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Infectious type C viruses released by normal cat embryo cells
VIROLOGY
66,
506-515
Infectious GEORGE
Viral
(1973)
Type
C Viruses
J. TODARO,
Leukemia
Released
by
Normal
RAOUL E. BENVENISTE, DAVID $1. LIVINGSTON
and Lymphoma
MICHAEL
Branch, National Cancer Institute, Bethesda, Maryland 20014 Accepted
July
Cat
Embryo
Cells
M. LIEBER,
Sational
Institutes
AND
of Health
10, 1973
Type C viruses with antigenic and host range properties similar to the RD114 virus and to a virus from a continuous line of cat kidney cells, CCC, have been isolated from six of ten diploid fetal cat cell cultures. Each of the new viruses replicates readily in human and rhesus monkey cells, but not in most other cat cell strains tested. However, a cat cell strain, FFcSOWF, was found that is permissive for replication of the RD/CCC group of type C viruses produced by other cat cell strains. The host range of these endogenous type C viruses includes bat, dog, mink, and rabbit cells, as well as primate cells. Type C viruses of the FeLV group could not, be detected in any of these ten fetal cat cell cultures. Viral RNA from one of the new isolates, from the CCC virus, and from RD114 virus each specifically anneal SI to an [3H]DNA transcript of the RD114 genome.
INTRODUCTION Domestic cats of various breeds have been found to contain t.ype C viruses which can cause leukemias and sarcomas (Jarrett et al., 1964; Kawakami et al., 1967; Rickard et al., 1969). These viruses have been isolated from the tissues of both tumor-bearing and normal cats and can be transmitted experimentally to various species, such as cats, dogs, rabbits, and monkeys (Gardner et al., 1970; Deinhardt et al., 1970; Rabin et al., 1972). The great majority of the type C feline leukemia virus (FeLV) isolates possess a common group-specific antigen (Hardy et al., 1969; Oroszlan et al., 1971) and a common reverse transcriptase antigen (Parks et al., 1972; Scolnick et al., 1972). Each of these common markers is distinctive enough to define a group of related viruses isolated from cat tissues. Subsequently, a type C virus, RD114, was isolated after passing the RD human rhabdomyosarcoma cell line through a fetal cat (McAllister et al., 1972). This virus has immunologic and host range properties different from previously isolated feline type C viruses (McAllister et al., 1972,1973; Oroszlan et al., 1972). 506 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
Recently, it was found that a type C virus could be induced from virus-free cell clones of the CCC continuous line of cat kidney cells (Livingston and Todaro, 1973; Fischinger et al., 1973). The type C virus from the CCC cells has antigenic properties, host-range properties, and viral-int’erference patterns virtually indistinguishable from those of RD114 (Livingston and Todaro, 1973; Fischinger et al., 1973). Since this virus can be induced from single cell clones of virusfree cat cells, it was suggested that the RD114 virus was a member of a group of endogenous type C viruses of the cat. To test whether viruses of this RD/CCC group could be found in diploid cat cells as well as in an established line and to test whether potentially infectious type C virus of this group was widespread in the domestic cat, we have examined cultures derived from a variety of feline tissues. MATERIALS
AND
METHODS
Cells. All cells were grown on plastic surfaces (Falcon Plastics, Ontario, CA) in Dulbecco’s modification of Eagle’s minimal essential medium containing 10 % calf serum
TYPE
C VIIZUSES
FROM
