Isolation of an avian leukosis virus associated with the Harris strain of Rous sarcoma virus

DISCUSSION Isolation

of

an Avian

Associated of Rous

with

the

Sarcoma

Leukosis Harris

AND

PRELIMINARY

Virus Strain

Virus

The defectiveness of the Bryan high titer and standard strain of Rous sarcoma virus (BH-RSV, BS-RSV), and the control of antigenicity, host range, and interference with Rous sarcoma virus (RSV) by the helper virus have been well reviewed (1, 2). Hanafusa (S), although unable to reveal that the Schmidt-Ruppin strain of RSV was defective, was able to modify its antigenic characteristics and its response to interference. In addition, he has recently (4) isolated virus from the Schmidt-Ruppin RSV stock which may act as a helper virus. To the authors’ knowledge, there has been no report of a helper virus isolated from the Harris strain (HA-RSV). This brief communication describes the isolation and some characteristics of an agent associated with the Harris strain of RSV. This strain has been assigned to subgroup B of the avian tumor viruses on the basis of its host range and antigenicity (5, 6). During our attempts to propagate HARSV in vitro, the titer of recoverable chickembryo fibroblast (CEF) transforming HARSV diminished with each serial passage of supernatant growth fluid. One possible explanation for this observation is that the HA-RSV infection is influenced by an associated leukosis virus and that this virus multiplies at a faster rate than the HA-RSV, so that at each subsequent passage the interference effect becomes more and more pronounced. To test this hypothesis, an aliquot of supernatant growth fluid from CEF infected with HA-RSV beyond the focusforming end point was inoculated on cells resistant to subgroup A avian tumor viruses (C/A) and grown for 5 days, at which t’ime the supernatant growth fluid and frozenthawed cell extract were passaged onto fresh C/A cells. These were cultured for 4 days, and again growth fluids and frozen-thawed cell ext,racts were harvested. This material will be referred to as Rousassociated virus-6 (RAVB) in accordance with previous helper virus designations (4, 5). This virus was tested for transforming

363

REPORTS

activity in three serial transfers of infected cells and none was detected. Day-old susceptible chicks inoculated in the wing web with RAV-6 and held for 32 days failed to develop tumors. The chorioallantoic membranes (CAM) of g-day embryonated eggs were inoculated with RAV6. No pocks were observed on examination of the membrane 9 days later. The resistance-inducing (RIF) property of this virus was tested using cells susceptible to both subgroup A and B viruses. These cells, previously infected for 3 days with RAV-6, were challenged with a number of subgroup A and B avian tumor viruses. The results are shown in Table 1. Interference with all subgroup B viruses, but no significant interference with the subgroup A viruses, was observed, thus indicating that the agent present belonged to subgroup B of the avian tumor viruses. The RAV-6 was tested for its ability to activate BH-RSV non-virus-producing (NP) cells to produce fully infective cell transforming virus (?‘). Chicken embryo fibroblast NP cells were obtained by limit dilution and single-focus cloning techniques using duck embryo fibroblast cells as a feeder layer. The RAV-6 activated these NP cells to produce CEF-transforming-virus. This virus induced foci morphologically characteristic of BH-RSV but not HA-RSV. The agent was designated BH-RSV (RAV-6) in the manner of Hanafusa et al. (7). The morphology of foci in CEF cultures has been shown to be helper independent; TABLE INTERFERENCE

RSV strain

WITH

or pseudotype

Subgroup A BH-RSV (RAV-1) SR-RSV-1 BH-RSV (AMV-1) BS-RSV Subgroup B BH-RSV (RAV-2) SR-RSV-2 BH-RSV (AMV-2) HA-RSV a One-tenth lated onto CEF

1 RSV

BY RAV-6~

Relative sensitivity

0.89 0.64 1.m 1.07 <0.015 <0.014 0.046 <0.031

ml of undiluted RAV-6 was inocu3 days prior to the RSV challenge.

364

DISCUSSION

AND

PRELIMINARY TABLE

NEUTRALIZATION

REPORTS

2

SPECTRUM OF Aw-RAVB Fraction

I

SER.<

of virus

survivors

Serum

Anti-RAV-6,

# 1

Anti-RAT’-6,

# 2

l/10 l/100 1/1000 l/10 l/10

Normal Normal

TABLE

CROSS

OF

21.00

21.00 2_1.00,

21.00 21.00

21.00 Ll.00

AND

HA-RSV

(RAV6)*

I Antisera

21.00

3

NEUTRALIZATION BH-RSV

21.00

Fraction of virus survivors Dilution HA-RSV

IAnti-HA-RSV, Turkey

BH-RSV

iRAV-6)

0.020 1.020

0.083 0.880

Anti-RAV-6, Chicken

No.

1

<0.009 0.009 0.058 0.510

<0.008 0.025 0.168 0.990

Anti-RAV-6, Chicken

No.

