J. gen.

ViroL (t978), 40, 6z3-633 623

Printed in Great Britain

O K 10 Virus, an Avian Retrovirus Resembling the Acute

Leukaemia Viruses
By N I L S O K E R - B L O M , L A R S H O R T L I N G , A N N I K K I K A L L I O ,
E E V A - L I I S A N U R M I A H O AND H A K O N W E S T E R M A R C K
Department of Virology, University of Helsinki and
College of Veterinary Medicine, Helsinki, Finland

(Accepted 27 April ~978)

SUMMARY
The OK Io virus complex was isolated from a liver tumour of a chicken which,
as an embryo, had been inoculated intravenously with a field isolate of an avian
leukosis virus. The OK IO virus complex contains at least two viruses: the inter-
ference assay and serum neutralization test indicate that the helper virus belongs
to subgroup A. One of the viruses, OK IO V, induces distinct foci in chick embryo
cells under agar overlay and cells from the foci form colonies in soft agar. These
properties allow in vitro assay of the virus. Injection of virus or infected cells into
chicks induces acute leukaemia but no local tumours. Another virus, OK IO AV
(associated virus), comprises about 99 % of the OK IO complex. This virus does not
induce foci in chick embryo cells. In chickens it causes leukosis 17 months after
injection. Electron micrographs of OK io virus stocks show typical C type virus
particles. These particles have a density of 1-16 g/ml and contain 7oS RNA which,
after heat denaturation, releases type b RNA subunits. The OK Io virus complex
apparently represents a strain of acute leukaemia viruses.

INTRODUCTION
It has recently been suggested that avian retroviruses can be divided into three main
groups (Hanafusa I977): (i) the sarcoma viruses Rous sarcoma virus (RSV), avian sarcoma
virus B77 and the Fujinami viruses, (ii) the lymphoid leukosis viruses, the different resistance-
inducing factor viruses (RIF) and Rous associated viruses (RAV) and (iii) the acute leukae-
mia viruses of which avian myeloblastosis virus (AMY), avian erythroblastosis virus (AEV)
and avian myelocytomatosis virus MC 29 are well studied. This corresponds well to earlier
classifications by Temin (i97I) and Coffin (1976).
The avian sarcoma viruses cause distinct foci in chick embryo cells in vitro. The leukosis
viruses have generally been considered non-transforming because none of the established
laboratory strains of the different subgroups of helper viruses cause transformation of
cultured chick embryo cells (see review by Pont6n, I97r). Many of these viruses do, on the
other hand, cause a slight cytopathic effect in vitro (Dougherty & Rasmussen, 1964; Graf,
I972; Kawai & Hanafusa, I972; Oker-Blom et al. I975). A simple plaque test based on this
property has been developed for members of subgroups B and D (Graf, 1972; Kawai &
Hanafusa, 1972). After prolonged cultivation of cells infected with many of these viruses,
morphological changes in the cultures (' conversion') occur (Calnek, I964; Oker-Blom et al.
624 N. O K E R - B L O M AND OTHERS

I975). These changes are different from transformation since converted cells do not exhibit
loss of contact inhibition, do not form colonies in soft agar and do not induce solid tumours
at the site of inoculation in chickens (Oker-Blom et al. I975).
Of the viruses classified as acute leukaemia viruses, AMV, originally isolated in I94r,
(Hall et aL I94I) induces transformation of haematopoietic cells in culture (Beaudreau et al.
I96o; Baluda & Goetz I96I; Moscovici & Vogt I968; for review see Moscovici T975).
Avian erythroblastosis virus (AEV; Engelbreth-Holm & Rothe-Meyer, i932 ) has recently
been shown to transform both chicken erythroblasts and fibroblasts in vitro (Graf et al.
I976). The myelocytomatosis virus (MC 29), isolated in I964 (Ivanov et al. I964) transforms
bone marrow cultures (Todorov & Yakimov I967; Langlois et aL I969) as well as chick
embryo cells (Langlois et al. I967). All these viruses contain a transforming virus and an
excess of non-transforming helper virus. In the case of AMV, the myeloblastosis associated
viruses MAV-~ and MAV-2, belong to subgroups A and B respectively (Moscovici & Vogt,
1968). The AEV helper virus from the two strains studied belongs to subgroup B (Graf et al.
I976). Stocks of MC 29 virus have been found to contain helper viruses of subgroups A
and B (Graf, I972 ).
Another virus which has recently attracted attention is the MH 2 virus (Begg, I927). The
virus belongs to subgroup C and induces endotheliomas in chickens and foci in chick
embryo cells in vitro (Payne & Biggs I97o).
We have isolated (Oker-Blom et al. I97o) a group A avian retrovirus OK Io which induces
rapid transformation and focus formation of chick embryo cells in vitro as well as rapid
tumour formation in new-born chicks. The lesions induced in chicks resemble those of the
MH 2 virus. Here we describe the isolation and preliminary characterization of OK Io virus.

