l l
.In t h i s
respect
to co n s id e r the p ro d u c tio n o f
I t was n e v e r d i f f i c u l t
which n e u t r a l i z e d v i r u s a t d i l u t i o n s o f
e v e r , , a t no t i m e - d i d b i r d s
i nduced t h e r e l e a s e o f
it
is
in te r­
immune -serum
t o p r e p a r e serum a g a i n s t RSV(B)
1:100 o r g r e a t e r ( F i g u r e 9) •
How­
i n f e c t e d w i t h RSV(SR) y i e l d a n t i serum, w h ich
wo u ld n e u t r a l i z e v i r u s even a t d i l u t i o n s a s low as - I j l O .
p o r t e d t h a t he had d i f f i c u l t y
in
i n d u c i n g apt i- RS V( SR)
Vogt a l s o r e ­
antibodies
in
: c h i c kens ( V o g t , .1964):) .
'■ . D e s p i t e t h e - f a c t t h a t t h e m a j o r p a t h o j o g i c a l ■d l f f e r e n c e s
in the i n t a c t
b i r d were t h e t i m e o f t umor d ev el op men t and t h e l a c k o f humoral
p r o d u c t i o n . a g a i n s t RSV(SR),
d iffe re n tly
to the 2 v ir u s e s .
■p r odu ce d by v i r u s
foci
being
infecte d chick fib r o b la s t s
being sharp,
As e v i d e n c e d
responded q u i t e
in F i g u r e s 2 and 3,
i n f e c t e d c e l l s were - q u i t e - d i s t i n g u i s h a b l e ,
d i s t i n c t a r e a s on t h e p e t r i
r a t h e r vague,
d i f f u s e areas o f v i r u s
t h i s d i f f e r e n c e may be f o u n d
dish,
the f o c i
t h e RSV(B)
and t h e RSV(SR)
in fectio n .
i n F i g u r e s 4 and 5 .
antibody
foci
An e x p l a n a t i o n o f
Here i t may be seen t h a t
RSV(B)
foci
( F i g u r e 4) we re composed o f clumps o f rounded c e l l s w h i c h had
s t a c k e d on t op -of one a n o t h e r due t o t h e l o s s o f c o n t a c t
c r o m b i e and Heaysman,
so n e a r l y
1 95 4 )„
(Aber­
F i g u r e 5 shows t h a t RSV(SR)
foci
r esembled t h e m o n o l a y e r t h a t t h e y c o u l d n o t be e a s i l y d i s c e r n e d
m i c r o s c o p i c a l l y f r o m normal
contact
.In c o n t r a s t ,
in h ib itio n
in h ib itio n
p h o l o g y o f RSV(SR)
c e lls .
i n RSV(SR)
foci
I nde ed ,
t h e r e was v e r y
little
even a f t e r 9 days in c u l t u r e .
f o c i ■resembl ed t h e " m o r p h f "
loss o f
The mor­
f o c i o f Temin ( 1 960)
in
t h a t b o t h t y p e s o f f o c i we re composed o f c e l l s whose m o rp h o l o g y was o n l y
s l i g h t l y a l t e r e d and b o t h showed v e r y l i t t l e
ever,
app ear ed t o be l a r g e r t ha n " mor ph f "
Further d iffe re n c e s
stacking.
tact
ever,
foci,
how­
fo ci.
in t h e b e h a v i o r o f RSV(B) and RSV(SR)
c e l l s were f o u n d upon e x a m i n a t i o n o f e s t a b l i s h e d RSV(B)
RSV(SR)
RSV(SR)
infected
and RSV(SR) c e l l s .
i n f e c t e d c e l l s e v e n t u a l l y became rounded and showed l o s s o f co n­
in h ib itio n
RSV(B)
gia nt c e lls
(Figure 6 ).
Ver y f ew RSV(SR)
i n f e c t i o n always re s u lt e d
(Figure 7 ) .
Serial
g i a n t c e l l s were f o u n d .
i n t h e f o r m a t i o n o f l a r g e numbers o f
passage o f RSV(B)
c e l l s was o n l y p o s s i b l e
as I ogg as t h e m a j o r i t y o f c e l l s were n o t g i a n t c e l l s .
in c u l t u r e ,
How­
. A f t e r abo ut 60 days
g i a n t c e l l s became p r e d o m i n a n t and f u r t h e r t r a n s f e r o f RSV(B)
c e l l s became i m p o s s i b l e .
RSV(SR) c e l l s
remained t r a n s f e r r a b ' l e f o r a bo ut
90 days a f t e r w h i c h t i m e t h e y und erwe nt t h e normal d e g e n e r a t i v e changes
w h i c h accompany t h e l o s s o f n on - es t ab ' l i shed c e l l
D i f f e r e n c e s between RSV(SR) and RSV(B)
lin e s.
p a r t i c l e s we re -found upon e x ­
p o s u r e o f b o t h v i r u s e s t o 37 C f o r e x t e n d e d p e r i o d s o f t i m e .
a p p r o x i m a t e l y 3 t i m e s more s t a b l e t h a n RSV(SR)
a c t i v a t i o n c o n s t a n t f o r RSV(B)
(Figure 8 ).
RSV(B) was
The h ea t
in­
(Kg) was c a l c u l a t e d t o be 2 . 9 6 x IO"3 m i n - ' .
O t h e r w o r k e r s have o b t a i n e d v a l u e s f or . Kg '©f 2 . 9 x
l;9@l) and 4 . 5 x 10*3 min~^ i (Rub i n ,
1 9 5 5 ) . ' The d i f f e r e n c e s
were p r o b a b l y due t o ' t h e ^ u s e -e,f d i f f e r e n t
used were a l l
min- ^ i (Dougherty,
o f t h e Brya n h i g h t i t e r
in these values
su spe ndi ng m ed ia, as t he v i r u s e s
s tra in .