(Colorado Swum Co., Denver, CO) at 37°C. Cell cultures at either the third or the sixth passage were ohtaintbd as frozen ampoulcs from the Cell Culture Laboratory, Naval Biomrdical Research Laboratory, Oakland, CA. The fetal ccl1 strains includrd t\vo from kidwy (P‘Fc’LK and P’E’cSK), one from lung (I:I:&Lu), t,wo from thymus (IcE’c2Th and E’FcJTh), ow from tongue (FFc3Tg), thrw from whole c>mbryo (F’FcSWF; F’FcGOWF; and orw mixc>d spleen, thymus, liver, and boric marrow (FFcli3RES)). CCC is a clonal lirw of feline kidney fibroblasts isolated and charactNcrizedby Crandcll et al., CCC-%4 is a non-virus-producing subclonr of CCC kindly provided by Dr. Peter E’ischingcr (National Cancer Institutc). FEC is a strain of feline ~~mbryonic wlls (Lw et al., 1972). A’204 is a continuous lirw of human rhabdomyosarcoma cells dcvclopcld in this laborat’ory, and DBS-FRhL-1 (DBS-1) is a continuous line derived from fetal rhwus monkey lung fibroblasts and was kindly provided by Dr. Roslyn Wallace, Lederlc Laboratories, Pearl River, KY (Wallace et al., 1973). The various other cell cultures used in host range studies were obtainod from the> American Type Cult’ure Collcc+ion, Itockville, MD, c>xwpt,for a fetal caninn thymus liw, FCf2Th, which was obtaiwd in it,s 42nd passagefrom the Naval Biomndical Rwarch Laboratory, Oakland,
c.4. I’iw,ses. The> ICirsten strain of murine lruk(mia virus (Iii1JuLV) was grown in mouse NIH/BT3 cells (Jainchill et al., 1969). V-NRli is an cwdogenous rat typ(l C virus spontaneously wlcased from certain subclonts (Liebcr et al., 1973) of the NRIi lint (Due-Nguyen et al., 1966) of rat kidney fibroblasts and is continuously produced by thaw subclones. Thr woolly monkey type C virus (Wolf(, et al., 1972) was grown in this labortory in p1204 cells. Pig type C virus is cwntinually producrd by the porcine kidney c~pithcliul cell lint: (PI<-1.5) (Armstrong e( al., 1971; Licxber et al., 1973); this cell lint was obtained from the American Type Culture Collection (Rockville, MD). RD114 virus was grown in the RD linct of human Ihahdomyosarcorrla cells, while CCC virus \vas continually wcrt+>d by thcl Crandcll
FETAJ,
i-f,7
C:AT CKI,I,S
(CCC) fcliw kidncy ccl1 line. Vivian myeloblast’osis virus was provided by Dr. Jowph Brard t8hrough tht> Rcsourcw and Logistics Ofhw, Viral Caner Program, Sationdl Canwr Instit,utc. Thcl Gardner-Arnsbrin strain of f&w sarcoma virus (GA-P&V) was obtjairwd from El(~ct,ro-Sucl~onicv Laborat oriw, Bcthcsda, AID. Snyd~~r-Thc~ilcnstrain f&w kwlwmia virus was ohtaitwd from Universit,~ Laboratories, Highland Park, S.J. I’I’r7~s
tletecfifw
by superriafaul
wwrse
lmrr-
assay. This was pcrformcvt by the mc$thod of Ross ef. al. (1971). I:iftcw millilitws of mcsdium from each tissw caultjure flask was claritic~d by wntrifugation at 1‘2,OOO~q for 10 min. The supcrnatnilt was thaw tranxferwd to a wn- tubv and p(~lletc~d by wntrifugation through 20 “; gl~~ccrcllin 0.05 31 TrissHCl, pH 7.S; 0.10 JI KC1 at 10.5,OOOc/ for 90 miti at 4°C. Tlic, pcill(lt 11as rcsuspcndt>d in 0.1 ml of 0.0.5 .II Tris HCl, pH 7.S; 0.10 ilf SaCl; lo-” 11Ldithiothrc~itol (DTT) buffw wntainingO.l 5’;Triton X-100. Virus suspension (0.01 ml) \v:is :rdd(ld to a. O.Oli-ml rc~achionmixt,urcl containing 0.05 :I/ Tris-HCl, pH 73; 0.06 M IiCl; 2 X IO-:’ ;11 DTT; 5 X lo-? 111manganrw uwtatc~; 0.012 ‘4zs,,units poly(rA) (1lilrs Laboratoricu, 151khart, IS) ; 0.02 A2G0units oligo (dTp:-,,) (Collaborativcb Restawh, Waltham, ;\LZ 1; and 3 X 1O-6 :l[ (“H]TTI’ (40,000 (‘pm; p~nol~~i (KU\- I~:iigland Xwlwr Corporation, Best on. 11.4). Rcwults :w t~xprwwd as pic~omol(3of [aH]TXI I’ inc~orporatc~d illto radioact iw poly(dT) product in a GO-min iwubation at 37°C. 6-lotlot/eolryu;~jffi/~e (Id1 ‘) irdurfiotr f!i’ ljype C r+w. .~-Iodod(,ox~uridiI~(~ was ~nrrc~tlast~d from Calbiochr~m, Los Angelw, (‘.\. ‘I%~ iododtaoxyuriditwtreatcd culture UXYY’pwparrd by twating 6 X 10” wlls of ~(~1~st8rain lvith 100 pg/ml of drug in complt~tc~media for 2%hr at 37°C. Th(l mcxdinwas c~hangctit\z.iw \vith a 30.min intwval at 37Y’. and that culturw incubatc>d for 2 morr days. .1t that t)irncl,th(> wlls \vc:rcatrypsinizcd. mtl approximatc,ly .5 X IO” wlls \v(‘r(’ :tdd(~d to t’rwh indicator wll wlturw which had bwu sc~~icd thr day Irwfow at 3 X 10: wlls pc’t’ 7.1,cm2 flasli in cornpl(+cxmedia wmtailliiig pol!~twcwc~ (2 pg/ml).
