Archives of Virology

Archives of Virology 53, 9--23 (1977)

© by Springer-Verlag 1977

Isolation and Charaeterisation of Oneornavirus from Primary Cultures of Tissues from Cattle with Leukemia By

3/[. I. PARFANOVICtt, •. I). KASAK, A. E. BAI~A~ov, T. L. NAPASTNIKOVA, T. A. ~IKOLSKAYA~ and V. M. ZIrlDANOV The D. I. Ivanovsky Institute of Virologsz, Academy of Medical Sciences, Moscow, U.S.S.I~. With 15 Figures Accepted July 21, 1976

Summary The purpose of this investigation was to search for oneornavirus irt primary cell cultures obtained from leukemic cattle organs and lymphoeytes and to study their molecular-biological properties and role in the etiology of cattle leukemia. The investigation was carried out on 25 primary trypsinized cell cultures of lymph nodes, spleens, kidneys and lymphocytes from cattle with acute and chronic leukemia. I t was demonstrated that all cell cultures from leukemic cattle (in contrast to cell cultures from healthy cattle) released oncornavirus into the culture medium. The virus possesses the main properties of oncornaviruses: it has a virion of C-type structure with a density of 1.16--1.i8 g/ml in a 20--60 per cent sucrose gradient, which may be induced by 5-bromodeoxyuridine, inhibited by Aetinomyein D, has reverse transeriptase activity, contains 60S RNA, that is annealed in the reaction of molecular hybridization with DNA of lymph nodes of cattle with leukemia. The propagation of the isolated oneornavirus in continuous cell lines of calf kidney culture was demonstrated. Experimental inoculation of purified oneornavirus was carried out on 60 baby calves and 15 lambs from leukosis free herds or flocks. Several of the calves later showed evidence of virus infection. Introduction

intensive investigations on the viral etiology of carried out in recent years. Electron microscopic C-type particles in the lymphocyte cultures of cattle The ability to induce the experimental leukemia in

bovine leukemia have been studies have demonstrated with leukemia (3, 4, 13, 23). baby calves and lambs was

10

M . I . PA1a!eANOVICF[et al. :

also d e m o n s t r a t e d w i t h d i f f e r e n t m a t e r i a l s - - b l o o d , l y m p h o e y t e s , s u s p e n s i o n of l y m p h n o d e s cells, l y m p h o c y t e s c u l t u r e s , e t c . - - (25). H o w e v e r , a t t h e b e g i n n i n g of o u r i n v e s t i g a t i o n in 1972 t h e r e w e r e no p u b l i s h e d d a t a o n t h e i s o l a t i o n of o n c o r n a v i r u s f r o m c a t t l e w i t h l e u k e m i a u s i n g b i o e h e m i e M - b i o p h y s i e a l m e t h o d s , n o r on t h e e x p e r i m e n t a l i n d u c t i o n oI l e u k e m i a in eMves w i t h p u r i f i e d o n e o r n a v i r u s . T h e s e t w o q u e s t i o n s a r e t h e m a i n p u r p o s e of t h i s r e p o r t .

Materials and Methods Cell Lines T h e investigation was carried out on cell-cultures f r o m cattle with acute and chronic leukemia. The organs and blood from leukemic cattle were kindly supplied by Prof. V. N. Sjurin and p r i m a r y trypsinized cell cultures of l y m p h nodes, spleens, kidneys and culture of leukocytes were p r e p a r e d separately. The cells were p r o p a g a t e d in Eagle's m e d i u m - - 1 9 9 m e d i u m (equal volumes), w i t h 0.5 per cent h y d r o l y s a t e of l a e t a l b u m i n and 10 per cent of calf serum. Cell cultures in the first to fifth passages ~zere used.

