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RNA tumor viruses of pheasants: Characterization of avian leukosis subgroups F and G
VIROLOGY
60, 558-571
(1974)
RNA Tumor Viruses
Of Pheasants:
Leukosis DONALD
J. FUJITA,2
Subgroups
Characterization
of Avian
F and G ’
YOUNG C. CHEN, ROBERT PETER K. VOGT
R. FRIIS,3
AND
Department of Microb[ology. C’niversity of Southern California, School of Medicine, Los Angeles. California 900.3'3 Accepted
April 26. 1974
Endogenous leukosis-like viruses of ring-necked pheasants (Phasianus cokhicus) and golden pheasants (Chrysolophus pictus) have been isolated and characterized. The majority of the normal pheasant embryo cultures contain helper activity for the defective Bryan high titer strain of Rous sarcoma virus. The Rous sarcoma pseudotypes produced with endogenous helper activity from ring-necked pheasants belong to subgroup F. The pseudotypes from golden pheasant cells constitute subgroup G. Subgroup F and G pseudotypes can infect all known genetic types of chicken fibroblasts as well as pheasant and Japanese quail cells, but do not plate on goose cells. Duck cells are resistant to subgroup G but not to F. The subgroup F and G helper viruses isolated from Rous sarcoma viral pseudotypes show interference with their homologous subgroup. RAV-61, a standard of subgroup F, interferes with pseudotypes produced with endogenous helper activity from ring-necked pheasant cells but not with subgroup G pseudotypes. Subgroups F and G do not cross-react with subgroup A to E in neutralization tests. Some normal ring-necked pheasant sera have anti-F activity. Subgroup F and probably also G leukosis-like viruses can undergo genetic recombination with nondefective avian sarcoma viruses. INTRODUCTION
The existence of viral genetic information in normal avian cells has been documented through several different lines of evidence. All normal chicken embryos and embryo tissue cultures tested to date contain DNA sequences homologous to avian leukosis viruses (Rosenthal et al., 1971; Varmus et al., 1972; Baluda, 1972; Yoshikawa-Fukuda and Ebert, 1969; Neiman, 1973). In some embryos viral gene products ‘Supported by U.S. Public Health Service Grant No. CA 13213 and Contract No. NOl-CP-43242 from the National Cancer Institute. 2Fellow of the Leukemia Society of America. Present address: Department of Microbiology, University of California Medical School, San Francisco, California 94143. 3Present address: Robert Koch Institut, Nordufer 20, 1 Berlin, Germany.
such as group specific (gs) antigen or “chicken helper factor” (chf) are synthesized, and their intracellular levels seem to be at least in part regulated by genetically determined patterns of transcriptional control (Weiss, 1969; H. Hanafusa et al., 1970; Payne and Chubb, 1968; T. Hanafusa et al., 1970; Weiss and Payne, 1971; Hayward and Hanafusa, 1973; Bishop et al., 1973). Endogenous avian RNA tumor viruses are released spontaneously from the cells of certain chicken lines (Vogt and Friis, 1971; Crittenden et al., 1973; Crittenden et al., 1974). They can be activated by infection of the cell with RNA tumor viruses (T. Hanafusa et al., 1970), and they can be induced by treatment of normal fibroblasts with physical or chemical carcinogens or mutagens (Weiss et al., 1971). All endogenous chicken viruses, (with one possible excep-
RNA TUMOR
VIRUSES
tion) possess envelope specificities characteristic of the avian tumor virus subgroup E (Weiss, 1974). Recently normal ring-necked pheasant (Phasianus colchicus) cells have also been shown to harbor viral genetic information, and a leukosis virus with the envelope specificity and host range of a new avian tumor virus subgroup, F, has been isolated from such cells (Hanafusa and Hanafusa, 1973). The possible presence of endogenous virus in ring-necked pheasant cells had also been suggested by Weiss et al. (1971), who noted that in some cases leukosis viruses induced in normal chicken cells acquired a new host range after passage in ring-necked pheasant cells. In this report we describe the isolation of leukosis viruses from cells derived from another genus of the pheasant family, the golden pheasant (Chrysolophus pictus) and confirm the occurrence of subgroup F viruses in ring-necked pheasant cells. Host range, viral interference, and antiserum neutralization indicate that the endogenous golden pheasant virus (GPV) has envelope characteristics of a new avian tumor virus subgroup, G, which is distinct from previously described subgroups A through F. MATERIALS
AND
METHODS
Cells Preparation of primary and secondary cultures from avian embryos and formulation of growth media followed published techniques (Vogt, 1969). Fertile eggs of white leghorn chickens, phenotypes C/E and C/BE, were generously supplied by Mr. R. Raymond, H & N Farms, Redmond, Washington. Chicken eggs of the C/A and C/AB phenotypes came from line 7 x line 15 crosses originally supplied by the U.S.D.A. Regional Poultry Laboratory, East Lansing, Michigan. Eggs of ringnecked pheasant (Phasianus colchicus torquatus) and of the golden pheasant (Chrysolophus pictus) were purchased from B & B Mini Farms, Eureka, California and Marsh Farms, Garden Grove, California. Duck (Anas platyrhynchos) and
OF PHEASANTS
559
goose (Anser anser) eggs came from Ward Duck Farm, La Puente, California. Japanese quail eggs were supplied by Life Sciences Inc., St. Petersburg, Florida under contract with the National Cancer Institute. Infections were carried out in the presence of 2 pg/ml polybrene (Toyoshima and Vogt, 1969). Long term cultures were kept in “primary growth medium” (Vogt, 1969) containing 8% calf serum, 2% inactivated chicken serum and 1% dimethylsulfoxide (DMSO). DMSO (1%) was also present in agar overlays, and overlays for pheasant, quail, goose and duck cultures also contained 1% inactivated chicken serum. The presence of avian tumor virus group specific (gs) antigens and of chicken helper factor (chf) in normal primary cultures was checked as described previously (Vogt and Friis, 1971; Weiss et al., 1973). R(&)Q (line No. 3) is an established Japanese quail cell line which releases defective, noninfectious Bryan high titer Rous sarcoma virus referred to as RSV( ~ ). The origin of this cell line has been described (Friis, 1972). All cell cultures were incubated in polyethylene bags as described by Vogt and Harris (1974). Viruses The nondefective sarcoma viruses and the leukosis viruses had been cloned in chf negative chicken cells, except for RAV-0 which was derived from gs+ chf+ cells. A survey of nondefective sarcoma and of leukosis viruses used in this study is given in Table 1. RAV-61 was a generous gift from Dr. H. Hanafusa. Pseudotypes of the Bryan high titer strain of RSV are notated conventionally with the helper virus in parenthesis. Their origin has been detailed in previous publications (Duff and Vogt, 1969; Toyoshima et al., 1970; Weiss et al., 1971). Sendai-Virus Induced Cell Fusion and Cocultiuation of Cells Chicken, ring-necked pheasant, golden pheasant cells were trypsinized,
or and
560
FUJITA
TABLE 1 AVIAN LEUKOSIS AND SARCOMA VIRUSES USED IN THIS STUDY
Virus
Nondefective sarcoma viruses: Prague strain RSV Prague strain RSV Prague strain RSV Avian sarcoma virus BY7 Schmidt Ruppin RSV Leukosis viruses: Rous associated virus 1 Row associated virus 2 Row associated virus 0 Row associated virus 50 Rous associated virus 61 Transformation defective B77 Carr Zilher associated virus Induced leukosis virus
Subgroup
Abbreviation
A B C C D
PR-A PR-B PR-C B57 SR-I)
A B E D F C D E
RAV- 1 RAV-2 RAW) RAV-50 RAV-61
tdE377 CZAV IL\
ET AL.
ate leukosis virus of one of the subgroups A-D. After 3 days in culture at 37”, cells were passed and challenged with sarcoma viruses of various subgroups. (ii) Cells were infected with leukosis virus at a low multiplicity and passaged between three and eight times at 2-5 day intervals before challenge with a sarcoma virus. At alternate passages, an equal number of fresh, uninfected cells was added to the culture in the presence of 2 pg/ml polybrene. Chicken C/E or C/BE chf- cells were used in all interference tests except those involving subgroup E viruses, which were performed on ring-necked pheasant or quail cells. Growth of Transformed Foci
Cells from Single
RSV’(O) was plated at high dilution on us+ ring-necked pheasant cells, overlaid and incubated at 37” for 10 days. Single foci were aspirated with a capillary pipette 1 x lo6 cells were mixed with 5 x lo5 from plates containing l-10 foci, and were R( -)Q cells in test tubes in a total volume seeded individually in cloning medium of 0.2 ml primary growth medium. Approximately 800 hemagglutinating units of @- (Duesberg and Vogt, 1973) in 35 mm plates. Supernatant medium was assayed propiolactone-inactivated Sendai virus periodically in focus assays on quail and were added, and mixtures were incubated with occasional agitation for 30 min at 4”, chicken C/BE or C/E cells. followed by a 20 min incubation at 37”. by Antiserum After incubation 4 ml primary growth me- Virus Neutralization Virus neutralization by antiserum foldium were added, and the suspended cells lowed published techniques (Duff and were centrifuged at 1,000 rpm for 5 min. The pellet was resuspended in 4.5 ml Vogt, 1969). A 10-l dilution of heat inactigrowth medium, plated on 60 mm dishes, vated serum was incubated with virus, and survivors were assayed after 40 min at 37 ‘. and incubated at 37” in primary growth medium with 1% DMSO. At 2-3 day intervals, about one-half of the medium in each Infective Center Assays plate was removed and replaced with fresh Infective center assays were performed medium. Cells were transferred at 4-8 day as described by Weiss et al. (1973). Initiaintervals. Medium was harvested perioditor cells were seeded in a 60 mm dish and cally, frozen and thawed, sonicated and infected with 100-500 FFU virus in the tested for infectious focus-forming virus by presence of 2 pg/ml polybrene. After 18-24 plating on quail cells and on chicken C/E hr incubation at 37”, 2 pg/ml mitomycin C or C/BE cells. was added, and cells were incubated for an The same procedure was followed in the additional 14-20 hr. The mitomycin C was co-cultivation method, except that Sendai removed, and cells were washed with 5 ml virus was omitted. of medium three times over a period of 3 hr. Indicator cells (8 > 105/plate) were then Interference Tests added in the presence of 2 pglml polybrene. Interference tests were performed in one Cells were overlaid with agar after an of two ways: (i) Cells were infected at high additional 12-24 hr. Control cultures consisted of direct focus assays in the absence multiplicity (m.o.i. = 1) with an appropri-
RNA TLIMOR
VIRUSES
of mitomycin C which were overlaid at 18-24 hr after infection. Focus counts were made 7-10 days after infection. RESULTS
Helper Activity for Bryan High Titer RSV in Normal Pheasant Fibroblasts The Bryan high titer strain of RSV (BH RSV) is defective in an envelope component (Scheele and Hanafusa, 1971). This component can be supplied by a helper virus or by a helper factor endogenous to the host cell (Hanafusa et al., 1963; Weiss, 1969; H. Hanafusa et al., 1970). BH RSV grown in the absence of helper agents is not infectious and is referred to as RSV( ~~). BH RSV grown in the presence of helper is infectious but remains genetically defective. It is called a pseudotype and has envelope properties identical to those of the helper agent. This defectiveness makes BH RSV a sensitive tool for the detection of complementing endogenous leukosis viruses. Recently it has been shown that endogenous viral genes present in ringnecked pheasant cells can complement BH RSV leading to the synthesis of infectious sarcoma virus. This new pseudotype is referred to as BH RSV (RAV-61) and represents a new subgroup (F) of the avian leukosis and sarcoma complex (Hanafusa and Hanafusa, 1973). The leukosis virus. RAV-61. which is responsible for the endogenous helper activity in normal ringnecked pheasant fibroblasts has also been isolated (Hanafusa and Hanafusa. 1973). These results may clarify previous observations that induced leukosis viruses of chickens sometimes acquire a new host range after passage in ring-necked pheasant cells (Weiss et al., 1971). We have used BH RSV transformed fibroblasts of the Japanese quail in our attempts to detect endogenous viral gene expression in cells derived from ringnecked pheasant and golden pheasant embryos. These quail cells, designated as R( ~-)Q, had been kept in tissue culture for over one year and released RSV( ~ ) (Friis, 1972). Two methods were used to reveal helper activity for RSV( -). In the first, R( - )Q cells and fibroblasts from individ-
OF PHEASANTS
361
ual ring-necked or golden pheasant embryos were fused with p-propiolactone inactivated Sendai virus. In the second method, R( - )Q cells were cocultivated with normal fibroblasts of ring-necked or golden pheasant embryos in the absence of a fusion enhancing agent. Table 2 summarizes the control experiments with chicken cells, which were chf, gs+ or chf- gs-. Both Sendai virusinduced fusion and co-cultivation with R( ~-)Q cells resulted in the production of infectious subgroup E pseudotypes of BH RSV, if the chicken cells were chf+ gsr. No infectious virus was detected at day 12 in cultures of R( ~ )Q cells fused or co-cultivated with chf- gs- chicken cells. However, small numbers of infectious virus were demonstrable in some of these cultures by day 23. This virus is probably the result of spontaneous activation of chf. which is known to occur occasionally in chf - cells during extended passage (Hanafusa et al., 1972). (In one supernatant sample of R( ~ )Q cells a few FFU were also seen; these may have been derived from live cells accidentally transferred with the inoculum. ) These control experiments establish that fusion as well as co-cultivation with R( )Q cells can detect endogenous viral gene expression. Under our experimental conditions, fusion produced about 20-40“~ heterokaryons; most of these did not survive more than 7-9 days in culture. After 7 days fused and co-cultivated populations were probably qualitatively similar. the latter showing a low level of spontaneous fusion between test and R( -)Q cells. The results with cells from golden pheasant embryos are summarized in Table 3. Although all golden pheasant embryos were gs -, 7 out of 9 showed infectious virus by day 23. Some cultures did not survive beyond this time or were discarded. On day 52 all of the remaining cultures from six embryos produced infectious virus. This pseudotype of BH RSV plated with equal efficiency on Japanese quail and C/E and C/BE chicken cells. Table 4 lists the results with cells from various ring-necked pheasant embryos. In-
562
FUJITA
ET AL.