METHODS

Chickens. Brown Leghorn chickens (C/O or C/E cell type) were used. This flock orig-
inated from eggs brought to this Institute in 1969 from Houghton Poultry Research Station,
Huntingdon, England. As evident from continuous RIF (resistance inducing factor),
COFAL (complement fixation avian leukosis) tests and, more recently, RIA (radio-
immunoassay) and antibody assays, it has remained leukosis-free (Sandelin & Estola, I975).
Cell cultures. Primary chick embryo cells (CEC) were prepared from Jo- to I I-day-old
embryos. The growth medium (Medium I99) was supplemented with Io°/o tryptose phos-
phate broth, 5 % foetal calf serum, penicillin (2oo International units/ml) and streptomycin
(2oo #g/ml). The cells were grown at 37 °C in a humified atmosphere (95 % air and 5 %
CO2) and passaged after 3 to 6 days.
Viruses. RAV-I, RAV-2 and RSV (RAV-I) were obtained from Dr H. Hanafusa, New
York, N.Y., U.S.A. RSV (RAV-2), RSV (RAV-7) and RSV (RAV-5o) were obtained from
Dr P. Vogt, University of Southern California, Los Angeles, Calif., U.S.A. OK Io virus
was isolated from an embryo of a leukotic hen (Oker-Blom et aL I97o). The passage history
of the virus is described below.
Interference assays. Interference assays were performed according to Rubin 0960).
RSV (RAV-I), RSV (RAV-2), RSV (RAV-7), RSV (RAV-5o) of subgroups A, B, C and D
respectively, OK Io and OK Io V were used as challenging viruses. OK Io V was obtained
from a continuous cell line derived from a tumour-bearing chicken which seems to produce
only OK IO V (Oker-Blom et al. unpublished data). The standard interfering viruses were
RAV-I, RAV-2, RAV-49 and RAV-5o of subgroups A, B, C and D respectively.
Colony formation in soft agar. The method used was that described by Macpherson (I969).
 New transforming avian leukosis virus OK 10 62 5
Primary chicken embryo fibroblasts (Io 6) were exposed to 2"5 x io 4 focus forming units
(f.f.u.) of O K Io virus for 2"5 h and colonies counted after incubation for seven days.
Neutralization tests. The tests were performed as described previously (Oker-Blom et al.
I975). Control serum was obtained from a leukosis-free Brown Leghorn hen. Anti RAV-r
serum (F42), anti RAV-2 serum, anti RAV-49 serum and anti RAV-5o serum were obtained
from D r P. M. Biggs, Houghton Poultry Research Station, Huntingdon, England. Anti
O K IO and anti O K Io AV sera were obtained from chickens infected intravenously at
2 to 4 days of age with I ml virus with titres of Io 4 f.f.u./ml (OK Io V) and Io 6 interfering