Labelling and Puri/ication o/ Virus in Sucrose Gradient F o r the s t u d y of g N A - c o n t a i n i n g viruses the cell cultures were labeled with alturidine ( l ( ~ - 2 0 m C i / m t , specific a c t i v i t y 26 Ci/mmol) for 24 hours. The culture m e d i u m was collected, cell debris was r e m o v e d b y centrifugation at 15,000 × g for 20 minutes, and t h e virus was s e d i m e n t e d at 100,000 × g for two hours. The pellet was collected, resuspended in T H E - b u f f e r (Tris-IIC1 0.01 ~ p H 7.4; NaC1 0.1 M, E I ) T A 0.01 ~), layered onto a linear sucrose density g r a d i e n t 2 0 - - 6 0 per cent (w/w) and centrifuged in a S W 27.1 rotor of a Spinco L-3 centrifuge at 25,000 r p m for 3 hours. The g r a d i e n t fractions were collected in a L X B fractionator and the r a d i o a c t i v i t y of t h e acid insoluble m a t e r i a l was m e a s u r e d in a P a c k a r d - T r i c a r b liquid scintillation counter. U n l a b e l e d virus for electron microscopy, reverse transcriptase reactions mad for e x p e r i m e n t a l inoculation of animals was also c o n c e n t r a t e d and purified as described above. Electron Microscopy The monolayers of cell cultures and pellets of sucrose gradient fractions of 1.16 to 1.18 g / m l and 1.20--1.24 g/ml density were fixed w i t h 2 per cent g l u t a r a l d c h y d e in phosphate-buffer of MILLONIG (16) for i 0 - - 6 0 minutes, t h e n with 1 per cent o s m i u m oxide for 1--1.5 hours. T h e n t h e monolayers and pellets were e m b e d d e d into a m i x t u r e of E p o n 812 and ara]dite (17). U l t r a - t h i n sections were contrasted with 4 per cent aqueous solution of u r a n y l acetate and lead citrate (24) for t - - 3 minutes. U l t r a - t h i n sections were placed onto coated F o r m v a r films. All preparations were studied in J E M - 5 Y and H i t a c h i . 11B electron microscopes. Use o/Inhibitors To s t u d y tile action of A c t i n o m y c i n D on virus production 0.5 ,~g/ml was added to the cell cultures t o g e t h e r w i t h 3H-uridine for 18---24 hours. To reveal the stimulation of virus production, t h e cell cultures were t r e a t e d w i t h 100 ~g/ml of 5 b r o m o d e o x y uridine for 18--24 hours. T h e r e a f t e r t h e d r u g was r e m o v e d and t h e cultures were labeled w i t h 3H-uridine (12). Isolation and Analysis o/ Viral R N A 3tt-uridine-labeted virus was purified in 2 0 - - 6 0 per c e n t sucrose density gradients. The fractions w i t h density of 1.16--1.18 g / m l were collected, diluted with t h e buffer to a density of 1.10 g / m l and t h e m a t e r i a l was pelleted at 15,000 × g for 1 hour. I~NA was e x t r a c t e d w i t h phenol a n d s o d i u m dodecyI sulfate a n d studied in sucrose density gradients 10----30 per cent (w/v).

Isolation of Oncornavirus from Leukemic Cattle

11

Reverse Transcriptase Reaction To study the enzymatic activity of the extracellular virus the latter was isolated from non-labeled cultures as described above. The material from sucrose gradient fractions with a density of 1.16--1.18g/ml was pelleted and used in the reverse transcriptase reaction. The reaction was performed with endogenous template and also with an exogenous template oligo (dT)-poly (rA). The incubation mixture for the reaction with endogenous template contained in I ml: 10 lzmole pH 8.3 Tris-HCl, 150 y.mole NaCl, 5 y.mole MnCl2, 5 I~mole dethiothreitol, 0.015 per cent non-ionic detergent NP-40, 0. i ~mo]e of each deoxynueleoside triphosphate, dATP, dGTP, dCTP, I0 y.Ci3I-I-TTP (specific activity 16 Ci/mmole), virus 35--50 ~zg of protein. The reaetion was stopped by the addition of i0 volumes of I0 per cent TCA in 0.I ~ pyrophosphate. The incubation mixture for the reaction with exogenous template contained in i ml: 50 y.mole Tris-HCl, pH 7.8, 60 Mmole KCI, 0.125 Bmole MnCI2, 2 ~mole dithiothreitol, 0.05 per cent Triton X i00, oligo (dT):poly(rA) 0.02 ~60, 1 BCi 3H TTP, virus 35--50 Bg of protein. We also have studied the intracellular viral reverse-transeriptase activity (synthesis of 8I-I-TTP product) associated with 70--60S RNA as described by SOHLO~I and S]PIEGELIVIAN (22).