.The medium used i n t h i s
l a b o r a t o r y was medium 1 9 9 , whereas t h a t used by Rubin w a s . E a r l e ' s p l u s 1.0
p e r c e n t h o r se serum
'was 0.01
M phosphate
. No pub I i shed h ea t
and c h i c k embryo
buffered
extract,
and t h a t used by Do ugh er ty
s a l i n e pH 7 . 0 p l u s 2p e r c e n t h o r se serum.
i n a c t i v a t i o n c o n s t a n t s f o r RSV(SR) we re a v a i l a b l e f o r
c o m p a r i s o n . '.Per haps
t h e g r e a t e r h ea t s t a b i l i t y o f RSV(B)
production o f la rg e r
amounts o f v i r u s were
important f a c t o r s
RSV(B) more p a t h o g e n i c f o r c h i c k e n s t h a n RSV(SR)
- o f humoral
antibodies
tests
rendering
in s p i t e -of t h e p re se nc e
i n d i c a t e d t h a t RSV(B) a n d : RSV(SR) possess
d i f f e r e n t n e u tra l I z a b l e -antigens (F ig u re 9 ) .
l i m i t e d due t o t h e absence o f a n t i -RSV(SR)
The v a l u e - o f t h i s
t e s t was
sera,,as r o n reciprocal
cross
has been shown t o t a k e p l a c e w i t h a n t i s e r a a g a i n s t many o f
t h e members o f t h e a v i a n l e u k o s i s complex ( A n d r e w s ,
However,
in
w ith the
i n "RSV(B)' i n f e c t e d b i r d s .
. Serum n e u t r a l i z a t i o n
n e u tra liza tio n
along
1933;
B e a r d , . 1:957) •
RSV(SR) and^RSV(B)uace n o t c o m p l e t e l y a n t i g e n ! c a l l y u n r e l a t e d ,
t h e y s h ar e common s o l u b l e o r t umor ■a n t i g e n s -.(Huebner e t 'a_L,
Vogt ( 1964b) has f o u n d t h a t a n t i -RSV(RAV 2)
sera w i l l
cross
1964) .
as
Also,
r e a c t t o some
e x t e n t w i t h RSV(SR).
.A p r o p e r t y o f RSV(B) w h i c h must be c o n t i n u a l l y . k e p t
discussion of t h i s v iru s
is
its
in. min<d in any
d e f e c t i v e n e s s (Hanaf usa e t a l _ . , 196 3) .
a t t e m p t s t o ‘f i n d e v i d e n c e - f o r d e f e c t i v e n e s s
i n RSV(SR)
fa ile d .
Al I
No h e l p e r
v i r u s c o u i d be d e t e c t e d ( T a b l e s . I I and
isolated
(Table
b u t t h e RSV(B)
III).
RSV(SR)
III),
and no 1N P ' c l o n e s c o u l d be
c o u l d s e r v e as a H e l p e r v i r u s f o r RSV(B),
in duce d by RSV(SR) was s t i l l
d efe ctive,
m a t u r e p a r t i c l e s when c o n t a m i n a t e d by RSV(SR)
foci
p r odu ce d by RSV(SR)
(Table s
I I and
ill).
,The
i nduced RSV(B) we re p h e n o t y p i c a i I y RSV(B); t h e r e b y
s u p p o r t i n g R u b i n ' s c o n t e n t i o n ,(1964)
t h a t t h e p he no ty p e o f t h e c o n v e r t e d
c e l l s was d e t e r m i n e d by t h e RSV genome and n o t
viru s,
and o n l y p roduced
. R u bi n -(1964) ■a l s o f o u n d thaf:RSV(SR)
i n f l u e n c e d by t h e h e l p e r
c o u I d muI t i p l y
in t h e absence
o f a h e l p e r v i r u s and was t h e r e f o r e n o t d e f e c t i v e .
The t i t e r
o f RSV(B)
by a t
o f R A V i h t h e RSV(B)
l e a s t TO f o l d
stocks
i n t h i s l a b o r a t o r y exceeded t h a t
( M a te r ia ls and-Methods).
T h i s means t h a t f o r
e v e r y RSV(B) p a r t i c l e p r o du c ed by an i n f e c t e d m o n o l a y e r ,
we re p r o d u c e d .
. C o n s i d e r i n g t h a t RSV(B)
100 t i m e s as much RSV as d i d RSV(SR)
10 RAV p a r t i c l e s
i n f e c t e d c u l t u r e s pro du ce d 10 t o
i n f e c t e d c u l t u r e s ( F i g u r e s Tl and 12),
and assuming t h a t t h e r e w e re TO i n f e c t i o u s u n i t s o f RAV f o r each FFU o f
RSV(B), t h e r e wo u l d be TOO t o TOGO i n f e c t i o u s u n i t s o f v i r u s
RAV)
r e l e a s e d f r o m RSV(B)
■by RSV(SR)
infecte d c e lls
infected c e l l s .
•discussing d iffe re n c e s
w i t h ' RSV(SR)
in fe ctio n
f o r each FFU o f RSV(SR)
infected c e lls
to v ira l
( F i g u r e TO)
is p a r t i c u l a r l y
and Hanafusa (1965)
in te re stin g
in h ib ito rs
Rubin '(1964)
in l i g h t o f
t h a t RAV i n t e r f e r e n c e was
most e f f e c t i v e a g a i n s t s t r a i n s - o f RSV w h i c h were a n t i g e n i c a l l y
i n t e r f e r i n g RAV.
in
i n f e c t i o n o f c h i c k f i b r o b l a s t s w i t h RAV i n t e r f e r e d
t h e r e p o r t s of. Rub i n , (1964)
the
plus
released
,These numbers may p r o v e t o be i m p o r t a n t
in the re sponse-of
,The f a c t t h a t p r i o r
(RSV(B)
reported th a t p r i o r
s im ila r to
in fe c tio n of c e lls
w i t h RAV I ,enhanced t h e i r
e v i d e n c e and t h e f a c t
s u s c e p tib ility
that p r io r
t e r f e r e n c e a g a i n s t RSV(RAV I )
that
t o RSV(SR).
On t h e b a s i s o f t h i s
i n f e c t i o n o f c e l l s w i t h RAV I
b u t n o t a g a i n s t R!>V(RAV 2 ) ,
i n t e r f e r e n c e was due t o t h e p r o d u c t i o n o f v i r a l
induced
in­
Rubin suggested
c o a t p r o t e i n wh ich
was a b l e t o p r e v e n t su pe r i n f e c t ion by a v i r u s w i t h s i m i l a r c o a t p r o t e i n .
Only v i r u s e s s h a r i n g n e u t r a l i z a b l e - a n t i g e n s were c o n s i d e r e d s u f f i c l e n t I y
'r e la te d -for
RSV(SR)
in te rfe re n c e t o
used i n t h i s
(F ig u re 9 ) , the
o ccur (Rubin , .1964).