units/ml ( O K Io AV). The chickens were bled at r month of age.
Test for Marek's disease virus A-antigen. The test was performed by Dr Bodil Norrild,
Institute for Medical Microbiology, University of Copenhagen (Norrild & Vestergaard,
I978 ). N o Marek virus A-antigen could be detected.
Labelling and purification of virus and RNA. O K Io virus infected CEC (thirty Io cm
culture dishes) were labelled with 2o mCi s2p-orthophosphate (Institute of Atomenergi,
Kjeller, Norway) for 2o h. The supernatants were clarified at 1oooo g for zo rain at 4 °C.
The virus in the supernatant was centrifuged in an SW 27 rotor at 24000 rev/min for 6o min
at 4 °C. The pelleted virus was resuspended overnight in T N (.o'o5 M-tris, p H 7"4, o ' I M-
NaC1) at o °C and 2 ml samples were layered on discontinuous sucrose gradients consisting
of: (from the bottom) 0"5 ml 65 % (w/w) sucrose, 2"5 ml + 2"5 ml 30 to 60 % (w/w) sucrose
and 4"5 m1+4"5 ml Io to 3o% (w/w) sucrose in TN.
Purified virus from the discontinuous sucrose gradient band was diluted with T N to a
final sucrose concentration of I o % and pelleted in a SW 27 rotor at 24ooo rev/min for
60 rain at 4 °C. The pellet was taken up in I ml of TSE containing ~ % SDS.
The R N A released with SDS was run in an SW 27 rotor on a 15 to 30% (w/w) sucrose
gradient in TSE-buffer at 240o0 rev/min for 6 h at 2o °C. The fractions containing R N A
were pooled and the R N A precipitated with ethanol at - 2 0 °C overnight. The precipitate
was pelleted and resuspended in Ioo/zl of 1% SDS. This R N A preparation was used for
gel electrophoresis. A part of the R N A probe was heat denatured for ~ min at xoo °C, and
was then cooled in ice. Cylindrical 2 % polyacrylamide-o.5 % agarose gels supplemented
with o.1% SDS were prepared according to Peacock & Dingman 0968). The gels were run
for 3 h at 2oo V 0 o V/cm), sliced into 2 m m pieces and the radioactivity solubilized with
NCS, tissue solubilizer (Amersham/Searle). The radioactivity was determined in a Packard
liquid scintillation counter.
Pathogenicity of OK Io V and OK IO A V in chickens. A group of nine new-born chickens
were infected intravenously with different dilutions of R K Io virus. Three chicks received
6 × Io 3, three 6 x io ~ and three 6 × io 1 f.f.u. In another experiment, Io 6 interfering units of
O K I o AV virus were injected intraperitoneally into 7 new-born chickens. The chickens were
bled at regular intervals, and they were studied for viraemia and neutralizing serum
antibodies.