soiptase
508
TODARO ET AL.
n&ant fluids from virus-producing cell cultures were filtered through 0.45 p Millipore filters (Millipore Co., Bedford, Mass.) and 1.5 ml was used to infect indicator cells plated the day previously at 3 X lo5 cells per flask in complete medium with polybrene (2 pg/ml). After a 1-hr adsorption period, complete media was added. Molecular hybridization technique. The production of reverse-transcriptase-catalyzed [3H]DNA transcripts of various types of viral RNA and the detection of their hybridization to purified viral 70s RNA have been reported elsewhere (Benveniste and Scolnick, 1973). The source of RD114 and CCC virus has been previously described (Livingston and Todaro, 1973). The virus from FFc2K cells was propagated in the human rhabdomyosarcoma cell line, A204; the supernatants were concentrated, and the virus isopycnically banded in sucrose. Polymeruse antibody-inhibition studies. Putative virus was concentrated, and standard reverse transcriptase reaction mixtures were used as described above. Antisera against viral polymerases were prepared as described previously (Parks et al., 1972). Reactions were initiated with a mixture of template, primer, and substrate. Detergent-disrupted virus (0.01 ml) diluted in 0.05 M Tris-HCl, pH 7.8; 0.10 M NaCl; lop3 M DTT was employed in each assay, and enzyme was added prior to IgG. In a standard reaction, a quantity of enzyme which led to incorporation of 0.184.20 pmoles of [3H]TMP (40,000 cpmjpmole) in 60 min was routinely added. Electron microscopy of cell mon.olayers.The cell monolayer was fixed in situ with buffered 2 % glutaraldehyde. The cells were then scraped off, pelleted, and post-fixed with 1% buffered osmium tetroxide. After incubation overnight in cold 0.5 % uranyl acetate: they were dehydrat,ed and embedded in Epon. Thin sections were doubly stained in uranyl acetate and lead citrate and examined with a SiemensElmiskop 1A electron microscope. RESULTS Virus production by primary cultures 4f feline cells. Ten fetal cat cell cultures were tested for spontaneous type C virus production by assaying their media for viral reverse
transcriptase activity. Two of the ten cultures, a fetal cat kidney strain (FFc2K) and a fetal cat strain from mixed spleen, thymus, liver, and bone marrow (FFc63RES), showed high levels of reverse transcriptase activity in their supernatant fluids and typical type C virus particles by electron microscopy. Figure 1 showselectron micrographs of budding and extracellular type C virus from the FFc2K cells. Immunologic relationship of the various feline viral polymerases. The reverse transcriptase activity in the supernatant fluids of these two cultures was not substantially inhibited by IgG from antiserum to the FeLV polymerase (Fig. 2). The enzymatic activity from both cell cult,ures, however, was markedly inhibited by antibody to the RD114 polymerase (> 50 % with 20 pg of anti-RD114 polymerase), suggesting that, like RD114 and CCC virus, these cells were producing a virus with properties quite different from FeLV. Host range properties of various feline type C viruses. The FFc2K and FFc63RES cells were cocultivated with a variety of cat cells (CCC-3A, FEC, FFc2Th, and FFcGOWF), a rhesus monkey cell line (DBS-FRhL-I), and a human rhabdomyosarcoma cell line (A204). As shown in Table 1, the virus grew readily in the two primate cell lines, but was unable to infect most of the feline cells. One exception is a cat cell line, FFcGOWF, derived from whole embryo tissue, which readily supports the growth of the FFc2K virus. Similar host range properties have been found for t,he CCC and RD114 viruses (Livingston and Todaro, 1973). Table 2 showsthat these viruses, whether growing in feline or in human cells, arc similarly inhibited by RD114 anti-polymerase IgG and not by control IgG or anti-FeLV polymerase IgG. Scolnick et al. (1972) have previously shown that there is no significant inhibition of FeLV polymerase by anti-RD114 polymerase IgG, or vice versa. Similarly, there is no detectable antigenic cross reaction between FeLV and CCC virus polymerase (Livingston and Todaro, 1973). Induction of infectious type C virus from nonvirus,producing feline cell strains. Lung cells (FFc2L) and thymus cells (FFc2Th) from the same cat fetus (FFc2) which had
510
ET AL.