R N A - D N A Hybridization RNA-DNA hybridization was carried out in the presence of formamide with the subsequent eentrifugation of the products in caesium sulfate gTadients. I n each experiment 0.5--10rag DNA from lymph nodes of cattle with leukemia (and as control DNA from lymph nodes of healthy cattle) was taken and denatured by boiling for 2--5 minutes and by rapid cooling in an ice bath. Radioactivity of 3I~-uridineR N A from isolated oncornavirus was 10,000--20,000 epm per DNA sample. Hybridization was carried out in 50 per cent formamide and 0.¢ iv1NaC1 in the volume of 0.2--0.4 ml at 37 ° C for 18--20 hours. Linear calcium sulfate gradients with densities of t .78 g/ml to 1.36 g/ml were prepared, and the gradients were centrifuged in a Ti50 rotor of the Spineo L 2 - - 6 5 centrifuge at 35,000 rpm for 60 hours (2).

Experimental Inoculation o] Baby Cattle and Sheep with Puri/ied Oncornavirus This was carried out in the Estonian Agricultural Academy (Tartu) and Uzbek Institute of Veterinary (Samarkand). Sixty calves, 1--15 days old, and 15 lambs, 2 days old, were infected by different methods: intravenously° intrasternally, in the area of preshoulder lymph-nodes, subcutaneously, per os, etc. For inoculation, oncornavirus (20 mg/ml protein) concentrated 2500--5000 times and purified by isopyenic centrifugation was used. All the calves were received from herds and farms without leukemia. Different stocks of cattle were used.

Results I t was d e m o n s t r a t e d t h a t all cell cultures from leukemic cattle as a rule release into the culture m e d i u m particles with d e n s i t y of 1.16--1.18 g/ml typieaI of oneornaviruses a n d most cultures also release particles with a d e n s i t y of 1.20 to 1.24 g/ml, typical of m y c o p l a s m a (Fig. 1). To stimulate the virus p r o d u c t i o n the cell cultures were t r e a t e d with 100 ~g/ml of 5-bromodeoxyuridine for 20 hours, the inhibitor r e m o v e d a n d the cultures labeled with 3tt-uridine. After p r e t r e a t m e n t with 5-bromodeoxyuridine the release of the particles with a d e n s i t y of 1.16--1.18 g/ml t h a t is typical for leukoviruses sharply increased, b u t the p r o d u c t i o n of the structures with a d e n s i t y of 1.20 to t.24 g/ml was strongly i n h i b i t e d (Fig. 2).

12

M . I . PAJaFA~rOWCH e t

al. :

Aetinomyein D (0.5 ~g/ml) inhibited the synthesis of particles with a, density of 1.16--1.18 g/ml in these cell cultures. As a control, cell cultures of lymph nodes, spleens and kidneys of healthy cattle were prepared and also labeled with aH-uridine. In contrast to cell cultures from cattle with leukemia these produce into eel] medium only the particles with density 1.20--1.24 g/ml (Fig. 3).

+0

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Fig. ] Fig. 2 Fig. 1. D e n s i t y distribution in 2 0 - - 6 0 per cent sucrose gradient of aH-uridine labelled structures f r o m p r i m a r y cell culture m e d i u m of k i d n e y from cattle w i t h leukemia after c e n t r i f u g a t i o n in SW27.1 rotor of Super speed-65 at 25,000 r p m for 12 hours Fig. 2. D e n s i t y distribution (in 20 --60 per cent sucrose gradient) of 3H-uridine labelled structures from p r i m a r y cell culture m e d i u m of kidney f r o m cattle w i t h l e u k e m i a after p r e t r e a t m e n t of these cell culture w i t h 5-bromdeoxyuridine. The conditions of eentrifugation as in F i g u r e 1 (g J

15

P

I0

5

l,IO

5 I0 15 ~O Fractions Fig. 3. D e n s i t y distribution (in 2 0 - - 6 0 per cent sucrose gradient) of SH-uridine labelled structures from p r i m a r y cell-cultures m e d i u m of k i d n e y f r o m h e a l t h y cattle. The conditions of een~rifugation as in Figures 1, 2

Isolation of Oncornavirus from Leukemic Cattle

13

To d e m o n s t r a t e t h a t the particles with a d e n s i t y of 1.16--1.18 g/ml from cell cultures are really oneornaviruses (and n o t t h e cell structures w i t h t h e same density) the R N A was isolated a n d s t u d i e d in 1.0--30 p e r cent sucrose gradients. I t was d e m o n s t r a t e d , t h a t t h e structures of 1.16--1.18 g/ml d e n s i t y c o n t a i n e d a high molecular weight I~NA w i t h a s e d i m e n t a t i o n coefficient of a b o u t 60S a n d also some slow sedimenting c o m p o n e n t s (Fig. 4). 60.S