RESULTS
Origin of OK Io virus
During a field study in I969 a leukosis virus was isolated from the embryonated egg of
a leukotic hen with lymphoid leukosis. Cell cultures from this embryo were passaged in
Roux bottles several times and also studied by electron microscopy. The cells contained
C type particles in abundance. In order to save the virus for later experiments the cells from
one bottle were frozen. About one year later cells were thawed, homogenized and the
supernatant used for infection of leukosis-free chick embryo cells. In order to study the
626 N. O K E R - B L O M AND OTHERS

effect of the virus in vivo some I2-day-embryonated eggs of Brown Leghorn chickens of
strain C / O or C / E were injected intravenously with the supernatant fluid from infected
cells. The embryos were allowed to hatch and kept in isolation. One of the chickens became
ill and was sacrificed as moribund at the age of I7 months. Autopsy revealed lymphoid
leukosis and a tumour in the liver. Histologically the liver tumour was a hepatic cell adenoma
or carcinoma. Between these cells lymphocytes and myeloid ceils were dispersed. A suspen-
sion was made from the liver tumour and inoculated on to monolayers of clhick embryo
cells from the same line of chickens. The virus obtained from such monolayers caused
distinct foci in chick embryo cells under agar overlay and is called O K Io.

Clone purified stocks of OK Io virus

For clone purification of O K IO virus, 3 ml of the stock liver suspension was inoculated
on to monolayers of secondary cultures of CEC of the C / O or C / E line containing about
6 million cells and the cells were passaged. F r o m the sixteenth cell passage the supernatant
was used to infect new cells which were overlaid with agar. Ten foci were picked, suspended
in 5 ml of growth medium and used to infect new CEC. After five clonings, using one or
two foci each time, stock virus was prepared.

Transformation and focus formation by OK Io virus

In early experiments, stock virus induced regions of morphologically altered cells in
monolayers of CEC after only one or two passages (Fig. I b). This contrasts with the much
later conversion of cultures induced by the non-transforming leukosis viruses (Oker-Blom
et al. I975). Under agar overlay the virus induced distinct foci (Fig. I c and d) which were
quite different from foci induced by several RSV strains tested in our laboratory (Fig. I e).
The borders of the O K Io foci were more irregular than those induced by RSV: the cell
looked less distinct but they never seemed to disappear as did the cells of the RSV (RAV-I)
foci in which a ' h o l e ' was often formed. With experience it was easy to distinguish the two
types of foci (Fig. I f ) .

Evidence for both transforming and non-transforming virus in stocks of

clone purified OK IO virus
In interference assays performed to define the subgroup of O K Io virus it was evident
that the virus stock contained both transforming and non-transforming virus. Interference
against challenge virus, RSV(RAV-I) was obtained about two logs beyond the end point
of the transforming titre of O K IO virus (Table I). We refer to these viruses henceforth as
O K Io V (transforming virus) and O K IO AV (associated virus, which is non-transforming);
O K lO virus refers to the complex.

Attempts to separate the transforming virus and the associated

virus of the OK IO virus complex
In order to separate O K IO V from O K Io AV the following experiments were per-
formed: the O K I o A V was isolated by end point titrations. Chick embryo cells were
infected with different dilutions of O K Io. Cells from the last dilution showing 5o % inter-
ference and no foci were passaged several times (Table I). Virus was collected and stored
at - 7 o °C to serve as O K Io AV stock. This virus did not cause any foci on CEC in several
experiments. However, after several passages it induced the 'conversion' of CEC, as de-
scribed earlier (Oker-Blom et al. I975). Intravenous inoculation of O K IO AV induced
leukotic lesions of transformed lymphoblasts in the Bursa of Fabricius, liver, bone marrow,
 New transforming avian leukosis virus OK 10 627

Fig. I. Morphological alterations in CEC infected with OK io virus and RSV(RAV-1) virus.
(a) Uninfected CEC monolayers. Magnification x 4o. (b) Morphological changes induced by un-
diluted OK io virus on CEC. Magnification x 8o. (c) Focus formation by OK IO virus under agar
overlay. Magnification ×4o. (d) OK lo virus focus at a greater magnification (x 140). (e)
RSV(RAV-I) induced focus, showing focus with empty centre. Magnification × 80. (f) OK lO
virus foci and RSV(RAV-I) foci on the same plate in an interference assay. Magnification x 2o.

h e a r t a n d muscle in two o u t o f 7 chicks after 17 a n d 19 m o n t h s ( H o r t l i n g , i978). The o t h e r

chickens are still h e a l t h y n e a r l y z years later.
A t t e m p t s to p u r i f y the t r a n s f o r m i n g c o m p o n e n t O K IO V f r o m its associated virus were
m a d e by infecting C E C w i t h stock virus u n d e r a g a r overlay a n d by isolating single loci. I n
one such experiment, 18 o u t o f 20 loci p r o d u c e d b o t h O K IO A V a n d O K i o V. T w o foci
628 N. O K E R - B L O M A N D O T H E R S

T a b l e I. Capacity of OK IO virus to interfere with R S V of subgroups A, B, C and D