TODARO
spontaneously yielded type C virus from its kidney cells were also tested for virus production by the supernatant reverse transcriptase assay. Neither produced detectable levels of type C virus spontaneously. Therefore, these cultures were treated with 5-iododeoxyuridine (IdTJ) (Lowy et al., 1971; Aaronson I
I
1
I 20
I
I
I 60
I
100 75 50 25
100 75 50 25 I 40 W
bd
FIG. 2. Inhibition of reverse transcriptase from supernatants of fetal cat cells by antisera IgG. Putative virus was concentrated, and standard reaction mixtures were used as described in Methods. Values given are percentage of [“H]TMP incorporation in the presence of IgG relative to that in its absence. (Top) FFc2K, (Bottom) FFc63RES. (0) Enzyme plus nonimmune IgG, (0) enzyme plus anti-FeLV polymerase IgG, (0) enzyme plus anti-R.Dll4 polymerase IgG.
et al., 1971; Klement et al., 1971) then cocultivated with various indicator cells. The IdU-treated feline lung cells and thymus cells produced an infectious, reverse-transcriptase-containing particle which grew readily in human rhabdomyosarcoma cells (A204) and in the rhesus monkey cell line, DBS-FRhL-1, but not in three of the four cat cells tested (Table 1). It, however, did grow well in FFc60WF. In each instance, the polymerase of the virus from the induced and cocultivated cultures was specifically inhibited by the anti-RD114 polymerase IgG (Table 2). The extent of polymerase inhibition of these four cat type C virus isolates grown in human cells, as tested with antiRD114 polymerase IgG, was comparable to that obtained with the induced or the spontaneously produced type C virus from the continuous line of cat kidney cells, CCC. Isolation of endogenousvirus by cocultivation with permissivecells. Although no significant reverse transcriptase activity was found in the culture supernatants from a number of fetal cat cell strains, this biochemical test is less sensitive than certain biological assays for detecting type C virus in other systems. To enhance the possibility of detecting lowlevel spontaneous producers of RD/CCC virus, a group of apparently type C virusnegative strains of cat cells were cocultivated with the permissive human host line, A204. Of the four strains tested, FFcSWF, FFcGOWF, FFcl04WF, and FFc3Tg, two
TABLE HOST Cell
RANGK
OF CAT TYPE
1 C VIRAL
Origin
Viruses FFc2Ka Supernatant
CCC-3A FEC FFc2Th FFcGOWF DBS-FRhL-1 A204 a Filtered supernatant b 5-Iododeoxyuridine c Not tested.
ISOLATES
Feline Feline Feline Feline Rhesus Human. fluids treated
monkey
<0.04 <0.04 <0.04 9.14 1.88 0.89
FFc2L* (IdU treated)
FFc63RES”
Reverse Transcriptase H3-TMP incorporated) <0.04 <0.04 <0.04 3.63 NT 1.20
used to infect as described in Materials and and cocultivated as described in Materials
Activity
<0.04 <0.04 NT NT 0.43 1.85 Methods. and Methods.
FFc2Thb (IdU treated (pmoles,
<0.04 NT” NT NT NT 1.52
TYPE TABLK
<: VIE:UHES
FROM
FETAL
CAT
CELLS
2
TABLI1:
C~CULTIVATION
OF
A204 Cat cell strain a? Inhibition of [3H]TMP incorporation in supernatant reverse transcriptase assay” Type
of IgG Added
Control
Virrls from cat (aells FPc2K 67
FFcYWF FFcGOWF FFcl04WF FFc3Tg
51 1 3
C.\T CELL CULTDKI:S KITEI THE HUM-IN CELL 1,1x1.; Supernatant polymerase activity (pmoles [zH]‘I’MP incorporated)* I.7
___u Assayed at 3 weeks and after two subcultures of t,he cocultivnted cells. By this time, t,he culture consists almost entirely of the more rapidly growing human rhabdomyosarroma cells, .420-t.