TABLE APPEARANCE
OF INFECTIOUS
Species and embryo number
Method”
Chicken (Callus domesticus) C/BE No. ‘:3 gs + C/BE No. 24 gs -~ C/A No. 2<5gs , C/E No. 26gs c/E No. 27 gs ~ No cells added to R(
2
SUBGROUP E RSV AFTER SENDAI VIRUS INDUCED FUSION OR COCULTIVATION CHICKEN CELLS WITH R( - ) IJ~~~~~~~ QUAIL CELLS
)Q
SF cc SF CC SF cc SF cc SF cc SF CC
Titer of infectious 12de
23d
1 x 103 2 x 103 0 0 1 X lo3 2 x lo3 0 0 0 0 0 0
5 x 103 5 x 10s 0 0 3 x 103 3 k 103 25 0 10 0 0 0
OF
RSV (FFU/ml)b
-
35d
52d
1 X 10’ 1 x 10’ 0 5 5 x 103 5 x 103 0 10 NT 0 NT 25
1 X 105 NTd 0 0 1 x 105 NT 0 0 NT 0 NT 0
“SF, Sendai virus-induced cell fusion; CC, cocultivation. “FFU plating on Japanese quail cells but excluded from chicken C/E cells c Days of mixed culture between R( ~ )Q and chicken cells. dNT, not tested. TABLE
3
APPEARANCE OF INFECTIOUS VIRUS AFTER SENDAI VIRUS-INDUCED FUSION OR COCULTIVATION AND R( 1 JAPANESE QUAIL CELLS
Soecies and embryo number
Met hod”
Titer of infectious
OF GOLDEN PHEASANT
RSV (FFU/ml)b
12d’
23d
35d
52d
0 0 0 0 50
0 5
NTd NT 10 l-.5 8 x lo3 8 x lo3 NT NT 100 NT 100 150 NT NT ti0 30 5 x IO3 500
NT NT 20 20 35x 10’ 2 x 10’ N’? NT 500 NT 30 50 NT KT 5 30 i x 10’ 5 x 10’
Golden pheasant
(Chrysolophuspictus) GOP No. 6gs GOP No. i gs GOP h’o. 8gs GOPNo.
9gs
GOP No. 10 gs ~ GOP No. 11 gs GOP No. 12 gs GOPNo.
13gs-
GOP No. 14 gs
SF cc SF cc SF CC SF CC SF CC SF CC SF cc SF cc SF CC
1 ii 103
0 0 0 0 10 0 0 0 0 0 6
I- 10
“SF, Sendai virus-induced cell fusion; CC, cocultivation. *FFU plating equally well on Japanese quail and chicken C/BE. c Days of mixed culture between R( -)Q and pheasant cells. dNT. not tested.
5 0 3 x 103 5 x 103 NT 5 0 NT 5 5 0 NT 0 l-10 400 15
RNA TUMOR
VIRUSES TABLE
APPEARANCE
OF INFECTIOUS
563
OF PHEASANTS 4
VIRUS AFTER SENDAI VIRUS-INDUCED FUSION OR COCULTWATION JAPANESE QUAIL CELLS PHEASANT AND R(-)
Species and embryo number
Titer of infectious
Method”
OF RING-NECKED
RSV (FFU/ml)’ 35d
12d’
23d
0 0 0 0
0
0
0 5 0
5 30
52d
Ring-necked pheasant (Phasianus colchicus) RNPNo.
Igs-
RNP No. 2 gsRNPNo.3gsRNP No. 4gss RNPNo.
5gst
SF cc SF cc SF cc SF CC SF cc
0 0 0 0 0 0
0 N7 0 5 N’l 10 NT
3 A 103 NT” 800 0 NT N? 0 N’l NT
NT
a SF, Sendai virus-induced cell fusion; CC, cocultivation. b FFU plating equally well on Japanese quail and chicken C/BE. c Days of mixed culture between R( -)Q and pheasant cells. dNT. not tested
fectious virus plating equally well on Japanese quail and on C/E or C/BE chicken cells was present in 4 out of 5 embryo lines at day 35. In contrast to the situation in chickens, the gs status of the ring-necked pheasant embryos did not appear to affect the helper activity in the cultures. This observation is in agreement with results reported by Hanafusa and Hanafusa (1973). As will be shown below, the infectious Rous sarcoma virus detected after cultivation with ring-necked pheasant or golden pheasant cells represents pseudotypes of BH RSV formed with endogenous ringnecked pheasant virus (RPV) or golden pheasant virus (GPV). These pseudotypes will be referred to as RSV(RPV) and RSV(GPV).
Isolation of RSWRPV) From Single Foci Produced by RSV(RAV-0) on Ringnecked Pheasant Cells RSV(RPV) was also isolated by plating a high dilution of RSV(RAV-0) on ringnecked pheasant fibroblasts, picking cells from individual transformed foci and cultivating these cells for several weeks. Such
cells were expected to release infectious virus only if they were accidentally coinfected with RAV-0 present in the inoculum, or if the pheasant cells supplied endogenous helper activity to RSV. These two possibilities could be distinguished by the host range of the resulting pseudotype. Table 5 shows the focus forming titer of single foci 23 days after isolation. At least three foci (Nos. 1-3) produced virus which plated efficiently on C/BE, C/E and Japanese quail cells, similar to RSV(RPV). Interference and neutralization tests detailed below showed these pseudotypes from single foci to be indistinguishable from RSV(RPV). One of the single foci, however, (No. 10) released subgroup E virus, indicating that it had been doubly infected with RSV and RAV-0. The low titer-virus present in the remaining foci was not characterized. These observations provide additional independent evidence for the presence of endogenous RNA tumor virus genes in ring-necked pheasant cells. Since the technique of single focus isolation used neither R( -)Q cells nor Sendai virus, these auxiliary viral agents can be excluded as possible sources of RPV.
564
FUJITA TABLE
5
VIRUS WITH EXPANDED HOST RANGE ISOLATED FROM SINGLE FOCI PRODUCED BY RSV(RAV-0) OK RING-NECKED PHEASANT CELLS Isolated focus
Titer (FFU/ml)” Japanese quail
Chicken
C/BE
Chicken
C/E
1
1.7 x 103
1.6 x 103
1.7 x 103
2 3 4
8.0 x lo3
5.5 x 103
6.0 x IO3
1.3 x 102
1.7 x 102
10 40 0
30
1.3 x 102 > 10 20
NTD NT
NT NT NT 0
5
6 7 8 9 10
30 >lO’
10
0
“Tested on day 23 after isolation b Not tested.
0 40 40 10 10 of the focus.
Isolation of Leukosis Viruses From RSV(RPV) and RSV(GPV) Stocks The infectious pseudotypes of RSV isolated from pheasant cells presumably contained induced pheasant leukosis viruses. Inoculation of the pseudotype stocks into chfchicken cells resulted in a large increase of focus forming titer which suggested the presence of helper virus in the pseudotype stocks. These helper viruses, RPV and GPV, were isolated from the pseudotypes by endpoint interference (Rubin and Vogt, 1962). The RSV(RPV) stock used in this experiment was obtained from a single focus of BH RSV in gs+ ring-necked pheasant cells, and the RSV(GPV) stock was obtained after Sendai virus induced fusion of golden pheasant and R( -)Q cells, as described in Table 3. Both pseudotypes had been passed only in ring-necked or golden pheasant cells respectively. Closely spaced dilutions of RSV(RPV) or RSV(GPV) extending over the endpoint of focus formation were inoculated into chf- C/BE chicken cells. After 3-8 transfers the cells were challenged with RSV(RPV), RSV(GPV) and unrelated control viruses and overlaid with agar. Unchallenged plates served as controls which indicated the endpoint of focus forming activity. The results of such an endpoint
ET AL.
interference test are compiled in Table 6. Specific interference was observed with the homologous challenge virus in cultures which had received dilutions of RSV(RPV) or RSV(GPV) beyond the endpoint of transformation. The interfering viruses RPV and GPV were obtained from the supernatant medium of the corresponding unchallenged control cultures and were purified by an additional dilution series in gs- chf- chicken cells.