. S i n c e t h e RSV(B) and
l a b o r a t o r y d id n ot share n e u t r a l i z a b l e a n tig e n s
i n t e r f e r e n c e w i t h b o t h RSV(SR) a n d .RSV(B)
i n f e c t i o n by
?RAV was c o n t r a r y t o R u b i n ' s h y p o t h e s i s , , s u g g e s t i n g ^ t h a t a n t i g e n i c s i m i ­
l a r i t y was n o t t h e o n l y p r e r e q u i s i t e f o r
p o s s i b l e t o a r g u e - t h a t t h e RAV i s o l a t e d
t r a l izable antigens
a p o s s ib ility
in te rfe re n ce .
in t h i s
Of c o u r s e , i t
.
l a b o r a t o r y possessed neu­
i n common w i th- b o t h RSV(B) and -RSV(SR),
is extrem ely
is
However,
such
remote., s i n c e t h e RAV used h er e was i s o l a t e d
f r o m RSV(B) c u l t u r e s w h i c h we re f o u n d t o be d e f e c t i v e ( T a b l e s
I I -and I I I)
and w h i c h were used as a n t i g e n s t o s t i m u l a t e t h e a n t i s e r u m used f o r t h e
n e u tra liza tio n te s ts
o f t h e RSV(B)
(Figure 9 ).
By v i r t u e - o f b e i n g d e f e c t i v e , , t h e c o a t
had t o be p r odu ce d und er t h e d i r e c t i o n o f t h e R A V. p, r e s e n t . I n
the c u l t u r e , a n d
s i n c e t h e a n t i s e r u m n e u t r a l i z e d - RSV(B)
and n o t RSV(SR),
. RSV1
( SR) , and RAV c o u l d n o t shar e any n e u t r a l i z a b l e a n t i g e n s .
. a l t h o u g h p r o t e i n c o a t -p r o d u c t i o n may be n e c e s s a r y f o r
-place,
.Therefore,
in te rfe re n c e to take
t h e c o a t b e i n g p r odu ce d need n o t be a n t i g e n i c a l l y l i k e
superin fe c tin g
viru s.
t h a t o f the
' Normal
the
g r o w t h - c u r v e s o f RSV(B)
and -RSV(SR) ' . ( F i g u r e 11)
i rid i c a t e d t h a t
i n t r a c e l l u l a r e v e n t s l e a d i n g t o t h e p r o d u c t i o n o f b o t h v i r u s e s were
probably s im i la r .
. However,
RSV(B)
p r o d u c t i o n a l wa y s exceeded t h a t o f
RSV(SR) and remained a t maximum l e v e l s f o r
•11,! . T a b l e -I V) ,
. Bot h v i r u se s we re • r e l e a s e d f r o m e s t a b l i shed Rous c e l l s a t
a p p r o x i m a t e l y t h e same r a t e ,
b u t once a g a i n RSV(B) p r o d u c t i o n exceeded
RSV(SR) p r o d u c t i o n ( F i g u r e 1 2 ) .
lim itin g
lo n g e r p e r io d s o f tim e (F ig u r e
.Rubin (ig 6 4 )
t h e r a t e and t h e amount o f RSV(SR)
o f p ro te in coats.
s ugges ted t h a t t h e f a c t o r
p r o d u c t i o n was t h e a v a i l a b i l i t y
T h i s s u g g e s t i o n was based upon h i s f i n d i n g
t h a t the in -
I
f e c t i o n o f RAV I c e l l s by RSV(SR)
g r e a t e r q u a n t i t i e s o f RSV(SR).
th is
laboratory,
resulted
in more r a p i d - p r o d u c t i o n o f
These r e s u l t s were n o t s u b s t a n t i a t e d
.As s t a t e d p r e v i o u s l y , .RAV i n f e c t e d c e l l s
' f o e i upon i n f e c t i o n - w i t h RSV(SR).
.Also,
s ig n ific a n t
T h i s f i n d i n g was espe­
in l i g h t o f an e x p e r i m e n t by -Hanafusa e_t aj_.
w h i c h showed t h a t t h e r a t e and t i m e o f RSV(B)
t h a t o f RAV,
d id not y i e l d
as may be seen in F i g u r e 11,
',RSV(SR) was p r odu ce d a t t h e same r a t e as RSV(B),
cially
to
the ra te
;
o f RSV(SR) p r o d u c t i o n t h a t were n o t a t t h e samb t i m e o p e r a t i n g
infected c e lls .
( 1964b)
p r o d u c t i o n was i d e n t i c a l
T h e r e f o r e , t h e r e appear ed t o be no s t e p s l i m i t i n g
i
in
T h i s w o u l d su gg e st t h a t
was l i m i t e d by t h e amount o f p r o t e i n ,
b l e ' f o r use in v i r u s s y n t h e s i s ,
in t h e m u l t i p l i c i t y
of
and t h a t t h e r a t e o f p r o d u c t i o n o f v i r a l
in f i n a l
in fe c tio n
t h e q u a n t i t y o f RSV i n f e c t e d c e l l s
n u c l e i c a c i d a n d / o r ribosomes a v a l Ta­
• p r e c u r s o r s was t h e same f o r b o t h RSV(B)
. That the d i f f e r e n c e
i n RSV(B)
viru s
a n d -RSV(SR).
t i t e r was n o t due t o d i f f e r e n c e s
i s shown in F i g u r e 12.
.,Even though t h e
number o f e s t a b l i s h e d RSV(B)
d u c t ion b y ; RSV(B)
and RSV(SR)
cel Is s t i l l
c e l l s was t h e - same, . v i r u s p r o ­
exceeded t h a t o f t h e RSV(SR)
c e l I s by> a
f a c t o r o f : 10 o r more.
The 3 f o l d d i f f e r e n c e in h ea t s t a b ! I i t y
v i r u s e s d id not appear
s u f f i c i e n t to account f o r
o f t h e -2
10 t o 100 f o l d d i f f e r e n c e
in v i r u s productio n.,
In a t t e m p t i n g t o , s t u d y t h e r o l e t h a t p r o t e i n
re p lic a tio n ,v iru s
untreated c e l l s .
growth
Fo r t h e s e ' s t u d i e s ,
and c e l l
i n RSV
in PU t r e a t e d c e l l s was compared w i t h t h a t
c e l I s were exposed t o j y k g / m l
, a s t h i s c o n c e n t r a t i o n p r odu ce d t h e g r e a t e s t
( F i g u r e 15)
synthesis plays
d iv is io n
reduction
( F i g u r e s 13 and 14) w i t h
in
o f PU,
in v i r u s p ro d u c tio n
the l e a s t c e l l
• damage,
The e f f e c t o f PU on 24 h r . . v i r u s p r o d u c t i o n was e s s e n t i a l l y t h e same
f o r b o t h ' v i r u s e s ; ( F i g u r e 16 ) .
n o t e d between 0 and 12
d u c t i o n ^appeared t o
hrs.
. Marked s u p p r e s s i o n o f v i r u s p r o d u c t i o n was
a fte r in fe c tio n ,
increase w it h
b u t a f t e r 12 h r s . , v i r u s p r o ­
t h e t i m e o f a d d i t i o n o f PU.