Capacity of virus from passage 4 of OK IO infected CEC
Dilution of to interfere with subgroups*:
OK lo used to Number of , ~
infect CEC OK IO foci A B C D
1o-3 233 i.c.t 1"19 i.c.t 0'92
lO-4 18 i.c.t 1"27 1"I8 0'92
10-5 -- O3~ 1'15' 0"97* 1"13,
lO-e -- 0"03 0"99 0"92 0"90
lO.7 -- 0'07 i-o6 i.oo 0'90
lO-a -- 0.92 1.oi i.o2 1-23
io -9 -- l.lO 1-o5 0-96 i.o3
io -1° -- i-o4 1.2o o'93 I'I 3

* The following dilutions were used: RSV(RAV-I) 10-15, RSV(RAV-2) 10-3, RSV(1L~.V-7) 10-3 and
RSV(RAV-5o) 10-3.
"~ The plates were almost confluent with a mixture of O K IO and RSV loci, which made it impossible to
count (i.e.) challenging virus foci.
Interference is expressed as the relative sensitivity to challenge virus infection of the OK IO cultures
compared to control cultures.

T a b l e 2. Capacity of R S V of subgroups A, B, C and D to interfere with OK IO virus

Challenging virus*
r A

RSV RSV RSV RSV

Interfering virust (RAV-I)~ (RAV-2);~ (RAV-7)* (RAV-5o):~ OK IO§ OK lO V§H
RAV-1 + - - - + +
RAV-2 - + - -
RAV-49 -- - + -
RAV-5o - - - +
OK lO A V + - - - + +
* + = Interference; - = n o interference.
t S e c o n d p a s s a g e of virus infected c h i c k e m b r y o fibroblasts.
:~ V i r u s e s diluted to lO -3.
§ V i r u s e s diluted to 1 o - L
[1 This virus was isolated from a continuous tumour cell line, which apparently does not contain
OK IO AV (N. Oker-Blom et al. unpublished data).

p r o d u c e d n e i t h e r virus. A t t e m p t s to find t r a n s f o r m i n g virus b y s u p e r i n f e c t i o n o f t h e s e

cultures with the different RAVs were unsuccessful.
T h u s it h a s n o t b e e n p o s s i b l e so f a r t o i s o l a t e in vitro t h e t r a n s f o r m i n g c o m p o n e n t o f
O K IO w i t h o u t t h e a s s o c i a t e d virus O K IO A V .

Identification of the subgroup of OK IO virus

Interference assay
W h e n the i n t e r f e r e n c e w i t h t r a n s f o r m a t i o n in f i b r o b l a s t s p r e v i o u s l y i n f e c t e d w i t h v a r i o u s
s u b g r o u p s o f a v i a n l e u k o s i s viruses was d e t e r m i n e d , O K IO p r o v e d to b e s u b g r o u p A
(Table 2).
Neutralization test
T h e n e u t r a l i z a t i o n test s h o w e d t h a t b o t h O K i o V a n d O K I o A V w e r e o f s u b g r o u p A.
T h e r e was n o n e u t r a l i z a t i o n o f O K IO virus b y a n y a n t i s e r u m o f s u b g r o u p s B, C o r D
( T a b l e 3).
 New transforming avian leukosis virus O K 10 629

Table 3. Neutralization test for typing of OK Io*

Serum RSV RSV RSV RSV
Serum dilution (RAV-I)t (RAV-2) t (RAV-7)'~ (RAV-5o)~ OK'~ OK IoVt~
Anti-RAV-l I : IOO o'356 o'973 I'O I'lO o'3o5 o'487
serum
Anti-RAV-2 I : Ioo I'O o't74 po I'o 0"909 I'O
serum
Anti-RAV-49 I : Ioo 1'o 0"856 0"579 0'876 I'o 0"986
serum
Anti-RA¥-5o I "20 I'O O'712 l'O 0"068 t'O 0"939
serum
A n t i - O K 1o s e r u m J :20 o 0"957 I'O I.O o o
A n t i - O K IO AV I : ioo o'oH o'769 I'O 0'924 o o

* The neutralization is calculated as the fraction of virus surviving after neutralization.

t The virus was diluted to to -a5.
~: This virus was isolated f r o m a continuous t u m o u r cell line, which does not contain O K Io AV in
detectable a m o u n t s (N. Oker-Blom e t al. unpublished data).