2

<0.009 <0.009 0.009 0.058

<0.008 <0.008 0.017 0.083

*

* BH-RSV

NP

cell

activated

by RAv-o.

i.e., it is dependent on the RSV genome (1, S) Our observations of the morphology of foci induced by HA-RSV and the BH-RSV (RAV 6) confirm these reports. The foci of the original HA-RSV are of the diffuse, monolayered, and rounded cell type, and they are very difficult t’o enumerate (8, 9). Foci induced by the new pseudotype are characteristic of BH-RSV; they are compact, multilayered, and composed of rounded cells and are easily enumerated (10). Immune sera, produced by the intravenous inoculation of the RAV-6 into

~1.00 11.00

1.02 L1.00 21.00

1.25 21.00 0.62

0.75 1.06 ~1.00

i

0.03 0.81 0.86

4-week-old chickens, preferentially neutralized HA-RSV with a lower activit,y against the other subgroup B viruses (Table 2). There was no neutralization of subgroup A viruses observed. The titers against the homologous virus (HA-RSV) are greater than 1: 1000, whereas.the titers against some of the other subgroup B viruses were only 1: 10. In addition, reciprocal neutralization tests, using the HA-RSV and BH-RSV (RAV-6) and antisera against the original HA-RSV and the newly isolated RAV-6, indicated that the two RSV’s are antigenically identical (Table 3). These results demonstrate the presence of a virus associated with HA-RSV, i.e., RAV-6. In serial propagation of HA-RSV the associated virus may interfere with HARSV and result in the decreasein recoverable focus-producing virus. The RAV-6 can act as a helper by activating NP cells; however, its role as helper t,o HA-RSV is not known. Work to establish the role of the RAV-6 is now being carried out in our laboratory. REFERENCES 1.

VOGT, P. K.,

Advan.

Virus

Res.

11, 293-385

Diseases

9, 127-145

(1965).

2. PURCHASE, H. G.,

Avian

(1965).

5. HANAFIJSA, H., Natl.

Cancer Inst. Monograph (1964). HANAFUSA, H., and HANAFUSA, T., Proc. Natl. Acad. Sci. U.S. 55, 532-538 (1966). VOGT, P. K., and ISHIZAKI, R., Virology 26, 664-672 (1963). 17, 543-556

4. 5.

DISCUSSION

AND

PRELIMINARY

REPORTS

365

essentialmedium (REM) enriched wit’h 10 % calf serum was used as medium. The transformed cell lines and the comparable normal lines were used at the same passagelevels, i.e., from the 12th to the 39th after the establishment of transformation. A transformed culture contained an average of 3 X lo6 cells and a normal culture 2 X lo6 cells. The bovine origin of the cells was demonstrated by chromosome analysis. Cancer Inst. 32, 579-589 (1964). The presence of infectious polyoma virus R.N. REAMER in the cultures was tested by inoculat’ing into W.0, OKAZABI mouse embryo cell cultures suspensionsof B.H. RISPENS~ cell debris from transformed cultures which Regional Poultry Research Laboratory, ARS had been frozen and thawed once. TransEast Lansing, Michigan 68828 formed cultures infected with FMDV (strain Accepted August 15, 1967 C virulent) were also tested. In t,hese tests the suspensionsof cell debris were treated 1 On secondment from Centraal Diergeneeskunwith hyperimmune anti-C serum prior to dig Instituut, AFD. Rotterdam, The Netherlands. inoculation. No polyoma virus was detected after 4 weeks of incubation. The transformed cells contained polyoma “tumor” antigen as Increased Susceptibility to Foot-and-Mouth shown by complement fixation test using Disease Virus of Bovine lung Cells serum from hamsters bearing polyoma-inTransformed by Polyoma Virus duced tumors. (The serum was purchased from Flow Laboratories, Scotland.) The It was previously reported (1) that a line presence of this antigen and the morphoof BHK21 cells transformed by polyoma logical appearance which was characteristic virus was less susceptible to certain strains for polyoma transformation constit’ute eviof foot’-and-mouth disease virus (FMDV) dence that the transformation was really than normal BHK21 cells. The altered re- induced by polyoma virus. sponsewas found to be due to an impaired Three virulent strains of the types 03, A+ ability of transformed cells to attach and C of FMDV, and three attenuated F?LIDV. In contrast, to this system, the derivates of these strains (for references, see present work shows that lines of polyoma- 3) were used. The virulent strains are desigtransformed bovine lung cells are more sus- nated Ov, Av, and Cv, and the attenuated ceptible to certain st’rains of FMDV than strains Oa, Aa, and Ca, respectively. All comparable lines of normal cells. Using a titrations of FMDV were performed on priC strain of FI\IDV it is demonstrated that mary cultures of calf kidney cells. only the transformed cells are capable of Strain Ca was inoculated in amounts of 10 attaching virus. TCDDOand the other st’rains in amounts of The following lines of bovine embryonic lo4 TCD,, into cultures of transformed or lung cells were used: BTr-1, which was a normal cells. At intervals after inoculation line of polyoma-transformed cells, and BN-1, samples were withdrawn from the culture a line of normal cells derived from t’he same fluids and titrated. The growth curves in embryo. BTr-2, BTr-3, and BTr-4 were Fig. 1 show that all strains except Ov mult,hree lines of polyoma-transformed cells detiplied in the transformed line BTr-1 whereas rived from another embryo and the comparable normal lines from t’his embryo were no strain multiplied in the normal line BN-1. BN-2, BN-3, and BN-4, respectively. Trans- In Fig. 2 the multiplication of Cv and Ca in formation was obtained as described before the other lines is demonstrated. It is seen that the transformed lines were more sus(2). The lines were split 1: 1 or 1: 2 by trypsinization twice weekly. Eagle’s minimal ceptible than the normal lines alt’hough the 6. ISHIZ~KI, R., and VOGT, P. K., Virology 30, 375-387 (1966). 7. ~~~~~~~~~~ H., H.lN.4FUS.4, T., and RUBIN, H., Proc. Natl. Acad. Sci. U.S. 49, 572-580 (1963). 8. DOUGHERTY, R.M., SIMONS, P. J., and CHESTERMAN, F. C., J. lvatl. Cancer Ins%. 31, 1285-1307 (1963). 9. VOGT, P. K., and ISHIZ~KI, R., Virology 30, 368-374 (1966). 10. PURCHASE, H. G., and OKAZAKI, W., J. Natl.