T h i s sug­
g e s t e d t h a t t h e r e -might be 2 PU s e n s i t i v e e v e n t s ; one o c c u r r i n g b e f o r d 12
h r s , . a f t e r i n f e c t i o n and t h e o t h e r o c c u r r i n g a f t e r 12 h r s .
, Experiments
s u b j e c t i n g the cel Is t o 4 h r. puls es o f H ^ -Ie u c in e a t v a r io u s times a f t e r
i n f e c t i o n c o n f i r m e d t h a t t h e r e were 2 c y c l e s o f p r o t e i n s y n t h e s i s
■i n f e c t e d c el I s
( F i g u r e s .26 and 2 7 ) ,
in newl y
. These eye I e s were app r o x i m a t eI y t h e
■same f o r b o t h v i r u s e s ,
e x c e p t t h a t t h e y were c omp let ed 4 h r s . . I a t p r
RSV(SR)
than
infected c e lls
t h e 'PU e x p e r i m e n t s ( F i g u r e
i n . RSV(B)
16),
in fecte d c e lls .
in
From t h e r e s u l t s o f
i t a p p ea r s t h a t b o t h c y c l e s o f p r o t e i n
s y n t h e s i s we re n e c e s s a r y ' f o r v i r u s p r o d u c t i o n ,
and t h a t t h e amount o f v i r u s
1 16
p r od uc e d between 12 and; 2 4 -fersu. a f t e r
■amount o f p r o t e i n
i nf e c i t i on was p r o p o r t i o n a l
s y n t h e s i z e d d u r i n g ' t h e - second c y c l e
t o t he
o f p r o te in synthesis
. T h e - f a c t t h a t PU i n h i b i t s v i r u s p r o d u c t i o n m a r k e d l y between 0 and 12 h r s .
a fte r
i n f e c t i o n would lead t o the s uggestion t h a t the p r o t e i n s synthesized
between 0 and 12 h r s . were t h o s e n e c e s s a r y ' f o r e a r l y e v e n t s such as un­
coating,
fact
n u c le ic acid
r e p l i c a t i o n , . a n d p r o t e i n c o a t c o d i n g ; whereas t he
t h a t v i r u s p r o d u c t i o n between 12 and 24 h r s .
allowed f o r p r o t e in , s y n t h e s is
in creased,
i n c r e a s e d as t h e t i m e
s u g g e s t s t h a t t h e ‘p r o t e i n s p r o ­
duced-.dor i ng t h e second c y c l e o f s y n t h e s i s w e r e the. c o a t p r o t e i n s o f t h e
-v iru s.
. Gp ld e and V i g i e r
(1963)
p e r f o r m e d e x p e r i m e n t s s i m i l a r t o t h e ones
m e nt i o ne d I iere e x c e p t t h a t t h e y used 5 - f I uor ou rac i I t o a f t e r RNA s y n t h e s i s .
; They o b t a i n e d . a
lin e a r
in cre ase
in v i r u s p r o d u c t io n w i t h t h e tim e o f a d d i ­
t i o n o f 5 - f I u o r o u r a c M f r o m z e r o t i m e t o 10 h r s . a t w h i c h t i m e v i r u s p r o ­
d u c t i o n by 5 - f I u o r o u r a c i I
co n tro ls.
24 h r .
t r e a t e d c e l l s was equal
T h i s showed t h a t t h e - 5 - f I u o r o u r a c i I
t o t h a t o f the u n tre a te d
s e n s itiv e event c o n t r o lli n g
v i r u s p r o d u c t i o n was c o m p l e t e by 10 h r s . a f t e r
s u l t s would i n d i c a t e - t h a t th e
genome i n t h e f i r s t
10 h r s .
i nf act I
da
.
i n f o r m a t i o n decoded f r o m t h e v i r a l
a fte r
i n f e c t i o n was s u f f i c i e n t
c y c l e s o f p r o t e i n s y n t h e s i s o b s e rv e d
i n F i g u r e s 26 and 27.
. Thei.r r e ­
o r host
t o conduct both
T h e i r data are
i n good agr ee me nt w i t h t h e - e s t i m a t i o n t h a t e a r l y p r o t e i - n s y n t h e s i s o c c u r s
between 0 and 12 h r s . a f t e r
is not necessary f o r
cycle o f p ro te in
10 h r s .
synthesis,
in fe c tio n .
f o r 24 h r .
If
th is
is t r u e ,
v iru s production,
and'. RNA s y n t h e s i s
t he n t h e second
shown i n F i g u r e s 26 and .27, must, o c c u r under
117.
t h e d i r e c t i o n o f e i t h e r a s t a b l e messenger o r -newly s y n t h e s i z e d v i r a l
■genomes.
.The-effect of
i n h i b i t o r s on 24 h r .
v i r u s p r o d u c t i o n has been d i s c o v e r e d
t o be d i f f e r e n t f r o m t h e i r e f f e c t on 48 h r .
V i g i e r , . 19 63 ; ■ Bader,
Therefore,
1 9 6 4 ; . V i g i e r and Go l de ,
t h e e f f e c t o f PU on 48 h r .
o f PU was added t o t h e
f e e t ion,
taken.
and remained
RSV(SR)
a d d itio n u n til
1964; T e m i n , . 1963 and 1 9 6 4 a ) .
v i r u s p r o d u c t i o n was s t u d i e d .
in c o n t a c t w i t h the c e l l s u n t i l
16 h r s .
v iru s
increased l i n e a r l y w i t h
samples were
RSV(SR) p r o d u c t i o n by PU t r e a t e d c e l l s exceeded t h a t
c e l l s by 5 t o 7 t i m e s .
. A l t h o u g h t h e r e was v a r i a b i l i t y
c e l l s by a t
. The - same t y p e o f e x p e r i m e n t p er f o r m e d u s i n g RSV(B)
gave m a r k e d l y d i f f e r e n t
re su lts.
t h e t i m e - o f PU a d d i t i o n u n t i l
in
t h e maximum v i r u s p r o d u c t i o n by
infected
Th er e appeared t o be a s l i g h t
in v i r u s p r o d u c t i o n between 0 and 8 h r s . , f o l l o w e d by a l i n e a r
w ith
in-
t h e " t i m e o f PU
PU t r e a t e d c e l l s was a l w a y s g r e a t e r t h a n t h a t o f t h e c o n t r o l
c e lls
.Again,
a t w h i c h time-maximum v i r u s p r o d u c t i o n was main­
th e r e s u l t s from experim ent t o expe rim en t,
le a s t 4 tim es.