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30

Fracfi0ns Fig. 4. Sedimentation profiles of aH-uridine labelled R N A extracted from 1.16 to 1.18 g/ml extracellular structures from primary cell cultures of kidney from cattle with leukemia in 10--30 per cent sucrose density gradient after centrifugation in SW27.1 rotor of Super-speed 65 at 17,000 rpm for 16 hours. Ttle position of 28S marker ribosomal R N A is shown by the arrow Studies on the e n z y m a t i c a c t i v i t y of reverse t r a n s c r i p t a s e have d e m o n s t r a t e d t h a t t h e D N A - s y n t h e s i s increases 5 - - 7 times when the d u r a t i o n of r e a c t i o n increases from 5 m i n u t e s to 1 hour. The a d d i t i o n of exogenous t e m p l a t e oligo (dT)p o l y ( r A ) increases the r a t e of the r e a c t i o n 2 - - 3 times (Table 1). Reverse transcriptase was also d e t e c t e d in intraeellular virus associated w i t h the 60S R N A in p r i m a r y cell cultures from leukemic cattle (Fig. 5a, b). Electron-microscopic s t u d y of u l t r a - t h i n sections of t h e cells of p r i m a r y cultures of l y m p h nodes, spleens a n d k i d n e y s of cattle w i t h l e u k e m i a r e v e a l e d i n t r a c e l l u l a r virions w i t h a dense nueleoid a n d an envelope t h a t is f o r m e d during t h e " b u d d i n g " of virions t h r o u g h the cell m e m b r a n e . The size of t h e virions is a b o u t 8 0 - - 1 5 0 n m a n d t h e y resemble C-type oneornaviruses. Electrort m i c r o s c o p y s t u d y of t h i n sections of t h e pellet of an oneornavirus from sucrose 2 0 - - 6 0 per cent g r a d i e n t fractions w i t h a d e n s i t y 1.16--1.18 g/ml also reveale~d t h e same particles (Fig. 6). The sucrose g r a d i e n t fractions w i t h a d e n s i t y of 1.20--1.24 g/ml c o n t a i n e d the structures of m y c o p l a s m a .

14

M . I . PAXFA:~OVlCI-Iet al. : Isolation of Oncornavirus from Leukemic Cattle

Table 1. The results of endogenous and exogenous reverse transcriptase reaction with oncornavirus isolated ]rom primary cell cultures o/ lymph nodes, kidney, spleen and leukocytes /rom cattle with leukemia The virus origin No. of cell culture

Inclusion of 3H TTP epm 5 min

The condition of reaction

60 min

1,035

with matrix oligo (dT)-poly (rA) without matrix

3,010 831

21,624 18,250

995

with matrix oligo (dT)-poly (rA) without matrix

1,769 880

9,013 5,055

1,012

with matrix oligo (dT)-poly (rA) without matrix

974 453

12,091 9,474:

987

with m a t r i x oligo (dT)-poly(rA) without matrix

12,152 19,234

65,788 30,360

with matrix oligo (dT)-poly (rA) without matrix

96 141

35,562 297

993

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Fig. 5. Sedimentation profiles of SH-TTP labelled product of the reverse transeriptase reaction with intracellutar structures of lymphocyte culture from leukemic cattle (a) and healthy cattle (b). The conditions of centrifugation of 3H-TTP labelled products in sucrose gradient are the same as in Figure 4. The position of 28S marker ribosomal I%NA is shown b y the arrow I t was d e m o n s t r a t e d t h a t virus-specific I~NA b a n d e d a t a d e n s i t y of 1.61 to 1.63 g / m l in caesium s u l p h a t e (Fig. 7). I n R N A - D N A h y b r i d i z a t i o n e x p e r i m e n t s no essential changes were o b s e r v e d in t h e d e n s i t y a f t e r a n n e a l i n g this I~NA w i t h D N A f r o m n o r m a l l y m p h nodes (Fig. 8). A f t e r annealing t h e v i r a l I~NA with D N A from l y m p h nodes of cattle w i t h leukemia, I ~ N A : D N A h y b r i d s were f o r m e d w i t h d e n s i t y of 1.54 to 1.58 g/ml (Figs. 9, 10). W e also s t u d i e d t h e p o s s i b i l i t y of p r o p a g a t i o n of t h e o n e o r n a v i r u s f r o m p r i m a r y cell cultures of leukemic c a t t l e l y m p h nodes (LN) in t h e continuous cell line of