Host Ranges of RSV(RPV) and RSV(GPV) The host ranges of RSV(RPV) and RSV(GPV) were determined by performing focus assays on several types of avian fibroblasts as shown in Table 7. Both RSV(RPV) and RSV(GPV) are capable of forming foci with a high efficiency on avian cells which are resistant to viruses of subgroups A, B, C, D and E. The high plating TABLE
6
ISOLATION OF RPV AND GPV FROM RSV PSEUDOTYPES BY ENDPOINT INTERFERENCES Dilution of pseudotype
Foci formed by challenge virus RSV(RPV)
RSV(RPV1 1: 3,200 1: 6,400 1: 12,800 1: 25,600 uninfected
RSV(GPV)
83
14
17 0
182
0
366
60
0
296
89
0
474
71
0
PR-A RSV(GPV) 1: 100 1:300 1:900
NO
challenge
RSV(GPV)
No challenge
30
34 1
0
1:2,700
45 62 70
%b
uninfected
96
356 400 488
0 0 0
’ C/BE, gs ~ chf- chicken cells were infected with RSV(RPV) or RSV(GPV1 at the dilutions listed in the table, passaged as described in Materials and Methods, and challenged at the eighth passage. ‘The end point of transformation was 1: 300, but in the course of several transfers foci disappeared from the unchallenged plates, presumably because of overgrowth of leukosis virus.
RNA TUMOR
VIRUSES TABLE
,565
OF PHEASANTS 7
RESISTANCE AND SUSCEITIBILITV OF VARIOUS AVIAN CELLS TO SUBGROUPF AND G PSEUDOTYPESOF BH RSV Virus
Cell type Species
-
Chicken CIE CIA CIAB C/BE c/c Japanese quail Ring-necked pheasant Golden pheasant Peking duck Goose
Viral subgroups excluded
E A A,B,D,E B,D,E C B,D B, D None A,B,D.E,G A,B,D,E,F,G
PR-A
S” R” R s S s S s R R
PR-B
PR-C
RSVt GPV)
S S R R S R R S R R
S s s S R SR” SR S S S
s s s S s S s SR” R R
RSV(RPV)
s S s s s S S S S R
OS. focus forming efficiency greater than 0.25 relative to type C/E chicken cells; R, focusefficiency less than 0.01 relative to type C/E chicken cells; SR. focus forming efficiency variable from 0.01 to 0.25 relative to type C/E chicken cells. This variability may reflect genetic or physiological differences between embryos.
efficiency on Japanese quail cells (EOP quail/chicken = 1) is also unlike that of subgroup C viruses which show reduced plating on Japanese quail cells (Duff and Vogt, 1969). The data on RSV(RPV) are in agreement with earlier observations which have established that the endogenous helper virus from the ring-necked pheasant belongs to a new subgroup, F, of the avian leukosis and sarcoma complex (Hanafusa and Hanafusa, 1973). The RSV(RPV) described in this report will therefore be included in subgroup F. This classification is also supported by interference tests described below. The host range of RSV(GPV) differed from that of RSV(RPV). RSV(RPV) plated on duck cells with an efficiency of 0.4 relative to chicken cells, whereas RSV(GPV) did not form foci on duck cells (EOP relative to chicken cells =
RSV(RPV) and RSV(GPV) were found to require polycations for effective adsorption to the host cell. In the absence of polybrene (2 pg/ml) plating efficiencies were reduced 50 to 200-fold. Because of the wide host range of RSV(RPV) and RSV(GPV), both viruses were tested for their ability to transform continuous cell lines of mammalian cells. Although these data are still preliminary, it appears that the transforming ability of subgroups F and G for Balb-C mouse 3T3 and normal rat kidney cells are not increased over those of subgroups C and D. Interference Patterns RSV(GPV)
of RSV(RPV)
and
Chicken embryo (C/E, gs-) were infected with avian leukosis viruses of subgroup A through D and subgroup F as well as with RPV or GPV. Ring-necked pheasant fibroblasts were infected with ILV of subgroup E. Interference by RPV and GPV was also tested by inoculation of quail embryo fibroblasts. Infected cultures were transferred at 2-5 day intervals, and at the first passage (cultures infected with leukosis viruses of subgroups A to D) or 3rd to 8th passage (cultures infected with RAV-0, RAV-61, RPV or GPV) the cells
566
FUJITA
ET AL.
were challenged with Rous sarcoma viruses of subgroups A through E and with RSV(RPV) or RSV(GPV). The results are presented in Table 8. Leukosis viruses of subgroups A through E failed to interfere with either RSV(RPV) or RSV(GPV), although they interfered with sarcoma viruses of homologous subgroups. RPV and GPV interfered with their respective pseudotypes but not with each other nor with sarcoma viruses of subgroups A to D. RPV did interfere, however, with a subgroup E challenge in quail cultures; but this interference was nonreciprocal as ILV failed to cause interference with subgroup F virus. No interference was seen between subgroups G and E. Occasionally RPV induced significant interference with subgroup A, but this observation was not consistently reproducible. RAV-61, a member of subgroup F, interfered with RSV(RPV) but not with RSV(GPV). These observations suggest that RPV belongs to subgroup F previously described by Hanafusa and Hanafusa (19X3), and that GPV belongs to a new subgroup, G.
Antigenic Properties RSV(GPV) Neutralization
RSV(RPW
and
tests were carried
out
of
with RSV(RPV) and RSV(GPV) using eight chicken sera with neutralizing activity against subgroups A, B, C or D. None of these sera neutralized either RSV(RPV) or RSV(GPV) to a significant extent, although they showed neutralizing activity against their homologous viruses. The results for 5 sera are summarized in Table 9. These data support the conclusion that RSV(RPV) and RSV(GPV) have envelope antigens distinct from those of subgroups A, B, C or D. In Table 10 neutralization results with antisubgroup E sera and with normal pheasant sera are compiled. Several sera from ring-necked pheasants neutralized RSV(RPV) but did not neutralize RSV(GPV). This observation supports the conclusion that RSV(RPV) and RSV(GPV) are not closely related with respect to their envelope antigens. The neutralizing activity of pheasant sera for RSV(RPV) was not correlated with neutralization of RSV(RAV-0), a subgroup E virus. A rabbit antiserum prepared against RAV-0 also failed to neutralize RSV(RPV) or RSV(GPV). We conclude that the new pheasant viruses are antigenically distinct from subgroup E. One out of 20 sera from nonimmunized ring-necked pheasants
TABLE CLASSIFICATION
Cell Species
OF
RPV
AND
8
GPV
HY VIKAL
Challenge virus
Interfering virus” Resistant to
Chicken C/E
E
Ring-necked pheasant dapanese quail
B, D B.D
none RAV-I (A) RAV-2 (B) tdEE7(C) CZAV (D) RPV (F) GPV (G) RAV-61 (F) none ILV (El none RPV (F) GPV (G)
INTERFERENCE
PR-A
PR-B
PR-C
SR-D
RSV (RAW))
RSV (RPV)
RSV (GPV,
S” R S s s s S S NT NT NT NT Xl’
s s H S R s s Nl N-1 NT NT NT IN?
s 5 s R s s s NT NT NT Iv.1 Nl N’l
NT’ NT NT Nl R s s NT Nl NT NT NT N’I
NT NT NT NT Nl N-l NT NT s R s R s
S s S s s R s R s s s R s
S s s s s s R s s s s s R
’ Viral subgroup in parenthesis. OS. EOP of challenge \zirus 0.2-1.0; R, EOP of’ challenge virus ~0.01 relative ‘NT. not tested.
to control cells.
RNA TUMOR
VIRUSES TABLE
FAILURE
9
AGAINST SUBGROUPS A-D
OF ANTISERA
567
OF PHEASANTS
TO NEUTRALIZE
RSV(RPV)
OR RSV(GPV)”
Number of foci subgroup and virus Serum specificity and number
C RSV(AV-11
Control, no serum Anti-A, 520 Anti-A*, pool Anti-C, 160 Anti-CD, 580 Anti-D, 265
RSV(&V-21
PR-C
BSI
SRDD
F RSV(RPV1
171
41
59
125
49
83’
77
0 3 NT NT NT
NT” 4 NT NT NT
NT NT 1 I NT
NT NT 2 NT NT
NT NT NT 0 5
;:h? 744 642 786 582
90 12 18 96 1’,5 UC
“Virus was incubated on C/E cells. b NT. not tested.
with a 10-l dilution
of antiserum
TABLE IMMUNOLOGICAL
Source of serum
DIFFERENTIATION
Serum number
for 40 min in a 37” waterbath.
Ring-necked pheasant
887 635 621 634 32171
Rabbit
showed neutralizing activity against subgroup F, perhaps indicating that ringnecked pheasants can be horizontally infected and are not immunologically tolerant to RPV.
of
RSV(RPV)
and
Throughout the course of this investigation we have assumed that RSV(RPV) and RSV(GPV) were defective pseudotypes of BH RSV with envelope antigens specified by RPV and GPV respectively. This interpretation is supported by results of infective center assays in which focus formation was dependent on the ability of RSV to produce infectious progeny in solitary infection (Weiss et al., 1973). In this experiment avian fibroblasts were infected with RSV(RPV) or RSV(GPV), and after one day the cells were treated with 2 pug of mitomycin C for 16 hr. The mitomycin C was then removed by washing, and an
were plated
AND RSV’(GPV1
Percent surviving
Immunizing antigen RSV(RAV-01 RSV(RAV-01 RSV(RAV-0) none RAV-O
Survivors
10
BETWEEN RSV(RPV)
RSV(GPV)
Defectiveness RSV(GPV)
G RSV(GPV1
74 120 120 126 67
RSV(RPV) 2.8 9 91 12 109
foci RSV(RAV-0) 0.2 9 14 102
additional cell layer was seeded on the cultures. The mitomycin C-treated cells will initiate foci in the secondary layers only if they release infectious virus but will not divide and grow into foci themselves. Under these conditions focus formation by RSV(RPV) or RSV(GPV) was reduced to less than 0.5%, in contrast to a nondefective control virus, PR-A, which produced foci equally well in direct assays and in infectious center tests (Table 11). These observations indicate that RSV(RPV) and RSV(GPV) are indeed defective viruses.
Probable Recombination with Host Range Markers of RSV(RPV) and RSV(GPV) Chicken C/E fibroblasts were mixedly infected with PR-B and RSV(RPV), and from the mixed harvests recombinants capable of infecting C/BE cells were isolated and cloned by the infectious center technique of Weiss et al. (1973). Several such recombinants of PR-B and RPV were char-
568
FIJJITA
acterized. They showed the host range of RPV, but in contrast to RSV(RPV) they were able to induce foci under conditions requiring release of infectious virus (Table 12) and appeared therefore to contain nondefective virus. This observation is in itself insufficient proof for recombination, because a pseudotype with RPV may give similar results if RPV was in great excess; but the presumptive recombinants listed in Table 12 had been cloned several times in TABLE
11
DEFECTIVENESSOF RSV(GPV) Virus
AND RSV(RP\‘,
Number of foci Direct assays
PR-A RSVCGPV) RSV(RPV)
Infectious
.____ centers”
C/E
C/HE
I”
II
85 446 358
86 280 291
78 1 0
80 0 0
“In the infectious center assay cultures with “initiator cells” are infected with 300-500 FFI1. Twelve to eighteen hours post infection the cells are treated with 2 &ml of Mitomycin C overnight. The drug is then removed, the cells are washed, and a second cell layer (“indicator cells”) is seeded, following which the cultures are overlaid. Focus formation occurs only if the initiators release virus which can infect the indicators as shown by LVeiss et al., 193 ‘I. C/E initiators. C/E indicators: II. C/BE initiators. Japanese quail indicators.
E7’ AL.
the infectious center assay at low multiplicities, making this second explanation unlikely. Presumptive recombinants have also been isolated from mixed infections of PR-B and RSV(GPV) but still require characterization. For these experiments pure pheasant leukosis viruses were not yet available; therefore the mixed infections were carried out with pseudotypes. Since the BH RSV component of these pseudotypes does not participate in host range recombination (Kawai and Hanafusa, 1972; Weiss et al., 19731, it seemed justifiable to assume that only the pheasant leukosis viruses interact with PR-B. The data of Table 12 suggest that endogenous pheasant leukosis viruses are capable of host range recombination with PR-B.