( Go ld e and
in f e c t e d c e l l s a t v a rio u s tim es a f t e r
v iru s production
t a i n e d ! ( F i g u r e -I 7 ) .
o f the c o n t r o l
v iru s production
20 h r s . , ( F i g u r e -I 7 ) .
lag
increase
Here a g a i n ,
the
r e s u l t s f r o m e x p e r i m e n t t o e x p e r i m e n t were v a r i a b l e b u t t h e r e was g e n e r a l l y
s lig h tly
l e s s v i r u s p r o d u c t i o n b y 'PU t r e a t e d c e l l s t ha n by c o n t r o l
. The I n c r e a s e
i n 48 h r .
.be due t o 2 t h i n g s .
the c o n t r o l
hrs. a f t e r
RSV(SR)
p r o d u c t i o n by PU t r e a t e d c e l l s c o u l d
. E i t h e r - t h e "PU s t i m u l a t e d a r a p i d
c e l l s d ecr e as e d
in fe c tio n ,
ra p idly
c e lls .
release o f v ir u s ,
or
i n v i r u s p r o d u c t i o n between 24 and 48
and PU p r e v e n t e d t h i s drop f r o m o c c u r r i n g
in t h e
experimental
c e lls .
- T a b l e 1IV i n d i c a t e s
t h a t t h e r e was i n d e e d . a
large-de­
c r e a s e i n v i r u s p r o d u c t i o n by; RSV(S-R) ' i n f e c t e d cel I s bet ween 24 and 48 h r s .
, wh i ch ' Wa s n o t f o u n d
dica te d th a t
in RSV(B)
infected c u ltu re s .
t h e r e was a - d e c r e a s e i n RSV(SR) p r o d u c t i o n ;
o u t t h e p o s s i b i l i t y o f a sudden i n c r e a s e
treated c e lls .
, Experiments
of v iru s
showed t h a t t h e i n c r e a s e d
in d ica tin g
i n an i n c r e a s e
whereas c o n t r o l
c r e a s e d , , f o l I owed by d e c r e a s e d ,
case . w i t h RSV(B)
,Also,
in c o n t r o l
. . T he r e f o r e ,
a
fe e d in g the con­
v ir u s production
t o t h o s e o f PU t r e a t e d cel I s-,.(F i g u res :I 8 and -19)
t h a t PU t r e a t m e n t s t a b i l i z e d
I e v e I s ( F i gu r e - 19 ) ,
rule
le ve ls of v iru s
( F i g u r e - 18) .
r e l e a s e d i d n e t seem l i k e l y .
c e l l s a t 48 h r s . . r e s u l t e d
: t o , l e v e l s n e a r l y equal
they d id not
i n RSV(SR) p r o d u c t i o n by PU
• p r o d u c t i o n we re m a i n t a i n e d . f o r a t l e a s t -96 : h r s .
sudden f l u r r y
in­
i n wh I c h 1RSV(SR)' p r o d u c t i o n b y /PU t r e a t e d c e l l s
was measured -aver a 4 day p e r i o d
tro l
T h e s e - r e s u lt s merely
RSV(SR) p r o d u c t i o n a t n ea r maximum.
c e lls
underwent a 48 h r . ,
v i r u s p r o d u c t ion.-
cycle of
in­
T h at such was n ot t h e
i n f e c t e d c e l l s may be seen in F i g u r e s 20 and 21.
. RSV(B)
p r o d u c t i o n a ppear ed t o . b e - c o n s i d e r a b l y more s t a b l e t h a n RSV(SR) p r o d u c t i o n
, ( F i g u r e s 19 and 2 1 ; ^Ta b le
IV).
. H o w e v e r , . PU d i d m a i n t a i n .96 h r .
d u c t i o n a t maximum l e v e l s w h i l e c o n t r o l
in te re stin g
RSV(B) p r o ­
v i r u s p ro d u c t io n decreased.
t h a t t h e r e a ppear ed t o be - some i n h i b i t i o n o f 48 h r .
It
is
RSV(B)
p r o d u c t i o n by ,-PU.
. T h e - s t a b i l i z a t i o n o f RSV(SR)
p r o d u c t i o n by PU c o u l d be - e x p l a i n e d by a
mechanism i n v o j v i n g c o m p e t i t i o n between t h e
The c e l l
RSV(SFi)
f o r amino a c i d s o r
ribosomes.
p r o d u c t i o n was o n l y 0 . 0 5 FFU
per
i n f e c t i n g v i r u s p a r t i c l e and
C o n s i d e r i n g t h e f a c t t h a t maximum
c e l l : (average va lu e o f Table
IV),
119
t h e p e -may •have -been ' r e l a t i v e l y
few s i t e s ,devoted . t o v i r a l
•eompared t o - t h o s e - d e v o t e d t o - c e l l u l a r p r o t e i n ^ s y n t h e s i s .
p ro te in
synthesis
. Keepi ng i n mind
t h a t min imum i n h i b ' it o. ry ' co n c e n t r a t i ons o f PU were used, . t h e "p.robabi I i t y
t h a t any p o l y s o m e - a f f e c t e d b y 'PU was i n v o l v e d
must have been g r e a t e r t h a n ' t h e - p r o b a b i l i t y
" p ro te in ,
. Im o th e r words, . i f
■the i n f e c t i n g v i rus and t h e
^amount o f v i r u s
that
i t :was s y n t h e s i z i n g i RSV(SR)
t h e r e ’was a n e q u i l i b r i u m e s t a b I i shed between
i n f e c t e d ce l I s , , i t m i g h t be r e f l e c t e d
in the
r e l e a s e d by t h e - c e l l , and c o u l d be a l t e r e d by e n v i r o n m e n t a l
'■f a c t o r s as shown in F i g u r e s ! 2 and 19 .
i n ' f a v o r o f th e cel I ,
w ith in
i n .ice I l u l a r p r o t e i m s y n t h e s i s
changes c a u s i n g s h o r t a g e s
t h e - e e l I . .would r e s u l t s
d i v i s i o n was h a l t e d ,
Tf t h i s e q u i l i b r i u m we re w e i g h t e d
in n u t r i t i o n a l 'fa c to rs -
in le ss v i r u s p r o d u c t io n .
However,
if
ce ll
and t h e r e f o r e t h e - s y n t h e s i s o f c e l l u l a r components
re ta r de d -, ■ i t m i g h t be p o s s i b l e t o m a i n t a i n t h e e q u i l i b r i u m e s t a b l i s h e d
between t h e v i r u s and t h e
infected c e ll
at
the-tim e o f
re tarda tio n.