Biochemical properties of OK I o virus

The budding particles seen in cells in the electron microscope, and the negatively stained
particles, are indistinguishable from other C type particles. Purified virus has a density of
I . I 6 g / m l in sucrose gradients. N o difference was seen between high and low titre virus,
i.e. O K IO and O K Io AV alone. The virus contained 7oS R N A and 35S R N A subunits
(Fig. 2), and as far as could be judged from gel electrophoresis of heated RNAs, the 35S
R N A subunits of O K Io virus belong to class b (Fig. 2). N o differences were detected in the
electrophoretic mobilities of the R N A s of the O K IO virus and O K Io AV or between their
R N A s and the R N A s of RAV-I or RAV-a. It has to be remembered, however, that the
transforming virus represents only about 1% of the total virus in the preparations used in
these experiments.
Growth of OK Io virus infected cells in soft agar
Monolayers of CEC were infected with different dilutions of O K IO virus and, after one or
two passages, the cells were seeded in soft agar. After seven days incubation, three types of
colonies were observed. Large colonies consisting of dispersed cells, medium sized fairly
compact colonies and very small colonies. RSV ( R A V - 0 infected cells give both the large
disperse type and the compact medium size colonies, B H K cells also give rise to two types
of colonies, and uninfected CEC may yield small compact colonies containing up to ten or
twenty cells. The very small colonies therefore should be regarded with caution. This may
also hold true for the two or three types of colonies formed by different transformed cells,
although the possibility that different types of colonies represent different cells or cells
infected by different viruses has to be kept in mind. The colony forming efficiency was
6.2 × 102 colonies in I million cells.

Pathogenity of OK Io virus for chicken

In order to study the effect of the different components of O K IO virus in chicken, 2 to
4-day-old chicks were injected intravenously with O K Io or O K Io AV virus. O f the nine
chicks injected intravenously with 6 x io 3, 6 x to 2 and 6 × to I f.f.u, of O K Io, 8 came down
with an acute leukotic-like disease I to 2 months after injection. The only negative was one
of three chickens receiving the smallest dose of virus. The lesions were very similar to those
described for the M H 2 tumour (Campbell I969; the histological examination was kindly
40 VXR 4O
630 N. G K E R - B L O M AND OTHERS

(a)

100 30

5O 15

? ?
>( X

6 E

(b)
2 o 8

1 ~ 4

10 20 30 40
Fraction number
Fig. 2. (a) Isolation of 7oS R N A from purified OK IO virus. The virus was treated with I ml of
2 % SDS in TSE-buffer supplemented with 5 ml of o'o5 M-EDTA, layered on a 15 to 3o % (w/w)
sucrose gradient, and was centrifuged for 6 h at 2z °C, and 24ooo rev/min, using a Beckman SW
rotor. Semliki Forest virus 42S R N A was used as a marker. 0----0, ~P-labelled OK ~o virus: © - - ©,
3H-uridine labelled Semliki Forest virus RNA. (b) Gel electrophoresis of s~P-labelled OK lo virus
RNA, using 2 % polyacrylamide-o-5 ~ agarose gels containing o ' 1 % SDS. The R N A was heated
for I min at zoo °C prior to electrophoresis. The gels were run for 3 h at 2oo V (so V/cm), sliced into
2 mm pieces and treated with o'7 ml NCS. The radioactivity was determined in a Packard liquid
scintillation counter. © - - © , 3H-uridine labelled PR-C RNA; O--Q, 32P-labelled OK iovirus RNA.

performed by Dr L. N. Payne, Houghton Poultry Research Station, Huntingdon, England).

OK IO AV has so far, after nearly two years of observation, resulted in a disease in only
two out of seven chickens. These chickens were sacrificed when moribund and showed
leukotic lesions similar to those induced by RAV-I.