Co-cultivation or fusion of RSV( ) producing quail cells with normal fibroblasts of ring-necked or golden pheasants led to the appearance of infectious RSV pseudotypes. Such pseudotypes were also obtained by direct infection of ring-necked pheasant cells with RSV(RAV-0) at low multiplicities. Normal pheasant cells provide a helper function for the defective RSV. This helper function appears to reflect the presence of leukosis virus genetic information in pheasants. The pseudotypes obtained from various ring-necked pheas-
NONDEPECTILERECOMRI>AANTS RET)YEENHOST RANGE MAKKEKS OF RI’L AND ‘THE‘I‘KANSFOKMATIO\IMAKKEKS OF PR-I3 Number of FFL;
Virus
Direct assays __~___ Ui Expt 1 PR-B PR: RPV PR: RPV PR: RPV Expt 2 PR-A PR: RPV PR: RPV 0 See footnote”
20 I
(‘/HE
Infectious centers” with C/HE initiators and indicators
clone No. 1 clone No. 2 clone No. 3
0 1’ 50 “07
0 ti 11 50
clone No. 2 clone No. 7
:i:iti :i0 :100
277 15 :w;
of Table I I.
RNA TIblOR
VIRI.SES
ant embryos are all related to each other and to RAV-61 of subgroup F. This observation and the occasional detection of subgroup F antibody in pheasant sera confirm the widespread natural occurrence of this avian leukosis subgroup in pheasants as first described by Hanafusa and Hanafusa (1973). The RSV pseudotypes produced with endogenous viral information of several golden pheasant embryos are also related to each other, but distinct from all other avian leukosis and sarcoma virus subgroups in their host range, susceptibility to interference, and envelope antigens. These viruses have been designated subgroup G. Subgroups F and G do not cross interfere with each other, and antisera neutralizing one but not the other have been found. Although both subgroups appear unable to infect goose cells, duck cells are selectively resistant against subgroup G. In chicken cells the endogenous helper activity for RSV( -) is correlated with the presence of gs viral antigens in the cells (Weiss, 1969; H. Hanafusa et al., 1970; Vogt et al., 1973; Chen and Hanafusa, 1974). In contrast to chicken cells, there was no clear-cut correlation between presence of gs antigen in pheasant cells and helper activity for RSV( -). Similar observations have also been made by Hanafusa and Hanafusa (1973). Lack of helper activity in gs+ cells could be explained if only a partial viral genome, not including genetic information for envelope proteins, was present in some pheasant embryos, or if the production of envelope components could not always be activated by the superinfection of RSV( --). Analogous explanations may be offered for gs - chf + pheasant cells, but here it is also quite likely that the relatively insensitive complement fixation test used in our studies for the detection of gs antigens may have been unsuitable for low concentrations of this viral gene product. Endpoint interference tests have revealed the presence of independently replicating helper leukosis viruses in stocks of subgroup F and G BH RSV pseudotypes. These leukosis viruses grow well in a vari-
OF PHEASANTS
569
ety of avian cells. Since they are derived from a BH RSV pseudotype, the possibility exists that some of their genetic information has been acquired from BH RSV. Although BH RSV is not thought to recombine in the host range marker (Kawai and Hanafusa, 1972; Weiss et al., 1973), observations have been reported which suggest that recombination for other markers may occur (Hanafusa, 1970). Support for the suggestion that RPV and GPV may contain RSV genetic information comes from the observation by Kang and Temin (1973) that the RNA of RAV-61, which appears very closely related to RPV, is almost entirely homologous to the genome of chicken derived leukosis and sarcoma viruses. Yet Neiman (1973) using such a chicken derived virus (RAV-0) as a probe to detect endogenous viral sequences in normal chicken and ring-necked pheasant cells found that in contrast to the chicken, which shows complete genomic representation of RAV-0, gs+ or g’s- ring-necked pheasant cells carry sequences that are homologous to only a small portion of the RAV-0 genome. These data could be interpreted to mean that either the pheasant embryos studied by Neiman contain only a portion of an integrated viral genome, or they contain a complete genome but on11 part of this is homologous to RAV-0. It will now be of interest to use nucleic acid probes prepared from subgroup F and G viruses to search for endogenous sequences in pheasant cells. No reliable information is available on the incidence of leukosis in the pheasant flocks from which we obtained fertile eggs. However, preliminary experiments suggest that RPV is able to cause lymphoid leukosis in chickens (Dr. Graham Purchase. 1973, private communication). More experimentation on the possible leukemogenie activity of subgroups F and G is clearly required.
The authors thank William
S. Mason for helpful
suggestions.Martin C. Alevy for goose and duck cells. and Joseph Green, Hsiao-Ching and Barbara Miles for technical
Pang. Annie Chung assistance.
570
FUJITA
REFERENCES BALUDA, M. A. (1972). Widespread presence, in chickens, of DNA complementary to the RNA genome of avian leukosis viruses. Proc. Nat. Acad. Sci. USA 69, 576-580. BISHOP, J. M., JACKSON, N., QUINTRELI, N., and VARMUS, H. E. (1973). Transcription of RNA tumor virus genes in normal and infected cells. In “Possible Episomes in Eukaryotes,” (L. G. Silvestri, ed.), North Holland, Amsterdam, pp. 61-73. CHEN, J. H. and HANAFUSA, H. (1974). Detection of protein of avian leukovirus in uninfected chick cells by radioimmunoassay. J. Viral. 13, 340-346. CRITTENDEN, L. B., WENDEI, E. J.. and MOTTA, J. (1973). The interaction of genes controlling resistance to RSV(RAV-0). Virolog.v 52, 373-384. CR~TTENDEN, L. B., SMITII, E. J., WEISS, R. A., and SARMA, P. S. (1974). Host gene control of endogenous avian leukosis virus production. Virology 57, 128138. DUESBERG,P. H. and VOGT, P. K. (1973). RNA species obtained from clonal lines of avian sarcoma and from avian leukosis virus. Virology 54, 2077219. DUFF, R. G. and VOGT. P. K. (1969). Characteristics of two new avian tumor virus subgroups. Virology 39, 1830. FRIIS, R. R. (1972). Abortive infection of Japanese quail cells with avian sarcoma viruses. Virology 50, 701-712. HANAFUSA, H. (1970). Virus production by Rous sarcoma cells. Cur. Top. Microbial. Immunol. 51, 114-123. HANAFUSA, H., HANAFUSA. T., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 49, 572-580. HANAFUSA, H., MIYAMOTO. T.. and HANAFUS~ T. (1970). A cell-associated factor essential for formation of an infectious form of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 66, 314-321. HANAFUSA, T. and HANAFUSA, H. (1973). Isolation of Ieukosis-type virus from pheasant embryo cells: Possible presence of viral genes in cells. Virology 51, 247-251. HANAFUSA. T., HANAFUSA, H., and MIYAMOTO, T. (1970). Recovery of a new virus from apparently normal chick cells by infection with avian tumor viruses. Proc. Nat. Acad. Sci. USA 67, 179771803. HANAFUSA, T., HANAFUSA, H., MIYAMOTO, T.. and FLEISSNER, E. (1972). Existence and expression of tumor virus genes in chick embryo cells. Proc. Nat. Acad. Sci. USA 66, 314-321. HAYWARD, W. S. and HANAFUSA, H. (1973). Detection of avian tumor virus RNA in uninfected chick embryo cells. J. Virol. 11, 15-167. KANG, C. and Temin, H. M. (1973). Sequence homoogy among RNAs of avian leukosis-sarcoma viruses,
ET AL. reticuloendotheliosis viruses, and chicken endogenous RNA-directed DNA polymerase activity. J. Viral. 12, 1314-1324. KAWAI, S. and HANAFUSA, H. (1972). Genetic recombination with avian tumor virus. Virology 49, 37-44. NEIMAN, P. E. (1973). Measurement of endogenous leukosis virus nucleotide sequences in the DNA of normal avian embryos by RNA-DNA hybridization. Virology 53, 196-204. PAYNE, I,. N. and CHUBB, R. (1968). Studies on the nature and genetic control of an antigen in normal chick embryos which reacts in the COFAL test. J. Gen. Viral. 3, 379-391. ROSENTHAL, P. N., ROBINSON, H. L., ROBINSON, W. S., HANAFUSA, T., and HANAFUSA, H. (1971). DNA in uninfected and virus-infected cells complementary to avian tumor virus RNA. Proc. Nat. Acad. Sci. USA 68, 2336-2340. RUBIN, H. and VOGT, P. K. (1962). An avian Ieukosis virus associated with stocks of Rous sarcoma virus. Virology 17, 1844194. SCHEELE, C. M. and HANAFUS~ H. (1971). Proteins of helper-dependent RSV. Virology 45, 401-410. TOYOSHIMA, K., FRIIS, R. R., and VOGT, P. K. (1970). The reproductive and cell-transforming capacities of avian sarcoma virus B77: Inactivation with UV light. Virology 42, 163170. TOYOSHIMA, K. and VOGT, P. K. (1969). Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 38, 414-426. VARMUS. H. E., WEISS, R. A., FRIIS, R. R., LEVINSON, W., and BISHOP, J. M. (1972). Detection of avian tumor virus-specific nucleotide sequences in avian cells DNA. Proc. Nat. Acad. Sci. USA 69, 20-24. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology,” (K. Habel and N. P. Salzman, eds.). Academic Press, New York, pp. 198211. VOGT, P. K. and FKIIS, R. R. (1971). An avian leukosis virus related to RSV(0): Properties and evidence for helper activity. Virology 43, 223-234. VOGT. P. K. and HARRIS, P. (1974). Use of plastic bags to maintain a humidified atmosphere for animal cell cultures, Appl. Microbial. 27, 618-619. VOGT. P. K., FRIIS. R. R., and WEISS, R. A. (1973). Cell genetics and growth of endogenous viruses. Proc. 7th Nat/. Cancer Conf. pp. 55X9. WEISS, R. A. (1969). The host range of Bryan strain Rous sarcoma virus synthesized in the absence of helper virus. J. Gen. Viral. 5, 511-528. WEISS. R. A. ( 1974). Ecological genetics of RNA tumor viruses and their hosts. In “Analytic and Experimental Epidemiology of Cancer,” (T. Hirayama, ed.), University of Tokyo Press, Tokyo, in press. WEISS, R. A., FRIIS, R. R., KATZ, E., and VOGT, P. K. (1971). Induction of avian tumor viruses in normal
RNA TUMOR
VIRUSES
cells by physical and chemical carcinogens. Viralogy 46, 920-928. WEISS, R. A., MASON, W. S., and VOGT, P. K. (19’73). Genetic recombination and heterozygotes derived from endogenous and exogenous avian RNA tumor viruses. Virology 52, 535-552. WEISS, R. A. and PAYNE, L. N. (1971). The heritable
OF PHEASANTS
571
nature of the factor in chicken cells which acts as a helper virus for Rous sarcoma virus. Virology 45, 508-515. YOSHIKAWA-FUKUDA, M. and EBERT, J. D. (1969). Hybridization of RNA from Rous sarcoma virus with cellular and viral DNA’s Proc. Nat. Acad. Sci. USA 64, 870-877.