sarcoma ccl1line, ,4204, \vas uscldsince it had previously been shown to ho a pcrmissivr: host for the type C viruses from the CCC cat cell lincx (Livingston and Todnro, 1973) and from thtl spontaneously type C virusreleasing fetal cat rcllls (FFc2Ii and FI~cMRES). Ability of emlogel~,ous virus of the cat to ~/ww in clistady related cell hosts.Table 5 shows a more extensive study of the host range of the virus being spontaneously produced by the fetal cat kidney strain, I~I’c2Ii. It is compared with the host) rang{’ of two related viruses, RD114 and CCC. Xonc~ of the viruses successfully infect, cat CCC-SA cells, but all can grow in the fetal cast strain, FI’cGOWI~‘. The mouse, rat, and raccoon cells uscbdin this study did not support virus replicat,ion, but, the viruses all rradily repliyirldrld infec*tious polymctrasc:-cont,aining cated in fetal dog thymus cells (ICf2Th) and particlcs after 3 weeks and two suhcultivain bat lung cells (TblLu). Thexcl cell strains tions of tht> mixed cell cultures (Table 3). In appear comparable in their ability- to support both inst’ances, tho enzymes were inhibited virus replication to the primate (rhesus by anti-RD114 polymerasc IgG, but not monkey and human) cells that wcrc prclI&(; directc>d against the I~cLV enzyme viously described as permissive hosts (Liv(Table 2). ingston and Todaro, 1973). Thr two cndogTablr> 4 summarizes the stud& used to cnous type C viruses growing in human detect’ type‘ C viruses released by fetal feline cells, IJFc21< virus in A204 cells and RD114 cc&. Six of the ten lines test,ed produce a virus, arc’ also able to grow in mink and t\.pc‘ C virus related to CCC and RD114. buffalo lung cells, \vhile the endogcnous virus TR.Oof these>strains product enough virus to from CCC cells appears unable to grow. This bc dr:tc>ctcd directly in t’he supcrnatant fluids may rrflect a diffcrcnco in the three isolates, of tho cultures themselves by the reverse or, perhaps, R host’ modificat,ion resulting transcriptasr> assay; four others rcyuire co- from the replication by RD114 and FFc2K cultivation with a permissive host cell. In in human cells. The viruses in the info&d thcxsct ctxpfriments t,hc human rhabdomyo- buffalo lung ~~11sand dog thymus ~~11s havtl
512
TODARO
ET
a polymerase with the antigenic properties of those of the RD/ ‘CCC group. The four fetal ck cell strains that are apparently virus-negative have not been exhaustively tested by induction and cocultivation with each of the potentially permissive host cells shown in Table 5. Nucleic acid homology of RNA from various
AL.
type C viruses with the DNA transcript of RDll4 RNA. Viral RNA extracted from the RD114, CCC, and FFc2lX viruses was annealed to the [3H]DNA product of an “endogenous” reverse transcriptase reaction with RD114 RNA as template. Using a singlestrand specific nuclease digestion method to generate specific RNA *DNA hybrids, sub-
TABLE
4
SUMMARY OF CAT CELL CULTURES RELEASING TYPE C VIRUSES OF THE RD/CCC Tissue
Strain
Infectious Spontaneously
FFc2K FFc3K FFc2Lu FFc2Th FFc3Th FFc3Tg FFc9WF FFcBOWF FFcl04WF FFc63RES
a Not
Kidney Kidney Lung Thymus Thymus Tongue Whole fetus Whole fetus Whole fetus Mixed spleen-thymusliver-bone marrow
type
C virus
GROUP release
Cocultivation
NTa -
f -
NT NT + + -
NT NT -
f
I
Inductioncocultivation
+ -
NT NT -
+ NT
NT NT
tested. TABLE
COMPARISON OF HOST RANGE OF RD114, Cell line
CCC-3A FFcGOWF MDCK FCf2Th MDBK NRK MHlG NIH/3T3 SIRC Bu (IMR-41) Mv 1 Lu Tb 1 Lu Pl 1 ut IgH-2 DBS-FRhL-1 A204
Species
Cat Cat Dog Dog cow Kat Rat Mouse Rabbit Buff alo Mink Bat Raccoon Iguana Monkey Human
Tissue
5 CCC, type
AND THE VIRUS FROM FFc2K Supernatant (pmoles
transcriptase activity incorporated)*
FFc2Kb
ccc
RD114
<0.04 1.2 0.2 7.3 0.4 <0.04 <0.04 <0.04 <0.04 0.2 1.1 5.5 <0.04 <0.04 7.5 2.6
<0.04 1.4 0.1 3.4 0.2 <0.04 <0.04 <0.04 0.7 <0.04 <0.04 8.3 <0.04 (0.04 5.6 0.6
<0.04 0.9 1.2 5.6 1.3 <0.04 <0.04 <0.04 0.6 0.9 3.6 2.4 <0.04 <0.04 4.9 1.7
Kidney Fetus Kidney Thymus Kidney Kidney Hepatoma Fetus Cornea Lung Lung Lung Uterus Heart Lung Sarcoma
Q Culture fluids were assayed 10 days after infection. b Virus was obtained from human A204 cell cultures lated from the cat FFc2K cells.
reverse [!HlTMP
CELLS
chronically
producing
the virus
originally
iso-
TYPE
C VIRUSES
FROM
stantial homology (> 60 %) was detected between the grnomes of RD114, CCC, and FFc2K virus, as shown in Table 6. Less than 2 % of the RD114 transcript anneals to RNA from mouse, rat, woolly monkey, pig, and avian type C viruses. Previous studies have shown that RD114 and FeLV are not significantly related (< 5 %) by DNA-RNA hybridization (Bctnveniste and Todaro, 1973). Sincr the CCC and FFc2Ii RNA is only able to sat#urate 60-X0? of the RD114[3H]DNA product’ as compared to the RD114 RNA, it would appear t,hat these viruses, while clearly relatt>d to one another, may not be identiral.