..PU at.
. l o w c o n c e n t r a t i o n s c o u l d s e r v e as t h e a r r e s t i n g a ge n t and s t o p >cell u l a r
and v i r a l
s y n t h e t i c p r o c e s s e s a t o r n e a r t h e e q u i l i b r i u m ; l e v e l s e s t a b i i shed
by t h e t i m e o f PU a d d i t i o n . ,
.PU c o u l d . then m a i n t a i n t h e e.qui I i b r i u m b y p r e ­
v e n t i n g t h e - e s t a b l i s h m e n t . o f new s i t e s o f v i r a l
sis.
o r c e l l u l a r p-rotein 'synthe­
, T h i s .would a c c o u n t f o r t h e m a i n t e n a n c e o f n ea r maximal
d u c t i o n b y t h e - a d d i t i o n o f PU a t 16 t o .24 h r s .
sign i f ic a n t a t t h i s p o in t th a t
a fte r
stable o r s l i g h t l y
RSV(SR) p r o -
in fe c tio n .
• -RSV(B)
behavior
- the-above-hypothesis..
is
increasing v ir u s produc­
t i o n was f o u n d o v e r a .96 h r . p e r i o d when PU was added to.'RSV(SR)
c e l I s 12 h r s . . o r more a f t e r
It
infected
i n f e c t i o n " ' ( F i g u r e -19) .
i n t h e p r e s e n c e - o f PU m a y - a l s o be a c c o u n t e d - f o r by
.However, d i f f e r e n c e s
in ' t h e . b e h a v i o r o f RSV(B) ; and
120
RSV(SR) 'we,re ' f o u n d - w h i c h m i g h t be e x p l a i n e d b y c o n s i d e r i n g t h a t RSV(B)
T n f e c t e d -cel I s '-produced 40 t o 400 t i m e s morb - v i r u s t han RSV(SR) ' c e T l s
.-(Tpb'l e - TV) . "T h er e -we re -ev i d e n t I y more s i t e s o f v i rus . synt he s i s i n RSV(B)
i n f e c t e d c e l I s •t h a n i n RSV(SR)
some v l r a ! ' i n h i b i t i o n
maximal
le ve ls.
24 h r s , a f t e r
in fe c te d cells-;
therefore.,
one m i g h t e x p e c t
i f 'PU was added as RSV(B) p r o d u c t i o n ,was r e a c h i n g
' This,
in f a c t ,
in fe c tio n
i s .what was .found I f PU was added a t 20 o r
( F i g u r e '2 1 ).
. However, RSV(B) p r o d u c t i o n was .able
t o -i n c r e a s e - t o maximum e qu i I i b r i urn I eve-1 s i n t h e p r e s e n c e -of PU by 72 h r s *
and m a i n t a i n t h i s
level
indicate
t h r o u g h 96 - h r s . . ( F i g u r e s 20 and, 21) .
o f ■PU was s u f f i c i e n t
Tb: s .would
t o s t a b i l i z e environmental
c o n-
d itio n s a t.a
l e ve l '. wh er e -ma x imu m v i r u s p r o d u c t i o n c o u l d b e - e s t a b l i s h e d and
malntaIned;
G o l de (1962)
occurs w i t h i n 4 hrs. b f t e r
z a tio n process.
in fe c tio n
has shown t h a t r a p i d
r e l e a s e of- f r e e - n u c l e o t i d e s
i n f e c t i o n w i t h RSV(B),
i n d i c a t i n g ;a dep ol yme r-i -
A r i s e -in v i r u s p r o d u c t i o n b e t w e e n -24 and 48 h r s . a f t e r
in c e l l s
t r e a t e d . w i t h PU a t 4 t o 16 h r s . . ( F i g u r e 21)
i n d i c a t i v e -of t h e e x p a n s i o n o f v i r a l
could.be
s y n t h e t i c p r o c e s s e s t o n o n - f u n c t i o n ing
r i b o s o m e s , . e s t a b l i s h i n g maximum v i r u s p r o d u c t i o n a t w h a t e v e r l e v e l s were
possible
If
i n t h e p r e s e n c e o f PU.
t h e m a i n t e n a n c e o f maximal ; l e v e l s of. RSV(SR) p r o d u c t i o n -were d u e - t p
a f o r t u i t o u s . s e l e c t i o n o f a c o n c e n t r a t i o n o f PU w h i c h a l l o w e d e s t a b ' l ishment
and m a i n t e n a n c e - o f a p h y s i o l o g i c a l
p ro te in
s y n t h e s i s , ,t hen h i g h e r c o n c e n t r a t i o n s o f PU m i g h t be -e xp ec t ed t o
i n h i b i t c e l l u l a r and v i r a l
p rotein
nance e q u i l i b r i u m was u p s e t .
?
e q u i l i b r i u m between c e l l u l a r and v i r a l ,
s y n t h e s i s t o a p o i n t w he re t h e m a i n t e ­
, T h i s was t h e - c a s e when 2y t g / m l
o r more o f PU
12(1
w e r e ' a d d e d t o c e l l s a t .16 h r s . . a f t e r
hr.
in fe c tio n
(Figure 22).
.F o rty-e ig ht
v i rus p r o d u c t i o n :was i n h i b i t e d by $5 t o -99 p e r c e n t .
.Because f U a d d i t i o n d u r i n g
s t a b i l i z e RSV(SR)
the f i r s t
24 . h r s ., o f
i n f e c t i o n was a b l e t o
and t o some e x t e n d RSV(B) p r o d u c t i o n ,
e x p e r i m e n t s were
d e s i g n e d t o d e t e r m i n e t h e e f f e c t o f PU o n - e - s t a b I i shed RSV(B) a n d " RSV(SR)
cel I s .
.PU had no e f f e c t on v i r u s p r o d u c t i on by RSV(SR) ■ce l I s ^ F i g u r e -24) ..
■However,
RSV(B)
it
had q u i t e a n o t i c e a b l e e f f e c t on v i r u s p r o d u c t i o n by e s t a b l i s h e d
c e l l s . ( F i g u r e -23) .
was a d d e d , , b u t
was o n e - f i f t h
Not o n l y was RSV(B)
the-maximum amount o f v i r u s p roduced
that' o f
t h e c o n t r o l s.
Also,
in 't he p r e s e n c e o f 'PU.