60, 558-571
(1974)
RNA Tumor Viruses
Of Pheasants:
Leukosis DONALD
J. FUJITA,2
Subgroups
Characterization
of Avian
F and G ’
YOUNG C. CHEN, ROBERT PETER K. VOGT
R. FRIIS,3
AND
Department of Microb[ology. C’niversity of Southern California, School of Medicine, Los Angeles. California 900.3'3 Accepted
April 26. 1974
Endogenous leukosis-like viruses of ring-necked pheasants (Phasianus cokhicus) and golden pheasants (Chrysolophus pictus) have been isolated and characterized. The majority of the normal pheasant embryo cultures contain helper activity for the defective Bryan high titer strain of Rous sarcoma virus. The Rous sarcoma pseudotypes produced with endogenous helper activity from ring-necked pheasants belong to subgroup F. The pseudotypes from golden pheasant cells constitute subgroup G. Subgroup F and G pseudotypes can infect all known genetic types of chicken fibroblasts as well as pheasant and Japanese quail cells, but do not plate on goose cells. Duck cells are resistant to subgroup G but not to F. The subgroup F and G helper viruses isolated from Rous sarcoma viral pseudotypes show interference with their homologous subgroup. RAV-61, a standard of subgroup F, interferes with pseudotypes produced with endogenous helper activity from ring-necked pheasant cells but not with subgroup G pseudotypes. Subgroups F and G do not cross-react with subgroup A to E in neutralization tests. Some normal ring-necked pheasant sera have anti-F activity. Subgroup F and probably also G leukosis-like viruses can undergo genetic recombination with nondefective avian sarcoma viruses. INTRODUCTION
The existence of viral genetic information in normal avian cells has been documented through several different lines of evidence. All normal chicken embryos and embryo tissue cultures tested to date contain DNA sequences homologous to avian leukosis viruses (Rosenthal et al., 1971; Varmus et al., 1972; Baluda, 1972; Yoshikawa-Fukuda and Ebert, 1969; Neiman, 1973). In some embryos viral gene products ‘Supported by U.S. Public Health Service Grant No. CA 13213 and Contract No. NOl-CP-43242 from the National Cancer Institute. 2Fellow of the Leukemia Society of America. Present address: Department of Microbiology, University of California Medical School, San Francisco, California 94143. 3Present address: Robert Koch Institut, Nordufer 20, 1 Berlin, Germany.
such as group specific (gs) antigen or “chicken helper factor” (chf) are synthesized, and their intracellular levels seem to be at least in part regulated by genetically determined patterns of transcriptional control (Weiss, 1969; H. Hanafusa et al., 1970; Payne and Chubb, 1968; T. Hanafusa et al., 1970; Weiss and Payne, 1971; Hayward and Hanafusa, 1973; Bishop et al., 1973). Endogenous avian RNA tumor viruses are released spontaneously from the cells of certain chicken lines (Vogt and Friis, 1971; Crittenden et al., 1973; Crittenden et al., 1974). They can be activated by infection of the cell with RNA tumor viruses (T. Hanafusa et al., 1970), and they can be induced by treatment of normal fibroblasts with physical or chemical carcinogens or mutagens (Weiss et al., 1971). All endogenous chicken viruses, (with one possible excep-
RNA TUMOR
VIRUSES
tion) possess envelope specificities characteristic of the avian tumor virus subgroup E (Weiss, 1974). Recently normal ring-necked pheasant (Phasianus colchicus) cells have also been shown to harbor viral genetic information, and a leukosis virus with the envelope specificity and host range of a new avian tumor virus subgroup, F, has been isolated from such cells (Hanafusa and Hanafusa, 1973). The possible presence of endogenous virus in ring-necked pheasant cells had also been suggested by Weiss et al. (1971), who noted that in some cases leukosis viruses induced in normal chicken cells acquired a new host range after passage in ring-necked pheasant cells. In this report we describe the isolation of leukosis viruses from cells derived from another genus of the pheasant family, the golden pheasant (Chrysolophus pictus) and confirm the occurrence of subgroup F viruses in ring-necked pheasant cells. Host range, viral interference, and antiserum neutralization indicate that the endogenous golden pheasant virus (GPV) has envelope characteristics of a new avian tumor virus subgroup, G, which is distinct from previously described subgroups A through F. MATERIALS
AND
METHODS
Cells Preparation of primary and secondary cultures from avian embryos and formulation of growth media followed published techniques (Vogt, 1969). Fertile eggs of white leghorn chickens, phenotypes C/E and C/BE, were generously supplied by Mr. R. Raymond, H & N Farms, Redmond, Washington. Chicken eggs of the C/A and C/AB phenotypes came from line 7 x line 15 crosses originally supplied by the U.S.D.A. Regional Poultry Laboratory, East Lansing, Michigan. Eggs of ringnecked pheasant (Phasianus colchicus torquatus) and of the golden pheasant (Chrysolophus pictus) were purchased from B & B Mini Farms, Eureka, California and Marsh Farms, Garden Grove, California. Duck (Anas platyrhynchos) and
OF PHEASANTS
559
goose (Anser anser) eggs came from Ward Duck Farm, La Puente, California. Japanese quail eggs were supplied by Life Sciences Inc., St. Petersburg, Florida under contract with the National Cancer Institute. Infections were carried out in the presence of 2 pg/ml polybrene (Toyoshima and Vogt, 1969). Long term cultures were kept in “primary growth medium” (Vogt, 1969) containing 8% calf serum, 2% inactivated chicken serum and 1% dimethylsulfoxide (DMSO). DMSO (1%) was also present in agar overlays, and overlays for pheasant, quail, goose and duck cultures also contained 1% inactivated chicken serum. The presence of avian tumor virus group specific (gs) antigens and of chicken helper factor (chf) in normal primary cultures was checked as described previously (Vogt and Friis, 1971; Weiss et al., 1973). R(&)Q (line No. 3) is an established Japanese quail cell line which releases defective, noninfectious Bryan high titer Rous sarcoma virus referred to as RSV( ~ ). The origin of this cell line has been described (Friis, 1972). All cell cultures were incubated in polyethylene bags as described by Vogt and Harris (1974). Viruses The nondefective sarcoma viruses and the leukosis viruses had been cloned in chf negative chicken cells, except for RAV-0 which was derived from gs+ chf+ cells. A survey of nondefective sarcoma and of leukosis viruses used in this study is given in Table 1. RAV-61 was a generous gift from Dr. H. Hanafusa. Pseudotypes of the Bryan high titer strain of RSV are notated conventionally with the helper virus in parenthesis. Their origin has been detailed in previous publications (Duff and Vogt, 1969; Toyoshima et al., 1970; Weiss et al., 1971). Sendai-Virus Induced Cell Fusion and Cocultiuation of Cells Chicken, ring-necked pheasant, golden pheasant cells were trypsinized,
or and
560
FUJITA
TABLE 1 AVIAN LEUKOSIS AND SARCOMA VIRUSES USED IN THIS STUDY
Virus
Nondefective sarcoma viruses: Prague strain RSV Prague strain RSV Prague strain RSV Avian sarcoma virus BY7 Schmidt Ruppin RSV Leukosis viruses: Rous associated virus 1 Row associated virus 2 Row associated virus 0 Row associated virus 50 Rous associated virus 61 Transformation defective B77 Carr Zilher associated virus Induced leukosis virus
Subgroup
Abbreviation
A B C C D
PR-A PR-B PR-C B57 SR-I)
A B E D F C D E
RAV- 1 RAV-2 RAW) RAV-50 RAV-61
tdE377 CZAV IL\
ET AL.
ate leukosis virus of one of the subgroups A-D. After 3 days in culture at 37”, cells were passed and challenged with sarcoma viruses of various subgroups. (ii) Cells were infected with leukosis virus at a low multiplicity and passaged between three and eight times at 2-5 day intervals before challenge with a sarcoma virus. At alternate passages, an equal number of fresh, uninfected cells was added to the culture in the presence of 2 pg/ml polybrene. Chicken C/E or C/BE chf- cells were used in all interference tests except those involving subgroup E viruses, which were performed on ring-necked pheasant or quail cells. Growth of Transformed Foci
Cells from Single
RSV’(O) was plated at high dilution on us+ ring-necked pheasant cells, overlaid and incubated at 37” for 10 days. Single foci were aspirated with a capillary pipette 1 x lo6 cells were mixed with 5 x lo5 from plates containing l-10 foci, and were R( -)Q cells in test tubes in a total volume seeded individually in cloning medium of 0.2 ml primary growth medium. Approximately 800 hemagglutinating units of @- (Duesberg and Vogt, 1973) in 35 mm plates. Supernatant medium was assayed propiolactone-inactivated Sendai virus periodically in focus assays on quail and were added, and mixtures were incubated with occasional agitation for 30 min at 4”, chicken C/BE or C/E cells. followed by a 20 min incubation at 37”. by Antiserum After incubation 4 ml primary growth me- Virus Neutralization Virus neutralization by antiserum foldium were added, and the suspended cells lowed published techniques (Duff and were centrifuged at 1,000 rpm for 5 min. The pellet was resuspended in 4.5 ml Vogt, 1969). A 10-l dilution of heat inactigrowth medium, plated on 60 mm dishes, vated serum was incubated with virus, and survivors were assayed after 40 min at 37 ‘. and incubated at 37” in primary growth medium with 1% DMSO. At 2-3 day intervals, about one-half of the medium in each Infective Center Assays plate was removed and replaced with fresh Infective center assays were performed medium. Cells were transferred at 4-8 day as described by Weiss et al. (1973). Initiaintervals. Medium was harvested perioditor cells were seeded in a 60 mm dish and cally, frozen and thawed, sonicated and infected with 100-500 FFU virus in the tested for infectious focus-forming virus by presence of 2 pg/ml polybrene. After 18-24 plating on quail cells and on chicken C/E hr incubation at 37”, 2 pg/ml mitomycin C or C/BE cells. was added, and cells were incubated for an The same procedure was followed in the additional 14-20 hr. The mitomycin C was co-cultivation method, except that Sendai removed, and cells were washed with 5 ml virus was omitted. of medium three times over a period of 3 hr. Indicator cells (8 > 105/plate) were then Interference Tests added in the presence of 2 pglml polybrene. Interference tests were performed in one Cells were overlaid with agar after an of two ways: (i) Cells were infected at high additional 12-24 hr. Control cultures consisted of direct focus assays in the absence multiplicity (m.o.i. = 1) with an appropri-
RNA TLIMOR
VIRUSES
of mitomycin C which were overlaid at 18-24 hr after infection. Focus counts were made 7-10 days after infection. RESULTS
Helper Activity for Bryan High Titer RSV in Normal Pheasant Fibroblasts The Bryan high titer strain of RSV (BH RSV) is defective in an envelope component (Scheele and Hanafusa, 1971). This component can be supplied by a helper virus or by a helper factor endogenous to the host cell (Hanafusa et al., 1963; Weiss, 1969; H. Hanafusa et al., 1970). BH RSV grown in the absence of helper agents is not infectious and is referred to as RSV( ~~). BH RSV grown in the presence of helper is infectious but remains genetically defective. It is called a pseudotype and has envelope properties identical to those of the helper agent. This defectiveness makes BH RSV a sensitive tool for the detection of complementing endogenous leukosis viruses. Recently it has been shown that endogenous viral genes present in ringnecked pheasant cells can complement BH RSV leading to the synthesis of infectious sarcoma virus. This new pseudotype is referred to as BH RSV (RAV-61) and represents a new subgroup (F) of the avian leukosis and sarcoma complex (Hanafusa and Hanafusa, 1973). The leukosis virus. RAV-61. which is responsible for the endogenous helper activity in normal ringnecked pheasant fibroblasts has also been isolated (Hanafusa and Hanafusa. 1973). These results may clarify previous observations that induced leukosis viruses of chickens sometimes acquire a new host range after passage in ring-necked pheasant cells (Weiss et al., 1971). We have used BH RSV transformed fibroblasts of the Japanese quail in our attempts to detect endogenous viral gene expression in cells derived from ringnecked pheasant and golden pheasant embryos. These quail cells, designated as R( ~-)Q, had been kept in tissue culture for over one year and released RSV( ~ ) (Friis, 1972). Two methods were used to reveal helper activity for RSV( -). In the first, R( - )Q cells and fibroblasts from individ-
OF PHEASANTS
361
ual ring-necked or golden pheasant embryos were fused with p-propiolactone inactivated Sendai virus. In the second method, R( - )Q cells were cocultivated with normal fibroblasts of ring-necked or golden pheasant embryos in the absence of a fusion enhancing agent. Table 2 summarizes the control experiments with chicken cells, which were chf, gs+ or chf- gs-. Both Sendai virusinduced fusion and co-cultivation with R( ~-)Q cells resulted in the production of infectious subgroup E pseudotypes of BH RSV, if the chicken cells were chf+ gsr. No infectious virus was detected at day 12 in cultures of R( ~ )Q cells fused or co-cultivated with chf- gs- chicken cells. However, small numbers of infectious virus were demonstrable in some of these cultures by day 23. This virus is probably the result of spontaneous activation of chf. which is known to occur occasionally in chf - cells during extended passage (Hanafusa et al., 1972). (In one supernatant sample of R( ~ )Q cells a few FFU were also seen; these may have been derived from live cells accidentally transferred with the inoculum. ) These control experiments establish that fusion as well as co-cultivation with R( )Q cells can detect endogenous viral gene expression. Under our experimental conditions, fusion produced about 20-40“~ heterokaryons; most of these did not survive more than 7-9 days in culture. After 7 days fused and co-cultivated populations were probably qualitatively similar. the latter showing a low level of spontaneous fusion between test and R( -)Q cells. The results with cells from golden pheasant embryos are summarized in Table 3. Although all golden pheasant embryos were gs -, 7 out of 9 showed infectious virus by day 23. Some cultures did not survive beyond this time or were discarded. On day 52 all of the remaining cultures from six embryos produced infectious virus. This pseudotype of BH RSV plated with equal efficiency on Japanese quail and C/E and C/BE chicken cells. Table 4 lists the results with cells from various ring-necked pheasant embryos. In-
562
FUJITA
ET AL.