Of t,hc t,rn frtal cat cell cultures studied, six have> been found to produce type C viruses of the RD/CCC group. Two strains spontaneously produce high levels, and the virus can bc visualized by electron microscopy. Four other lines yield infectious virus upon cocult,ivation with a permissive host ~~11. Trt>atmcant of t,he cells with IdU prior to coc>ultivation facilitates t’he detection of typcx C virus rclcaac>. Infectious type C viruscbs have> b(L(>n found in caat crll cultures d(brivcld from kidney, thymus, and lung, as TABLH HTIIMDIZ.ITION
TO THE FROM
Viral
RI)114
6
DNA VIRAL
PKODUCT RNA
ICXA
It 1111-l CCC FFc2K FeLV (Snyder-Theilen Ki -RZuLV Rat (V-NIZK) Woolly monkey Pig (PK.15) Avian myeloblastosis
PREP.IRED
‘2 Hybridizationa
strain)
100 75 66 4.5 1.0 1.4 1.6 1.4 0.3
0 The percent hybridization values have been normalized with respect to the final percent hybridization obt,ained with RI1114 (7OG/;,). The values shown represent average final saturating percentage values obtained after adding varying amount,s of viral R.NA (up to 5 pg) to the RD114 [“H]I>XA product (Benveniste and Scolnick, 1073; Benveniste and Todaro, 1973).
FETAL
CAT
CELLS
5 13
well as from whole embryo cultures. It is of interest that three tissues from one cat fetus (FFc2) yielded virus; one from the kidncly was spontaneously producing virus, while the other two, from lung and thymus, produced tvpe C virus after induction and cocultiva&. On the other hand, thrccl cell strains from another cat fetus (FFc3) \v(bre apparently negative both spontaneously and after cocultivation. Whether still more sensitive methods or the use of diffrrtant pcbrmissive hosts will allow dctcction of typcl C viruses from thcscl cat cells as well as thtl others remains to be determined. The other cat ccl1 culture (l~l~cGOW1~) from which virus has so far not’ bt>cln dctc>ctrld even after induction and cocultivation is unusual among cat cells in being a permissive host for the replication of typt> C virus produced by the fetal cat cells. This cat cell lint: permits viral replication at least as ~~(111as any of the permissive primatcx ccl1 hosts \vc havcl so far tcstvd. This data suggclsts that, the rbndogctnous cat type C viruses may be analogous to cndogcnous mouscb viruscls where, generally, virus from one moustt strain is able to replicate much bc>ttrlr in (~11s from other mouse strains (Hartley pf rrl.. 1970; Aaronson et al., 1969). As in the, mouse’t!pct C virus systems, th(l host restrict,ion is not absolute; with high titrrs of endogcnous virus from th(l CCC cell lint, w(’ have>found that there is some viral production aftclr infection of other feline cells. How-cvr>r, itsis difficult to be certain, at this timch,that this is due to replication of the input virus rathclr than activation of th(l ondog~~noustype, C virus in the indicator cells. 1Iorcovc~r, the Sit0 of CT1iUhr rChktiOri t0 CWi~JgNlOUs type C virus growth in that (aat ~~11is not yclt und(lrstood. From the fntal ccl1 strains testcld \v(t have not bcm abl(t to obtain, cithrr spontanctously or by induction and cocultivation, pvidtqlce for thr production of a type C’ virus of the E’eLV group. A cell lint, ho\\-cvclr, tlcbrivcd from the boncbmarrow of an adult cat Gth lymphoma (E’lb), productbd high titchrs of type‘ C virus wit’h antigcnic and host- range properties similar to that of th(i l”clLV group (unpublished experiments). Th(> results dcscrihed here demonstrate that domestic cat’ cell culturc>s can spontja-
514
TODARO