(n t h e b e h a v i o r o f e s t a b l i s h e d RSV(B) and RSV(SR)
p r e s e n c e o f PU c o u l d be e x p l a i n e d I n -2 ways.
s e n s itiv e event
is not
in t h e p r e s e n c e - o f PU
t h e r a t e o f v-i rus p r o d u c t i o n
d ec re as e d more r a p i d l y and a t an e a r l i e r t i m e
-d.if f e r e n c e
r e l e a s e d more s l o w l y when .RU
F irs t,
r e q u i r e d f o r RSV(B) p r o d u c t i o n
r e q u i r e d f o r RSV(SR) p r o d u c t i o n .
Or,
c e lls
in the
t h e r e may be a PU
in e s t a b l i s h e d c e l l s
second,
.The
that
t h e p r o d u c t i o n o f TOO
t i m e s more i n f e c t i o u s u n i t s o f v i r u s p e r c e l I by RSV(B)
ce lls
t ha n by RSV(SR)
c e i l s . ( F i g u r e 12) means t h a t m o r e - p r o t e i n was used p e r u n i t o f t i m e f o r
RSV(B)
s y n t h e s i s t h a n was used . f o r RSV(SR)
synthesis.
c e l l s c o u l d c o n t i n u e t o - p r o d u c e v i r u s a t normal
Therefore,
RSV(SR)
l e v e l s ' ) : g n g e r under c o n d i ­
t i o n s o f I i mi t e d p-rote i n ^ s y n t h e s i s t ha n c o u l d RSV(B) c e l l s .
T h i s would
n e c e s s i t a t e a p r e m a t u r e decay in v i r u s p r o d u c t i o n by e s t a b l i shed RSV(B)
c e l l s due t o d e p l e t i o n o f
occur
in. RSV(SR)
r e s e r v e p r o t e i n w h i c h m i g h t n o t be e x p e ct e d t o
c e lls .
The r e s u l t s o f
in h ib itio n of v iru s
s y n t h e s i s by t h e -presence o f IyAgZml
I
" o f AD - i n t h e c u l t u r e s f o r 5 h r .
i n t e r v a l s d u r in g the f i r s t
24 h r s . a f t e r
122
i n f e c t i o n we re v e r y t e n t a t i v e .
ment w i t h Temin ( i g 6 3 )
and. V i g i e r and Goi de ( 1 9 6 4 ) ,
i r r e v e r s i b l e -AD s e n s i t i v e ,
t h e - f i r s t 35 hrs-. a f t e r
no J r r e v e r s i b l e
48 h r s .
a fte r
in h ib itio n
s t ea d - of RSV(B)
o f RSV(SR)
were not
. Whet her t h i s
in t h i s
,T he-results
in good a g r e e ­
tha:-t t h e r e -was an
t h e r e f o r e DNA d e p en de nt ,
in fe c tio n .
in fe c tio n ,
above a u t h o r s .
However,' T a b l e 'V i n d i c a t e s - ,
e v e n t o c c u r r i n g - w i t h in
i n Table V I, which
indicate
by O. I y t g / m l ■o f AD1d u r i n g t h e ' f i r s t
i n agr eement w i t h t h e r e s u l t s o f
the
d i f f e r e n c e was d u e - t o t h e use o f RSV(SR)
la b o ra to ry,
o r t h e use o f d i f f e r e n t
in­
tissu e c u ltu re
media i s n o t kn ow n . . However, t h e e f f e c t o f even low c o n c e n t r a t i o n s o f -AD
on b o t h
i n f e c t e d and normal
■r e v e r s i b l e .
c h i c k ^ f i b r o b l a s t s was s e v e r e and n o t a l wa ys
I t wo u l d a p p e a r t h a t AD, even i n small
t o x i c and . t h a t
i t wo u l d be - d i f f i c u l t
c i f i c a l l y / DNA d ep endent e v e n t s ,
r e s u l t o f the general
to t e l l
q ua n tities,
i s - e x t re m el y
w h e t h e r AD was a f f e c t i n g
o r whether v i r a l
spe­
i n h i b i t i o n was m e r e l y t h e
breakdown o f c e l l u l a r p r o c e s s e s .
The r e s u l t s o f t h i s
in ve stiga tio n
" d i f f e r e n c e s b e t w e e n , RSV(B)
t i v e n e s s , w h i c h was f o u n d
in d ic a te t h a t - t h e r e are d e f i n i t e
a n d -RSV(SR) .
. However,
i n RSV(B), b u t n o t
in isome w a y . f p r most o f t h e s e d i f f e r e n c e s ,
s ta b ility ,
a n t i g e n i c i t y and p a t h o g e n i c i t y .
considered
in d is c u s s in g the d i f f e r e n c e s
the p r o p e r ty o f defec­
i n RSV(SR), may be r e s p o n s i b l e
in clu ding d iffe re n c e s
.It
in hea t
i s a - p r o p e r t y ' w h i c h . m u s t be
in the t o t a l
amount o f v i r u s p r o ­
duced by i n f e c t e d c e l l s and t h e d i f f e r e n c e -in t h e response o f v i r u s p r o ­
d u c t i o n t o RU and AD.
The d i f f e r e n c e
independent o f d e f e c t iv e n e s s .
trol
o f t h e RSV genome -and n o t
i n f o c u s mo r p h o lo g y ,
. T h i s p r o p e r t y was d e f i n i t e l y
however, was
und er t h e c o n ­
i n f l u e n c e d by t h e p r e s e n c e o f a h e l p e r v i r u s .
123
Al t h ou gh t h e r e -were d i f f e r e n c e s between t h e '2 v i r u s e s ,
t h e r e were a l sep
p r o p e r t i e s w h i c h t h e y s har ed and w h i c h t h e r e f o r e . , must e i t h e r be shared
w i t h " RAV o r be -in de pe nd e n t o f
d u c t i o n by n ew l y
it.
.These i n c l u d e d t h e - r a t e o f v i r u s p r o ­
i n f e c t e d and - e s t a b l i s h e d c e l l s ;
t h e r e s p o n s e - o f 2.4 h r .
v i r u s p r o d u c t i o n t o P U ; :a nd t h e d e p e n d e n c e - o f v i r a l
s e n s itiv e event.
These s i m i l a r i t i e s
haps a l l
o f RSV r e p l i c a t e
t h ou gh i t
stra in s,
r e p l i c a t i o n on a n , AD
I n d ic a t e t h a t both s t r a i n s , a n d p e r­
in t h e same manner.