TABLE APPEARANCE
OF INFECTIOUS
Species and embryo number
Method”
Chicken (Callus domesticus) C/BE No. ‘:3 gs + C/BE No. 24 gs -~ C/A No. 2<5gs , C/E No. 26gs c/E No. 27 gs ~ No cells added to R(
2
SUBGROUP E RSV AFTER SENDAI VIRUS INDUCED FUSION OR COCULTIVATION CHICKEN CELLS WITH R( - ) IJ~~~~~~~ QUAIL CELLS
)Q
SF cc SF CC SF cc SF cc SF cc SF CC
Titer of infectious 12de
23d
1 x 103 2 x 103 0 0 1 X lo3 2 x lo3 0 0 0 0 0 0
5 x 103 5 x 10s 0 0 3 x 103 3 k 103 25 0 10 0 0 0
OF
RSV (FFU/ml)b
-
35d
52d
1 X 10’ 1 x 10’ 0 5 5 x 103 5 x 103 0 10 NT 0 NT 25
1 X 105 NTd 0 0 1 x 105 NT 0 0 NT 0 NT 0
“SF, Sendai virus-induced cell fusion; CC, cocultivation. “FFU plating on Japanese quail cells but excluded from chicken C/E cells c Days of mixed culture between R( ~ )Q and chicken cells. dNT, not tested. TABLE
3
APPEARANCE OF INFECTIOUS VIRUS AFTER SENDAI VIRUS-INDUCED FUSION OR COCULTIVATION AND R( 1 JAPANESE QUAIL CELLS
Soecies and embryo number
Met hod”
Titer of infectious
OF GOLDEN PHEASANT
RSV (FFU/ml)b
12d’
23d
35d
52d
0 0 0 0 50
0 5
NTd NT 10 l-.5 8 x lo3 8 x lo3 NT NT 100 NT 100 150 NT NT ti0 30 5 x IO3 500
NT NT 20 20 35x 10’ 2 x 10’ N’? NT 500 NT 30 50 NT KT 5 30 i x 10’ 5 x 10’
Golden pheasant
(Chrysolophuspictus) GOP No. 6gs GOP No. i gs GOP h’o. 8gs GOPNo.
9gs
GOP No. 10 gs ~ GOP No. 11 gs GOP No. 12 gs GOPNo.
13gs-
GOP No. 14 gs
SF cc SF cc SF CC SF CC SF CC SF CC SF cc SF cc SF CC
1 ii 103
0 0 0 0 10 0 0 0 0 0 6
I- 10
“SF, Sendai virus-induced cell fusion; CC, cocultivation. *FFU plating equally well on Japanese quail and chicken C/BE. c Days of mixed culture between R( -)Q and pheasant cells. dNT. not tested.
5 0 3 x 103 5 x 103 NT 5 0 NT 5 5 0 NT 0 l-10 400 15
RNA TUMOR
VIRUSES TABLE
APPEARANCE
OF INFECTIOUS
563
OF PHEASANTS 4
VIRUS AFTER SENDAI VIRUS-INDUCED FUSION OR COCULTWATION JAPANESE QUAIL CELLS PHEASANT AND R(-)
Species and embryo number
Titer of infectious
Method”
OF RING-NECKED
RSV (FFU/ml)’ 35d
12d’
23d
0 0 0 0
0
0
0 5 0
5 30
52d
Ring-necked pheasant (Phasianus colchicus) RNPNo.
Igs-
RNP No. 2 gsRNPNo.3gsRNP No. 4gss RNPNo.
5gst
SF cc SF cc SF cc SF CC SF cc
0 0 0 0 0 0
0 N7 0 5 N’l 10 NT
3 A 103 NT” 800 0 NT N? 0 N’l NT
NT
a SF, Sendai virus-induced cell fusion; CC, cocultivation. b FFU plating equally well on Japanese quail and chicken C/BE. c Days of mixed culture between R( -)Q and pheasant cells. dNT. not tested
fectious virus plating equally well on Japanese quail and on C/E or C/BE chicken cells was present in 4 out of 5 embryo lines at day 35. In contrast to the situation in chickens, the gs status of the ring-necked pheasant embryos did not appear to affect the helper activity in the cultures. This observation is in agreement with results reported by Hanafusa and Hanafusa (1973). As will be shown below, the infectious Rous sarcoma virus detected after cultivation with ring-necked pheasant or golden pheasant cells represents pseudotypes of BH RSV formed with endogenous ringnecked pheasant virus (RPV) or golden pheasant virus (GPV). These pseudotypes will be referred to as RSV(RPV) and RSV(GPV).
Isolation of RSWRPV) From Single Foci Produced by RSV(RAV-0) on Ringnecked Pheasant Cells RSV(RPV) was also isolated by plating a high dilution of RSV(RAV-0) on ringnecked pheasant fibroblasts, picking cells from individual transformed foci and cultivating these cells for several weeks. Such
cells were expected to release infectious virus only if they were accidentally coinfected with RAV-0 present in the inoculum, or if the pheasant cells supplied endogenous helper activity to RSV. These two possibilities could be distinguished by the host range of the resulting pseudotype. Table 5 shows the focus forming titer of single foci 23 days after isolation. At least three foci (Nos. 1-3) produced virus which plated efficiently on C/BE, C/E and Japanese quail cells, similar to RSV(RPV). Interference and neutralization tests detailed below showed these pseudotypes from single foci to be indistinguishable from RSV(RPV). One of the single foci, however, (No. 10) released subgroup E virus, indicating that it had been doubly infected with RSV and RAV-0. The low titer-virus present in the remaining foci was not characterized. These observations provide additional independent evidence for the presence of endogenous RNA tumor virus genes in ring-necked pheasant cells. Since the technique of single focus isolation used neither R( -)Q cells nor Sendai virus, these auxiliary viral agents can be excluded as possible sources of RPV.
564
FUJITA TABLE
5
VIRUS WITH EXPANDED HOST RANGE ISOLATED FROM SINGLE FOCI PRODUCED BY RSV(RAV-0) OK RING-NECKED PHEASANT CELLS Isolated focus
Titer (FFU/ml)” Japanese quail
Chicken
C/BE
Chicken
C/E
1
1.7 x 103
1.6 x 103
1.7 x 103
2 3 4
8.0 x lo3
5.5 x 103
6.0 x IO3
1.3 x 102
1.7 x 102
10 40 0
30
1.3 x 102 > 10 20
NTD NT
NT NT NT 0
5
6 7 8 9 10
30 >lO’
10
0
“Tested on day 23 after isolation b Not tested.
0 40 40 10 10 of the focus.
Isolation of Leukosis Viruses From RSV(RPV) and RSV(GPV) Stocks The infectious pseudotypes of RSV isolated from pheasant cells presumably contained induced pheasant leukosis viruses. Inoculation of the pseudotype stocks into chfchicken cells resulted in a large increase of focus forming titer which suggested the presence of helper virus in the pseudotype stocks. These helper viruses, RPV and GPV, were isolated from the pseudotypes by endpoint interference (Rubin and Vogt, 1962). The RSV(RPV) stock used in this experiment was obtained from a single focus of BH RSV in gs+ ring-necked pheasant cells, and the RSV(GPV) stock was obtained after Sendai virus induced fusion of golden pheasant and R( -)Q cells, as described in Table 3. Both pseudotypes had been passed only in ring-necked or golden pheasant cells respectively. Closely spaced dilutions of RSV(RPV) or RSV(GPV) extending over the endpoint of focus formation were inoculated into chf- C/BE chicken cells. After 3-8 transfers the cells were challenged with RSV(RPV), RSV(GPV) and unrelated control viruses and overlaid with agar. Unchallenged plates served as controls which indicated the endpoint of focus forming activity. The results of such an endpoint
ET AL.
interference test are compiled in Table 6. Specific interference was observed with the homologous challenge virus in cultures which had received dilutions of RSV(RPV) or RSV(GPV) beyond the endpoint of transformation. The interfering viruses RPV and GPV were obtained from the supernatant medium of the corresponding unchallenged control cultures and were purified by an additional dilution series in gs- chf- chicken cells.
Host Ranges of RSV(RPV) and RSV(GPV) The host ranges of RSV(RPV) and RSV(GPV) were determined by performing focus assays on several types of avian fibroblasts as shown in Table 7. Both RSV(RPV) and RSV(GPV) are capable of forming foci with a high efficiency on avian cells which are resistant to viruses of subgroups A, B, C, D and E. The high plating TABLE
6
ISOLATION OF RPV AND GPV FROM RSV PSEUDOTYPES BY ENDPOINT INTERFERENCES Dilution of pseudotype
Foci formed by challenge virus RSV(RPV)
RSV(RPV1 1: 3,200 1: 6,400 1: 12,800 1: 25,600 uninfected
RSV(GPV)
83
14
17 0
182
0
366
60
0
296
89
0
474
71
0
PR-A RSV(GPV) 1: 100 1:300 1:900
NO
challenge
RSV(GPV)
No challenge
30
34 1
0
1:2,700
45 62 70
%b
uninfected
96
356 400 488
0 0 0
’ C/BE, gs ~ chf- chicken cells were infected with RSV(RPV) or RSV(GPV1 at the dilutions listed in the table, passaged as described in Materials and Methods, and challenged at the eighth passage. ‘The end point of transformation was 1: 300, but in the course of several transfers foci disappeared from the unchallenged plates, presumably because of overgrowth of leukosis virus.