neously produce type C virus with an immunologically related reverse transcriptase, host range for growth, and, at least in part, the nucleic acid sequence of RD114 or CCC virus. In each instance, these viral properties are distinct from those of the FeLV group of type C feline viruses, although it now appears that certain feline cells are permissive for the RD/CCC virus group. As has been shown, RD114/CCC-like virus can be activated either spontaneously or by chemical induction from several non-virus-producing feline cells, whether single cell clones or diploid strains. Importantly, as shox-n by the results of the A204 cocultivation experiments, certain feline cells spontaneously produce low levels of virus, which, except for the permissive cell amplification system, might have gone otherwise undetected. Thus, the presence of six additional isolates of RD114/ CCC like type C virus indicates the presence of a definable class of viruses that can be frequently expressed by normal cat cells. Since members of the FeLV and RD/CCC groups can readily be distinguished from one another, it will be of interest to clarify their relationship to normal feline physiology and to the diseases affecting cats. Whether the type C viruses of the RD/CCC group play a role in the naturally occurring leukemias, lymphomas, and other spontaneous tumors in cats is not yet resolved. The suggestion that FeLV is an endogenous virus of another species which is now endemic in cats (Todaro, 1973), spreading readily from cell to cell and from animal to animal in the manner of a more conventional infectious virus (Hardy, et al., 1973), should also be considered. The use of cat cells harboring a latent type C virus that readily grows in primate and human cells may constitute a potential risk for those working with such cells. Similarly, the use and handling of live virus vaccines prepared in cat cells which are spontaneously producing RD group viruses could also constitute a potential risk to exposed humans. ACKNOWLEDGMENTS We thank Charlote Meyer, Celeste Meade, Lou Fedele, and Don Stuart for their excellent assistance. The research was supported in part by a contract from the Special Virus Cancer Program
ET
AL.
of the National Cancer Institute tories, Springfield, Virginia.
to Meloy
Labora-
REFERENCES AARONSON, S. A., HARTLEY, J. W., and TODARO, G. J. (1969). Mouse leukemia virus: “spontaneous” release by mouse embryo cells after long term in vitro cultivation. Proc. Nat. Acad. Sci. USA 64, 87-94. AARONSON, S. A., TODARO, G. J., and SCOLNICK, E. M. (1971). Induction of murine C-type viruses from clonal lines of virus-free BALB/3T3 cells. Science 174, 157-159. ARMSTRONG, J. A,, PORTERFIELD, J. S. and onMADRID, A. T. (1971). C-type virus particles in pig kidney cell lines. J. Gen. Fz?oZ. 10, 19Fh98. BENVENISTE, R. E., and SCOLNICK, E. M. (1973). RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA. J. FiroZ. 51,370-382. BENVENISTE, R. G., and TODARO, G. J. (1973). Homology between type C viruses of various species as determined by molecular hybridization. Proc. Nat. Acad. Sci. USA (in press). CRANDELL, R. A., FABRICANT, C. G., and NELSONREES, W. A. (1973). The development, characterization and viral susceptibility of a feline (FeZis catus) renal cell line (ccc). Zn Vitro. (In press.) DEINHARDT, F., WOLFE, L. G., THEILEN, G. H., and SNYDER, S. P. (1970). ST-feline fibrosarcoma virus: Induction of tumors in marmoset monkeys. Science 167, 881-882. DUGNGUYEN, H., ROSENBLUM, E. M., and ZEIGEL, R. F. (1966). Persistent infection of a rat kidney line with Rauscher leukemia virus. J. Bacterial. 92, 1133-1140. FISCHINGER, P. J., PEEBLES, P. T., NOMURA, S., and HAAPALA, D. K. (1973). Isolation of an RD114-like oncornavirus from a cat cell line. J. ViroZ. 11, 978-985. GARDNER, M. B., RONGKY, R. W., ARNSTE:IN, P., ESTES, J. D., SARMS, P. S., HUEBNER, R. J., and RICKARD, C. F. (1970). Experimental transmission of feline fibrosarcoma to cats and dogs. Nature (London) 226, 807-809. HARDY, W. D., JR., GIXRING, G., OLD, L. J., DEHARVEN, E., BRODEY, R. S., and MCDONOUGH, S. (1969). Feline leukemia virus: occurrence of viral antigen in the tissues of cats with lymphosarcoma and other diseases. Science 166, 1019-1021. HARDY, W. D., JR., OLD, L. J., HESS, P. W., ESSEX, M., and COTTER, S. (1973) Horizontal transmission of feline leukemia virus. Nature (London) 224, 266-269. H,IRTLEY, J. W., Itown, W. P., and HUEBNER,