DISCUSSION
Many of the properties of the OK Io virus described in this paper seem to differ from
those of other transforming avian leukosis viruses, such as avian myelomatosis virus
(AMV), avian erythroblastosis virus (AEV) and the myelocytomatosis virus MC 29. It has,
however, some properties in common with the virus of the MH z endothelioma isolated by
Begg in 1927 and later described by Payne & Biggs (197o) and should apparently be placed
in the group of acute avian leukaemia viruses. These viruses have lately attracted con-
 N e w transforming avian leukosis virus O K 10 63 I
siderable interest as being probable representatives of the avian carcinoma viruses (Duesberg
et al. 1977).
Two possible origins of the virus have to be considered. The more plausible is that the
virus represents a field isolate like MC 29 and M H 2. The other possibility is that the
transforming agent is an endogenous virus obtained from chickens after stimulation with
a non-transforming leukosis virus.
O K IO virus belongs to subgroup A. Thus, O K IO also differs from the known acute
leukaemia viruses like MC 29, which seems to contain helper viruses of both subgroup A
and B, and M H 2, which seems to belong to subgroup C.
The question of the helper dependence of the transforming component of O K IO virus
is open, as is that of virus O K IO AV which is non-transforming in vitro. This latter virus
induced a leukotic disease in two out of seven chickens of the C / O line after 17 and 19
months of incubation. Thus, O K IO AV resembles the common non-transforming leukosis
viruses. Attempts to separate O K IO V from the complex in vitro have so far been unsuccess-
ful, as have attempts to enhance the titre of O K IO V by using a helper of any subgroups
available.
The pathology of the virus infection in chickens is described in detail in another paper
(Hortling, I978), but it has many features in common with the M H 2 virus. It differs, how-
ever, in that it does not cause lesions on the CAM of the fertile eggs of the C / O line and it
does not cause local turnouts in chicks of the same line if inoculated intramuscularly a n d / o r
intra and subcutaneously. When tumour cells induced by the virus in vitro are injected
intramuscularly into chicks no local tumours arise. Chicks infected with purified virus either
intraperitoneally, intravenously or intramuscularly do, however, succumb within a few
weeks to an acute leukaemoid disease with visceral turnouts.
Electron microscopy of infected cells as well as the stable line of tumour cells shows
budding C-type particles indistinguishable from conventional C-type particles. From such
particles 7oS R N A can be isolated. This RNA, when heated produces b-type subunits only.
Since virus isolated from monolayers of chick embryo cells contains the non-transforming
O K Io AV in excess, it cannot definitely be excluded that a-type subunits are also present.
The RNA of O K ~o may thus differ in size from the R N A of RSV. Whether it also differs
from the RNA of the non-transforming leukosis viruses, remains to be seen. It may be
similar to, for instance, the RNAs of M H 2 and MC 29, which are said to contain smaller
RNAs than other C-type viruses. Experiments to study this question have been
initiated.
The data so far available indicate that O K Io virus is related to M H 2 virus, or to the
acute leukaemia viruses of chicken. These viruses may turn out to be excellent models for
the study of the oncogenes of the transforming avian leukosis viruses and acute leukaemia
viruses, along the lines successfully used for study of the oncogenes of the sarcoma viruses
(Stehelin et al. t976a, b).

The original field work was supported by Medica Ltd. One of the authors (L.H.) was
supported by grants from Finska L~ikares~illskapet.
We are grateful to Dr Bodil Norrild for the performance of the test for Marek antigen,
to Dr Robin Weiss for the 3H-PR-C RNA, to Dr Per Andersson for performing the autopsies,
to Dr L. N. Payne for giving his opinion of the histological sections, to Dr P. M. Biggs for
supplying immune sera, to Dr Leevi K~i~iri~iinen for the aH-uridine labelled Semliki
Forest virus R N A and to Dr Ralf Pettersson and Dr Tapani Hovi for helpful discussions
and technical help. We also wish to extend our sincere thanks to Mrs Kaija Kettunen for
40-2
632 N. O K E R - B L O M A N D O T H E R S

excellent technical assistance. Finally, we wish to thank Professors H. Hanafusa and

L. Philipson for reviewing the manuscript.

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(Received 9 January 1978)