RNA TUMOR
VIRUSES TABLE
,565
OF PHEASANTS 7
RESISTANCE AND SUSCEITIBILITV OF VARIOUS AVIAN CELLS TO SUBGROUPF AND G PSEUDOTYPESOF BH RSV Virus
Cell type Species
-
Chicken CIE CIA CIAB C/BE c/c Japanese quail Ring-necked pheasant Golden pheasant Peking duck Goose
Viral subgroups excluded
E A A,B,D,E B,D,E C B,D B, D None A,B,D.E,G A,B,D,E,F,G
PR-A
S” R” R s S s S s R R
PR-B
PR-C
RSVt GPV)
S S R R S R R S R R
S s s S R SR” SR S S S
s s s S s S s SR” R R
RSV(RPV)
s S s s s S S S S R
OS. focus forming efficiency greater than 0.25 relative to type C/E chicken cells; R, focusefficiency less than 0.01 relative to type C/E chicken cells; SR. focus forming efficiency variable from 0.01 to 0.25 relative to type C/E chicken cells. This variability may reflect genetic or physiological differences between embryos.
efficiency on Japanese quail cells (EOP quail/chicken = 1) is also unlike that of subgroup C viruses which show reduced plating on Japanese quail cells (Duff and Vogt, 1969). The data on RSV(RPV) are in agreement with earlier observations which have established that the endogenous helper virus from the ring-necked pheasant belongs to a new subgroup, F, of the avian leukosis and sarcoma complex (Hanafusa and Hanafusa, 1973). The RSV(RPV) described in this report will therefore be included in subgroup F. This classification is also supported by interference tests described below. The host range of RSV(GPV) differed from that of RSV(RPV). RSV(RPV) plated on duck cells with an efficiency of 0.4 relative to chicken cells, whereas RSV(GPV) did not form foci on duck cells (EOP relative to chicken cells =
RSV(RPV) and RSV(GPV) were found to require polycations for effective adsorption to the host cell. In the absence of polybrene (2 pg/ml) plating efficiencies were reduced 50 to 200-fold. Because of the wide host range of RSV(RPV) and RSV(GPV), both viruses were tested for their ability to transform continuous cell lines of mammalian cells. Although these data are still preliminary, it appears that the transforming ability of subgroups F and G for Balb-C mouse 3T3 and normal rat kidney cells are not increased over those of subgroups C and D. Interference Patterns RSV(GPV)
of RSV(RPV)
and
Chicken embryo (C/E, gs-) were infected with avian leukosis viruses of subgroup A through D and subgroup F as well as with RPV or GPV. Ring-necked pheasant fibroblasts were infected with ILV of subgroup E. Interference by RPV and GPV was also tested by inoculation of quail embryo fibroblasts. Infected cultures were transferred at 2-5 day intervals, and at the first passage (cultures infected with leukosis viruses of subgroups A to D) or 3rd to 8th passage (cultures infected with RAV-0, RAV-61, RPV or GPV) the cells
566
FUJITA
ET AL.
were challenged with Rous sarcoma viruses of subgroups A through E and with RSV(RPV) or RSV(GPV). The results are presented in Table 8. Leukosis viruses of subgroups A through E failed to interfere with either RSV(RPV) or RSV(GPV), although they interfered with sarcoma viruses of homologous subgroups. RPV and GPV interfered with their respective pseudotypes but not with each other nor with sarcoma viruses of subgroups A to D. RPV did interfere, however, with a subgroup E challenge in quail cultures; but this interference was nonreciprocal as ILV failed to cause interference with subgroup F virus. No interference was seen between subgroups G and E. Occasionally RPV induced significant interference with subgroup A, but this observation was not consistently reproducible. RAV-61, a member of subgroup F, interfered with RSV(RPV) but not with RSV(GPV). These observations suggest that RPV belongs to subgroup F previously described by Hanafusa and Hanafusa (19X3), and that GPV belongs to a new subgroup, G.
Antigenic Properties RSV(GPV) Neutralization
RSV(RPW
and
tests were carried
out
of
with RSV(RPV) and RSV(GPV) using eight chicken sera with neutralizing activity against subgroups A, B, C or D. None of these sera neutralized either RSV(RPV) or RSV(GPV) to a significant extent, although they showed neutralizing activity against their homologous viruses. The results for 5 sera are summarized in Table 9. These data support the conclusion that RSV(RPV) and RSV(GPV) have envelope antigens distinct from those of subgroups A, B, C or D. In Table 10 neutralization results with antisubgroup E sera and with normal pheasant sera are compiled. Several sera from ring-necked pheasants neutralized RSV(RPV) but did not neutralize RSV(GPV). This observation supports the conclusion that RSV(RPV) and RSV(GPV) are not closely related with respect to their envelope antigens. The neutralizing activity of pheasant sera for RSV(RPV) was not correlated with neutralization of RSV(RAV-0), a subgroup E virus. A rabbit antiserum prepared against RAV-0 also failed to neutralize RSV(RPV) or RSV(GPV). We conclude that the new pheasant viruses are antigenically distinct from subgroup E. One out of 20 sera from nonimmunized ring-necked pheasants
TABLE CLASSIFICATION
Cell Species
OF
RPV
AND
8
GPV
HY VIKAL
Challenge virus
Interfering virus” Resistant to
Chicken C/E
E
Ring-necked pheasant dapanese quail
B, D B.D
none RAV-I (A) RAV-2 (B) tdEE7(C) CZAV (D) RPV (F) GPV (G) RAV-61 (F) none ILV (El none RPV (F) GPV (G)
INTERFERENCE
PR-A
PR-B
PR-C
SR-D
RSV (RAW))
RSV (RPV)
RSV (GPV,
S” R S s s s S S NT NT NT NT Xl’
s s H S R s s Nl N-1 NT NT NT IN?
s 5 s R s s s NT NT NT Iv.1 Nl N’l
NT’ NT NT Nl R s s NT Nl NT NT NT N’I
NT NT NT NT Nl N-l NT NT s R s R s
S s S s s R s R s s s R s
S s s s s s R s s s s s R
’ Viral subgroup in parenthesis. OS. EOP of challenge \zirus 0.2-1.0; R, EOP of’ challenge virus ~0.01 relative ‘NT. not tested.
to control cells.
RNA TUMOR
VIRUSES TABLE
FAILURE
9
AGAINST SUBGROUPS A-D
OF ANTISERA
567
OF PHEASANTS
TO NEUTRALIZE
RSV(RPV)
OR RSV(GPV)”
Number of foci subgroup and virus Serum specificity and number
C RSV(AV-11
Control, no serum Anti-A, 520 Anti-A*, pool Anti-C, 160 Anti-CD, 580 Anti-D, 265
RSV(&V-21
PR-C
BSI
SRDD
F RSV(RPV1
171
41
59
125
49
83’
77
0 3 NT NT NT
NT” 4 NT NT NT
NT NT 1 I NT
NT NT 2 NT NT
NT NT NT 0 5
;:h? 744 642 786 582
90 12 18 96 1’,5 UC
“Virus was incubated on C/E cells. b NT. not tested.
with a 10-l dilution
of antiserum
TABLE IMMUNOLOGICAL
Source of serum
DIFFERENTIATION
Serum number
for 40 min in a 37” waterbath.
Ring-necked pheasant
887 635 621 634 32171
Rabbit
showed neutralizing activity against subgroup F, perhaps indicating that ringnecked pheasants can be horizontally infected and are not immunologically tolerant to RPV.
of
RSV(RPV)
and
Throughout the course of this investigation we have assumed that RSV(RPV) and RSV(GPV) were defective pseudotypes of BH RSV with envelope antigens specified by RPV and GPV respectively. This interpretation is supported by results of infective center assays in which focus formation was dependent on the ability of RSV to produce infectious progeny in solitary infection (Weiss et al., 1973). In this experiment avian fibroblasts were infected with RSV(RPV) or RSV(GPV), and after one day the cells were treated with 2 pug of mitomycin C for 16 hr. The mitomycin C was then removed by washing, and an
were plated
AND RSV’(GPV1
Percent surviving
Immunizing antigen RSV(RAV-01 RSV(RAV-01 RSV(RAV-0) none RAV-O
Survivors
10
BETWEEN RSV(RPV)
RSV(GPV)
Defectiveness RSV(GPV)
G RSV(GPV1
74 120 120 126 67
RSV(RPV) 2.8 9 91 12 109
foci RSV(RAV-0) 0.2 9 14 102
additional cell layer was seeded on the cultures. The mitomycin C-treated cells will initiate foci in the secondary layers only if they release infectious virus but will not divide and grow into foci themselves. Under these conditions focus formation by RSV(RPV) or RSV(GPV) was reduced to less than 0.5%, in contrast to a nondefective control virus, PR-A, which produced foci equally well in direct assays and in infectious center tests (Table 11). These observations indicate that RSV(RPV) and RSV(GPV) are indeed defective viruses.
Probable Recombination with Host Range Markers of RSV(RPV) and RSV(GPV) Chicken C/E fibroblasts were mixedly infected with PR-B and RSV(RPV), and from the mixed harvests recombinants capable of infecting C/BE cells were isolated and cloned by the infectious center technique of Weiss et al. (1973). Several such recombinants of PR-B and RPV were char-
568
FIJJITA
acterized. They showed the host range of RPV, but in contrast to RSV(RPV) they were able to induce foci under conditions requiring release of infectious virus (Table 12) and appeared therefore to contain nondefective virus. This observation is in itself insufficient proof for recombination, because a pseudotype with RPV may give similar results if RPV was in great excess; but the presumptive recombinants listed in Table 12 had been cloned several times in TABLE
11
DEFECTIVENESSOF RSV(GPV) Virus
AND RSV(RP\‘,
Number of foci Direct assays
PR-A RSVCGPV) RSV(RPV)
Infectious
.____ centers”
C/E
C/HE
I”
II
85 446 358
86 280 291
78 1 0
80 0 0
“In the infectious center assay cultures with “initiator cells” are infected with 300-500 FFI1. Twelve to eighteen hours post infection the cells are treated with 2 &ml of Mitomycin C overnight. The drug is then removed, the cells are washed, and a second cell layer (“indicator cells”) is seeded, following which the cultures are overlaid. Focus formation occurs only if the initiators release virus which can infect the indicators as shown by LVeiss et al., 193 ‘I. C/E initiators. C/E indicators: II. C/BE initiators. Japanese quail indicators.
E7’ AL.
the infectious center assay at low multiplicities, making this second explanation unlikely. Presumptive recombinants have also been isolated from mixed infections of PR-B and RSV(GPV) but still require characterization. For these experiments pure pheasant leukosis viruses were not yet available; therefore the mixed infections were carried out with pseudotypes. Since the BH RSV component of these pseudotypes does not participate in host range recombination (Kawai and Hanafusa, 1972; Weiss et al., 19731, it seemed justifiable to assume that only the pheasant leukosis viruses interact with PR-B. The data of Table 12 suggest that endogenous pheasant leukosis viruses are capable of host range recombination with PR-B.