Fig. 6 a) Virus particles in primary celt cultures of lymph nodes of cattle with lymphatic leukemia. × 50,000 b) Virus particles from 1.16--1.18 g/ml fraction of sucrose gradient 20--60 per cent, isolated from culture medium of primary cultures of lymph nodes of leukemic cattle. × 50,000

16

M . I . PARFAlgO¥IOI-Iet al. :

calf kidney, CCLCK (this line was received from Dr. Kobseva) (10). The viruses were inoculated in cells of the 50th passage. CCLCK cultures inoculated with virus are indicated as COLCK + LN 1, CCLCK 4- L N 2 and CCLCK + LN3. I n a preliminary investigation we demonstrated that CCLCK cells did not release into

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Fig. 8

Fig. 7. Density distribution of ~H-uridine labelled t/.NA from oncornavirus :isolated from primary cell cultures of lymph nodes of cattle with leukemia after equilibrium centrifugation in caesium sulfate density gradients in Ti 50 rotor of Spinco L-3 centrifuge at 35,000 rpm for 60 hours Fig. 8. Density distribution of 3I:f-uridine labelled products of the molecular hybridization of oncornavirus R N A annealed with D N A from lymph nodes of healthy cattle. The condition of eentrifugation as in the Figure 7

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Figs. 9, t0. Density distribution of aH-uridine labelled products of the molecular hybridization of oncornavirus tZNA annealed with D N A from lymph nodes of eattle with leukemia. The condition of eentrifugation as in the Figures 7, 8

Isolation of Oneornavirus from Leukemic Cattle

17

the culture-medium particles with 1.16--1.18 g/ml density typical of oncornaviruses (Fig. t 1 a). 5-bromodeoxyuridine also did not stimulate the production of oncornavirus b y ~his cell-line (Fig. t l b). a)

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Fig. 1 l. Density distribution (in 20.--60 per cent sucrose gradient) of 3H-uridine labelled structures from medium of persistent ceil line from cattle embryo kidney a) before pretreatment wRh 5-bromdeoxyuridine b) after pretreatment with 5~bromdeoxyuridine The condition of centrifugation as in Figures 1, 2, 3 However, it was {omtd ~hat the vicusdnoctJlated cell-lines (CCLCK ~ - L N I , CCLCK ~ - L N 2 and CCLCK-~ LN3) produced a virus with properties typical of oncornaviruses. The virions had a density 1.16--1.18 g/ml in the 20--60 per cent sucrose density gradient (Fig. 12) with obvious reverse transeriptase aetivRy (Table 2), and contain 60S RNA. All these properties were revealed in the cell lines within one passage after infection.

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Fraefions Fig. 12. D e n s i t y d i s t r i b u t i o n (m 2 0 - - 6 0 p e r eer~t sucrose g r a d i e n t ) of ~t-I labelled s t r u c t u r e s f r o m m e d i u m of p e r s i s t e n t cell line f r o m c a t t l e e m b r y o k i d n e y a f t e r inoculat i o n w i t h o n e o r n a v i r u s i s o l a t e d f r o m l e u k e m i c c a t t l e l y m p h nodes. T h e c o n d i t i o n of c e n t r i f u g a t i o n as i n t h e F i g u r e s 1, 2, 3, l l Arch. Virol. 53/1--2

2

18

M . I . PA~FANOWCIt et al. :

To investigate biological properties of the isolated oncornavirus and its role in the pathology of cattle, we carried out an experimental inoculation of cMves together with the Moscow K. S. Scryabin Veterinary Academy (V. N. Sjurin, V. P. Shishkov), the Estonian Agricultural Academy, Tartu (E. M. NSmm, Jn. A. Simovart) and Uzbek Institute of Veterinary, Samarkand (H. S. Salimov, F. I. Table 2 A . T h e results o/ endogenous reverse transeriptase reaction with oncornavirus, isolated /rom C C L C K + L N 1 and C C L C K - ~ L N 2 cell lines The oneornavirus origin CCLCK+LN