Therefore,
even
i s n o t p o s s i b l e a t t h e p r e s e n t t i m e t o / s e l e c t t h e model o f r e ­
p l i c a t i o n which
i s used b y , RSV,
in t h e c y c l e o f v i r u s
it
is f a i r l y
re p lic a tio n w ill
c e r t a i n . t h a t the e a rly -e v e n ts
be t h e same in a l l
reg ardle ss o f whether o r not they c a r r y s u f f i c i e n t
t h e i r own c o a t p r o t e i n .
s t r a i n s o f RSV
in fo rm a tio n f o r producing
SUMMARY
'The - M o I b g i-ca I p r o p e r t i e s o f 2 . s t r a i n s o f 'Rous =Sercoma v i r u s . ,
Bryan h i g h = t i t e r s t r a i n , . RSV ( B:) , and -t he" Sehmi dt-Rupp I n s t r a i n . ,
w.e-.re compared wi t h
RSV(SR) ,
r e s p e c t t o _ij2 v i t r o and _i_n v i v o p a t h o g e n i c i t y , f o c u s
.morpihol o,gy, a n t i g e n i c p r o p e r t i e s . ,
f at es-,
the
v i r u s . p r o d u c t ion, . p r o t e in
heat s t a b i l i t y ,
synthesis a f t e r
defectiveness,
in fe c tio n ,
g ro wt h
reaction to
p u r o m y c i n (PU) and r e a c t i o n t o A et in o my c i n D ( A D ) .
■.RSV(B) was f o u n d t o
t h a n .RSV(SR).
Also,
in du ce -tumors and d e a t h more r a p i d ) y i n c h i c k e n s
RSV(B)
c u l t u r e as l o n g as RSV(SR)
g ia nt c e ils
i n RSV(B)
i n f e c t e d c e l l s were n ot m a i n t a i n e d
c e i l s due t o d eve lo pmen t o f
in t i s s u e
l a r g e numbers o f
cu ltu re s.
The -focus m o r p ho l og y o f
t h e 2 v i r u se s were m a r k e d l y d i f f e r e n t .
RSV(B)
■ f o c i , w e r e d i s t i n c t c l u s t e r s o f c e l l s on t h e . m o n o l a y e r showing marked l o s s
o f contact
In h ib itio n .
RSV(SR)
fo ci,
in c o n t r a s t , w e r e - d i f f u s e c l u s t e r s
'
o f cel. I s show i ng l i t t l e . - i f any -loss o f c o n t a c t
.RSV(B)
in fe c tio n
t ha n d i d R S V ( S R )
in h ib itio n .
s.t i m u i a t e d - a n t i body p r o d u c t i o n
in fe c tio n .
.The R S V ( B )
i n . c h i c k e n s much b e t t e r
p a r t i c l e s we re f o u n d t o c a r r y a
n e u t r a l i z a b l e - a n t i gen t h a t was n ot p r e s e n t on t h e R S V ( S R )
viru se s a ls o d if fe r e d
in t h e i r heat s t a b i l i t y . .
RSV(B)
p a rtic le s .
The
was 3 t i m e s more
- s t a b le t o t i s s u e c u l t u r e c o n d i t i o n s than R S V ( S R ) .
- . D e f e c t i v e n e s s was f o u n d t o ,be a p r o p e r t y o f RSV(B) , b u t n o t o f RSV(SR) .
.In t h i s
respect
i t was i n t e r e s t i n g
RSV(B) , p r o d u c t i o n .
e q u a l l y by p r i o r
(RAV) .
t h a t RSV(SR) c o u l d s e r v e as a . h e l p e r f o r
. Focus f o r m a t i o n by RSV(B)
andvRSV(SR) was i n h i b i t e d
i n f e c t i o n o f c h i c k f i b r o b l a s t s w i t h Rous a s s o c i a t e d v,i rus
.125
■:The ' r a t e - o f v-i rus p r o d u c t i o n
i n . bo th 'newl y i n f e c t e d .and . e s t a b l i shed
■ RSV(B) and RSV(SR) c e l I s was i d e n t i c a l ,
. However,
t h e - a m o u n t o f RSV p r e ­
duced by RSV(B)' i n f e c t e d -cel I s was a l w a y s -10 t o 100 t i m e s g r e a t e r t han t h a t
p r o du c ed b-y 'RSV(SR)
!con d itio ns,
c e lls .
b o t h RSV(B)
I f . g r o w t h c u r v e s were compared u n d e r i d e n t i c a l
and RSV(SR) e x h i b i t ed e q u a l ‘ l a t e n t p e r i o d s . , and
reached maximum v i r u s p r o d u c t i o n a t n e a r l y t h e same t i m e .
t i o n was a l w a y s more - s t a b l e t h a n . RSV(SR)' p r o d u c t i o n .
re p lica tio n ,
W,i t h i n
o f PU on .24 h r .
e ig h t hr.
With respect to v i r u s
b o t h s t r a i n s a ppear ed t o ’ r e q u i r e 2 c y c l e s o f p r o t e i n isynth.es!§
the f i r s t
e v e r , 48 h r .
. RSV(B)' p r o d u c ­
24 h r s . a f t e r
in fe ctio n .
v i r u s p r o d u c t i o n was t h e
The - e f f e c t o f
low c o n c e n t r a t i o n s
same f o r RSV( B)' and RSV(SR) .
v i r u s p r o d u c t io n r e a c te d d L f f e f enply'.'to t h e
in h ib itor.
I t was n o t u n t i l
7 2 . and -96 h r s . . a f t e r
.
at
. F or t y - !
RSV(SR) p r o d u c t i o n was s t a b i l i z e d a t maximum l e v e l s o f v,i rus p r o ­
d u c t i o n .byvRU; ;wheTeas, 48 h r . . RSV(B): p r o d u c t i o n was si i g h t l y
'PU.
How­
control
RSV(B)
p r o d u c t i o n began t o - d e c r e a s e -between
i n f e c t i o n t h a t RSV(B)
h igh c o n c e n t r a t i o n s .(I ^ A g / m l )
RSV(SR) p r o d u c t i o n
in h ib ite d hy
sta b iliz a tio n
was found t o
became-evident.
inhibit
i f added d u r i n g t h e - f i r s t 35 h r s . a f t e r
RSV(B} and
infection.
. E a r l y a d d i t i o n s o f a l e s s e r q u a n t i t y o f AD* ( O eJzA g Z m l 1)' had no e f f e c t on 48
■hr.
v i r u s p r o d u c t i o n by RSV(SR).
.Mechanisms, . u t i l i z i n g
lo g ica l
d e f e c t i v e n e s s and t h e a s s u m p t i o n t h a t a ••physio­
e q u i l i b r i u m e x i s t s b et we en v i r u s p r o d u c t i o n and c e l l u l a r r e p l i c a t i o n ,
we re proposed t o e x p l a i n d i f f e r e n c e s
i n t h e b e h a v i o r o f . RSV(B)
and, RSV(SR) .
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x
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Buck, C. A.
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