Co-cultivation or fusion of RSV( ) producing quail cells with normal fibroblasts of ring-necked or golden pheasants led to the appearance of infectious RSV pseudotypes. Such pseudotypes were also obtained by direct infection of ring-necked pheasant cells with RSV(RAV-0) at low multiplicities. Normal pheasant cells provide a helper function for the defective RSV. This helper function appears to reflect the presence of leukosis virus genetic information in pheasants. The pseudotypes obtained from various ring-necked pheas-
NONDEPECTILERECOMRI>AANTS RET)YEENHOST RANGE MAKKEKS OF RI’L AND ‘THE‘I‘KANSFOKMATIO\IMAKKEKS OF PR-I3 Number of FFL;
Virus
Direct assays __~___ Ui Expt 1 PR-B PR: RPV PR: RPV PR: RPV Expt 2 PR-A PR: RPV PR: RPV 0 See footnote”
20 I
(‘/HE
Infectious centers” with C/HE initiators and indicators
clone No. 1 clone No. 2 clone No. 3
0 1’ 50 “07
0 ti 11 50
clone No. 2 clone No. 7
:i:iti :i0 :100
277 15 :w;
of Table I I.
RNA TIblOR
VIRI.SES
ant embryos are all related to each other and to RAV-61 of subgroup F. This observation and the occasional detection of subgroup F antibody in pheasant sera confirm the widespread natural occurrence of this avian leukosis subgroup in pheasants as first described by Hanafusa and Hanafusa (1973). The RSV pseudotypes produced with endogenous viral information of several golden pheasant embryos are also related to each other, but distinct from all other avian leukosis and sarcoma virus subgroups in their host range, susceptibility to interference, and envelope antigens. These viruses have been designated subgroup G. Subgroups F and G do not cross interfere with each other, and antisera neutralizing one but not the other have been found. Although both subgroups appear unable to infect goose cells, duck cells are selectively resistant against subgroup G. In chicken cells the endogenous helper activity for RSV( -) is correlated with the presence of gs viral antigens in the cells (Weiss, 1969; H. Hanafusa et al., 1970; Vogt et al., 1973; Chen and Hanafusa, 1974). In contrast to chicken cells, there was no clear-cut correlation between presence of gs antigen in pheasant cells and helper activity for RSV( -). Similar observations have also been made by Hanafusa and Hanafusa (1973). Lack of helper activity in gs+ cells could be explained if only a partial viral genome, not including genetic information for envelope proteins, was present in some pheasant embryos, or if the production of envelope components could not always be activated by the superinfection of RSV( --). Analogous explanations may be offered for gs - chf + pheasant cells, but here it is also quite likely that the relatively insensitive complement fixation test used in our studies for the detection of gs antigens may have been unsuitable for low concentrations of this viral gene product. Endpoint interference tests have revealed the presence of independently replicating helper leukosis viruses in stocks of subgroup F and G BH RSV pseudotypes. These leukosis viruses grow well in a vari-
OF PHEASANTS
569
ety of avian cells. Since they are derived from a BH RSV pseudotype, the possibility exists that some of their genetic information has been acquired from BH RSV. Although BH RSV is not thought to recombine in the host range marker (Kawai and Hanafusa, 1972; Weiss et al., 1973), observations have been reported which suggest that recombination for other markers may occur (Hanafusa, 1970). Support for the suggestion that RPV and GPV may contain RSV genetic information comes from the observation by Kang and Temin (1973) that the RNA of RAV-61, which appears very closely related to RPV, is almost entirely homologous to the genome of chicken derived leukosis and sarcoma viruses. Yet Neiman (1973) using such a chicken derived virus (RAV-0) as a probe to detect endogenous viral sequences in normal chicken and ring-necked pheasant cells found that in contrast to the chicken, which shows complete genomic representation of RAV-0, gs+ or g’s- ring-necked pheasant cells carry sequences that are homologous to only a small portion of the RAV-0 genome. These data could be interpreted to mean that either the pheasant embryos studied by Neiman contain only a portion of an integrated viral genome, or they contain a complete genome but on11 part of this is homologous to RAV-0. It will now be of interest to use nucleic acid probes prepared from subgroup F and G viruses to search for endogenous sequences in pheasant cells. No reliable information is available on the incidence of leukosis in the pheasant flocks from which we obtained fertile eggs. However, preliminary experiments suggest that RPV is able to cause lymphoid leukosis in chickens (Dr. Graham Purchase. 1973, private communication). More experimentation on the possible leukemogenie activity of subgroups F and G is clearly required.
The authors thank William
S. Mason for helpful
suggestions.Martin C. Alevy for goose and duck cells. and Joseph Green, Hsiao-Ching and Barbara Miles for technical
Pang. Annie Chung assistance.
570
FUJITA
REFERENCES BALUDA, M. A. (1972). Widespread presence, in chickens, of DNA complementary to the RNA genome of avian leukosis viruses. Proc. Nat. Acad. Sci. USA 69, 576-580. BISHOP, J. M., JACKSON, N., QUINTRELI, N., and VARMUS, H. E. (1973). Transcription of RNA tumor virus genes in normal and infected cells. In “Possible Episomes in Eukaryotes,” (L. G. Silvestri, ed.), North Holland, Amsterdam, pp. 61-73. CHEN, J. H. and HANAFUSA, H. (1974). Detection of protein of avian leukovirus in uninfected chick cells by radioimmunoassay. J. Viral. 13, 340-346. CRITTENDEN, L. B., WENDEI, E. J.. and MOTTA, J. (1973). The interaction of genes controlling resistance to RSV(RAV-0). Virolog.v 52, 373-384. CR~TTENDEN, L. B., SMITII, E. J., WEISS, R. A., and SARMA, P. S. (1974). Host gene control of endogenous avian leukosis virus production. Virology 57, 128138. DUESBERG,P. H. and VOGT, P. K. (1973). RNA species obtained from clonal lines of avian sarcoma and from avian leukosis virus. Virology 54, 2077219. DUFF, R. G. and VOGT. P. K. (1969). Characteristics of two new avian tumor virus subgroups. Virology 39, 1830. FRIIS, R. R. (1972). Abortive infection of Japanese quail cells with avian sarcoma viruses. Virology 50, 701-712. HANAFUSA, H. (1970). Virus production by Rous sarcoma cells. Cur. Top. Microbial. Immunol. 51, 114-123. HANAFUSA, H., HANAFUSA. T., and RUBIN, H. (1963). The defectiveness of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 49, 572-580. HANAFUSA, H., MIYAMOTO. T.. and HANAFUS~ T. (1970). A cell-associated factor essential for formation of an infectious form of Rous sarcoma virus. Proc. Nat. Acad. Sci. USA 66, 314-321. HANAFUSA, T. and HANAFUSA, H. (1973). Isolation of Ieukosis-type virus from pheasant embryo cells: Possible presence of viral genes in cells. Virology 51, 247-251. HANAFUSA. T., HANAFUSA, H., and MIYAMOTO, T. (1970). Recovery of a new virus from apparently normal chick cells by infection with avian tumor viruses. Proc. Nat. Acad. Sci. USA 67, 179771803. HANAFUSA, T., HANAFUSA, H., MIYAMOTO, T.. and FLEISSNER, E. (1972). Existence and expression of tumor virus genes in chick embryo cells. Proc. Nat. Acad. Sci. USA 66, 314-321. HAYWARD, W. S. and HANAFUSA, H. (1973). Detection of avian tumor virus RNA in uninfected chick embryo cells. J. Virol. 11, 15-167. KANG, C. and Temin, H. M. (1973). Sequence homoogy among RNAs of avian leukosis-sarcoma viruses,
ET AL. reticuloendotheliosis viruses, and chicken endogenous RNA-directed DNA polymerase activity. J. Viral. 12, 1314-1324. KAWAI, S. and HANAFUSA, H. (1972). Genetic recombination with avian tumor virus. Virology 49, 37-44. NEIMAN, P. E. (1973). Measurement of endogenous leukosis virus nucleotide sequences in the DNA of normal avian embryos by RNA-DNA hybridization. Virology 53, 196-204. PAYNE, I,. N. and CHUBB, R. (1968). Studies on the nature and genetic control of an antigen in normal chick embryos which reacts in the COFAL test. J. Gen. Viral. 3, 379-391. ROSENTHAL, P. N., ROBINSON, H. L., ROBINSON, W. S., HANAFUSA, T., and HANAFUSA, H. (1971). DNA in uninfected and virus-infected cells complementary to avian tumor virus RNA. Proc. Nat. Acad. Sci. USA 68, 2336-2340. RUBIN, H. and VOGT, P. K. (1962). An avian Ieukosis virus associated with stocks of Rous sarcoma virus. Virology 17, 1844194. SCHEELE, C. M. and HANAFUS~ H. (1971). Proteins of helper-dependent RSV. Virology 45, 401-410. TOYOSHIMA, K., FRIIS, R. R., and VOGT, P. K. (1970). The reproductive and cell-transforming capacities of avian sarcoma virus B77: Inactivation with UV light. Virology 42, 163170. TOYOSHIMA, K. and VOGT, P. K. (1969). Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 38, 414-426. VARMUS. H. E., WEISS, R. A., FRIIS, R. R., LEVINSON, W., and BISHOP, J. M. (1972). Detection of avian tumor virus-specific nucleotide sequences in avian cells DNA. Proc. Nat. Acad. Sci. USA 69, 20-24. VOGT, P. K. (1969). Focus assay of Rous sarcoma virus. In “Fundamental Techniques in Virology,” (K. Habel and N. P. Salzman, eds.). Academic Press, New York, pp. 198211. VOGT, P. K. and FKIIS, R. R. (1971). An avian leukosis virus related to RSV(0): Properties and evidence for helper activity. Virology 43, 223-234. VOGT. P. K. and HARRIS, P. (1974). Use of plastic bags to maintain a humidified atmosphere for animal cell cultures, Appl. Microbial. 27, 618-619. VOGT. P. K., FRIIS. R. R., and WEISS, R. A. (1973). Cell genetics and growth of endogenous viruses. Proc. 7th Nat/. Cancer Conf. pp. 55X9. WEISS, R. A. (1969). The host range of Bryan strain Rous sarcoma virus synthesized in the absence of helper virus. J. Gen. Viral. 5, 511-528. WEISS. R. A. ( 1974). Ecological genetics of RNA tumor viruses and their hosts. In “Analytic and Experimental Epidemiology of Cancer,” (T. Hirayama, ed.), University of Tokyo Press, Tokyo, in press. WEISS, R. A., FRIIS, R. R., KATZ, E., and VOGT, P. K. (1971). Induction of avian tumor viruses in normal
RNA TUMOR
VIRUSES
cells by physical and chemical carcinogens. Viralogy 46, 920-928. WEISS, R. A., MASON, W. S., and VOGT, P. K. (19’73). Genetic recombination and heterozygotes derived from endogenous and exogenous avian RNA tumor viruses. Virology 52, 535-552. WEISS, R. A. and PAYNE, L. N. (1971). The heritable
OF PHEASANTS
571
nature of the factor in chicken cells which acts as a helper virus for Rous sarcoma virus. Virology 45, 508-515. YOSHIKAWA-FUKUDA, M. and EBERT, J. D. (1969). Hybridization of RNA from Rous sarcoma virus with cellular and viral DNA’s Proc. Nat. Acad. Sci. USA 64, 870-877.