1

CCLCK@LN2

I n c l u s i o n of S H - T T P in c p m T h e c o n d i t i o n of r e a c t i o n

1--2 rain

60 m i n

Full i n c u b a t i o n m i x t u r e With RN-ase With actinomycin D

227 136 184

7023 422 5897

Full incubation mixture W i t h t~N-ase With actinomycin D

299 104 226

1904 189 1435

o~ exogenous reverse transeriptase reaction with oneornavirus i s o l a t e d / r o m C C L C K t-.LN 1, C C L C K -~ L N 2 and C C L C K + L N 3 cell lines

B . T h e results

I n c l u s i o n of 3 H - T T P in c p m

I n d e x of reverse

T h e v i r u s origin

w i t h oligo (dT)p o l y (rA) m a t r i x

control without matrix

transcriptase reaction

CCLCK @ LN 1 CCLCK + LN 2 CCLCK+LN3

517 114 1214

55 49 164

9 2.3 8

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Fractions Fig. 13. D e n s i t y d i s t r i b u t i o n (in 2 0 - - 6 0 p e r c e n t sucrose g r a d i e n t ) of 3I-I-uridine l a b e l l e d s t r u c t u r e s f r o m p r i m a r y cell c u l t u r e s of m e s e n t e r i M l y m p h n o d e s f r o m calves experim e n t a l l y i n f e c t e d w i t h o n e o r n a v i r u s (one y e a r a f t e r e x p e r i m e n t a l i n o c u l a t i o n of o n c o r n a v i r u s ) . T h e c o n d i t i o n of c e n t r i f u g a t i o n as i n F i g u r e s t, 2, 3, 11, 12

I s o l a t i o n of O n c o r n a v i r u s f r o m L e u k e m i c C a t t l e

19

Ibadullaev, Sh. A. Asimov). Sixty 3[--15 day old calves and fifteen 2 day old lambs were infected by different routes as described in Materials a:~d Methods. A regular hematological, histo- and cytochemieal, virological and serological investigation of these calves experimentally inoculated with purified oncornavirus has been carried out. It was demonstrated that primary mesenteric lymph-node cell-cultures of two experimentally inoculated calves produced i~ culture medium 3H-uridine labeled structures with 1.16--1.18 g/ml density in 20--60 per cent sucrose gradient (Fig. 13). Reverse-transcriptase activity was associated with these particles. In addition, the investigation of leukoey~es from 10 exper;mentatty inoculated calves revealed intraeellular reverse-transeriptase activity associated with 70 ~o 60S I~NA in leukocytes of two animals (Fig. t4). Cell cultures of lymphocytes from the same two calves produced in culture medium 3H-uridine labeled particles with 1.16--1.18 g/ml density in 20--60 per cent sucrose gradient (Fig. i5).

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Fractions Fig. 14. Sedimezltation profiles o£ ~H-TTP labelled products of the reverse-transoriptase reaction with intracellular structures of lymphocytes from infected cattle 12 months after inoculation. The conditions of eentrifuga~ion of ~H-TTP labelled products in sucrose gradient are the same as in Figures 4, 5 2*

20

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Fr~eti0n3 Fig. 15. Density distribution of 8tt-uridine labelled structures from lymphocyte cultures of healthy control calves (a) and of calves experimentally inoculated with oncornavirus (b, e), one year after inoculation. The conditions of eentrifugation as in Figures 4, 5, 14

Diseussion

The results presented in this paper allow the conclusion that a n oneornavirus has been isolated from primary cell cultures of lymph nodes, spleens, kidneys and leukocytes of cattle with leukemia. The virus possesses the main properties of oneornaviruses: it has a virion with a density of 1.16--1.18 g/ml in the 20 to 60 per cent sucrose gradient, particle release m a y be enhanced b y 5-bromodeoxyuridine, inhibited by Actinomycin D, has reverse-transeriptase activity, contains 60S P~NA, which anneals in the reaction of molecular hybridization with DNA of lymph nodes of cattle with leukemia (19, 27, 28). These data are in aceordanee with our study on the isolation of teukovirus from the 30th passage of lymph node cell culture from cattle with leukemia (27).

Isolation of 0ncornavirus from Leukemic Cattle

21

I t is of interest that some authors have prepared a detergent-treated antigen from the 1.16--1.18 g/ml sucrose gradient fractions of medium of lymphocytes culture (containing C-type particles) from cattle with leukemia and have found an antibody to this antigen in the serum of leukemic cattle (1, 7, 15). We found in immunodiffusion tests that primary cell-cultures of lymph nodes, kidney, spleen and lymphocytes from cattle with leukemia and oncornavirus, isolated from these cultures, have no common group-specific antigen with MasonPfizer monkey virus. I~ESSANG et al. (21) have also reported, using fluorescent antibody technique and immunodiffusion tests, that C-type virus from cell cultures of leukemic cattle has no group-specific antigen with mammalian leukemiasarcoma viruses. Earlier the same results were obtained by }PERRER (6). I n our experiments the oncornavirus isolated from primary cell cultures of lymph-nodes, kidney, spleen and lymphocytes from cattle with leukemia, has reverse-transcriptase activity for endogenous and exogenous [with oligo(dT)poly(rA) template] reactions in the presence of Mn ions (19, 20). DUTZSCHOLD et al. (5) have also reported that C-type particles from primary cell cultures of lymphocytes from leukemic cattle have the strongest reverse-transcriptase reaction in the presence of exogenous template oligo(dT)-poly(rA), and very little activity in the endogenous reaction. However, GILDEI~-et al. (8) have detected revers-transcriptase activity of BLV in the presence of Mg ions. In order to establish a biological role of the isolated oncornavirus, experimental inoculations of 60 calves and 15 lambs was carried out using purified oneornavirus. Previously, such experiments have been carried out not with purified oncornavirus but with different materials (blood, the lymph-nodes and spleen suspension, lymphoeytes, containing C-type particles) from leukemic cattle (9, l l , 14, 18, 26). Our experiments on the inoculation of calves and lambs with oneornavirus were made only two years ago. The data obtained so far concerning positive oncornavirus infection (reverse-transeriptase activity associated with 60--70S I~NA) in the leukocytes of inoculated calves are insufficient to draw definite conclusions about oncornavirus disease relationships. These inoculation experiments will continue and we hope they will help to elucidate the etiology of bovine leukemia.

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to 540 (1968). 3. CALAFAT, J., HAGEMAN, P., RESSANG, A. A. : Structure of C-type virus particles in l y m p h o c y t e cultures of bovine origin. J. N a t . Cancer Inst. 52, 1251 1257 (1974). 4. DUTTA, S. K., LARSON, V. L., SORE~SE~, D. K., I:)ERMAN, V., VEBER, A. L., HAMMER, 1~. F., SHOPE, R. F. : Isolation of C-type virus particles from leukemic and l y m p h o c y t o t i e cattle. Bibl. H a e m a t o l . 36, 548--554 (1970). 5. DUTZSCIIOLD, B., KAADEN, O. R., UEBERSCttAER, S., WEISLA2~D~F., STRAUB, O. C. :

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Isolation of Oneornavirus from Leukemic Cattle

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26. WIa:TSIAN,W., Ut~BANEK, D., SELLS, H., BEYER, J. : Untersuchungen zur Atiologie der Rinderleukose. 10. Gesamtauswertung erster Ubertragungsversuche mit Blut leukosekranker t%inder auf Sehafl/~mmer unter besonderer Ber/ieksiehtigung klinischer und h/imatologischer Befunde. Arch. exp. Vet. Med. 25, 587--595 (1971). 27. Z]~DA~,'OV,V. M., PARFANOVlCt~,M. I., YEI~SI~OV,F. I., NIICOLSKAYA,T. A., KAZAK, 1N'. F., NITAVSt~AYA,S. D., I~'TAGAEVA,L. L , KLrKAIN, R. A. : Isolation of leukovirus from cell line of calf's lymph nodes. Veterinary 4, t 5 - - t 6 (1975) (Russian). 28. ZHDANOV,V. M., PARFANOVICH,M. I., YEI~SHOV, F. :L, NIKOLSKAYA,T. A., I(AZAK, N. F., I'~ITAVSKAYA,S. D., NAGAEVA, L. S., KUKAIN, 1~. A. : A leukovirus isolated from leukemic calf lymph nodes. Brit. Vet. J. 131, 499--503 (1975). Authors' address: M. I. PAI~FANOVICmThe D. I. Ivanovsky Institute of Virology, Academy of Medical Sciences, U.S.S.R., Gamaleya St., 16, Moscow 123098, U.S.S.R. Received December 1, 1975