TYPE
C VIRUSES
FROM
I:. J. (1970). Host range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Viral. 5, 221-225. J \TKCIITLL, J. I,., A.~RONSON, S. A., and TOD~RO, (;. J. (1969). Murine sarcoma and leukemia viruses: assay using clonal lines of cont,act inhibited cells. J. Viral. 4, 549. JIILRKTT, W. F., CUWFORD, E. M., MARTIN, W. B., and I)I\vIF:, F. (1964). A virus-like particle associated with leukemia (lymphosarcoma). Salure (Londott) 202, 567-569. K.\\v.~K.uvII, T. (+., THISILKN, (+. H., DUNCV-ORTH, I). 1,. , MUXS, li. J., and BEALL, S. G. (1967). “(:“-t,ype viral particles in plasma of cats with lerlkemia. Scie~rcr 1049, 158-159. KLK~~I.:NT, V., N~co~,so,u, M. O., HUETINER, Ii. J., (~ILDICN, 1~. V., 01toszL~4~, S., b~M.4, P. Fi., l~os~r:~, R. W., and (GARDNER, M. B. (1971). Rat C-type virus indured in rat sarcoma cells by 5.bromodeoxyuridine. .Vafrrre (Londo?z) 12, 234237. Lr,:b,, Ii. I,., XOMUR.\, S., BASSIN, It. H., and FISC’HIKGKI<, P. J. (1972). Use of an established cat rell line for investigation and quantitation of feline oncornaviruses. J. ~Vul. Cancer Ivat. $9, 5560. LIE~KI~, M. M., BENVENISTP:, R. E., LIVINGSTON, 1). M., and T~TMRO, G. J. (1973). Mammalian cells in cultsure frequently release type C viruses. Scieuce, in press. LIUSGSTON, 1). M., and TOD.\RO, Cr. J. (1973). k;ndogenous type C virus from a cat cell clone with properties distinct from previously described feline type C viruses. Virology 53, 142151. Low>-, 1). R., Kowlc, W. P., TEICH, N., and HURTLKY, J. W. (1971). Murine leukemia virus: High frequency activation i,/ vitro by 5.iododeoxyuridine and 5-bromodeoxyuridine. Science 174, 155-156. Mc~I,I,IsTI~:~~, 1~. M., NICHOLSON, M. O., (:ARDKER, M. B., RONGKY, R. W., RSHEID, S., S.II~M.~, 1’. S., HUEANKH, R. J., HATAN~KA, M., OaoszL\S, s., (;lLI)~N, I<. V., K3BIGTING, A. and VFXWON, L. (1972). C-type virus released from cldhlred human rhabdomyosarcoma cells. Naftrre (London) 23.5, 3-6. MCALLISTER, Ii. ill., NICHOLSON, M., GSRDNER, M. B., I~.ZSHPXI), S., RONGEY, 1~. W., HARDY, W. I)., and C~ILD~:N, I<. V. (1973). RD-114 virus compared with feline and murine type C viruses
FETAL
CAT
CELLS
released 75-78.
from
1~11 cells.
5 15 .Yafwp
(Ltm~toor~‘~
242,
S., H~KHNKR, li. J., and (TILDES, R. V. (1971). Species-specific and interspecies nntigenie determinants associated with the strnctural protein of feline C-type virrrs. Prrv, .Yat. Acad. Sci. USA 68, 901-904. OROSZL.\N, S., Bov.\, l)., M.\RTIN-WHITE, RI. IT., TONI, lt., FOIZF:M.\N, C., and (;ILDF:N, I(. V. (1972). Purifiration and immunologica characterixat,ion of t.he major internal prot,ein of the KD-114 virus. Prof. A1-n/. dco((. Sri. 7-R.f 69, 1211-1215. P.\Rss, w. P., 8C’0LNICK, E. M., I
OROSZL,\N,
66,
506-515
Infectious GEORGE
Viral
(1973)
Type
C Viruses
J. TODARO,
Leukemia
Released
by
Normal
RAOUL E. BENVENISTE, DAVID $1. LIVINGSTON
and Lymphoma
MICHAEL
Branch, National Cancer Institute, Bethesda, Maryland 20014 Accepted
July
Cat
Embryo
Cells
M. LIEBER,
Sational
Institutes
AND
of Health
10, 1973
Type C viruses with antigenic and host range properties similar to the RD114 virus and to a virus from a continuous line of cat kidney cells, CCC, have been isolated from six of ten diploid fetal cat cell cultures. Each of the new viruses replicates readily in human and rhesus monkey cells, but not in most other cat cell strains tested. However, a cat cell strain, FFcSOWF, was found that is permissive for replication of the RD/CCC group of type C viruses produced by other cat cell strains. The host range of these endogenous type C viruses includes bat, dog, mink, and rabbit cells, as well as primate cells. Type C viruses of the FeLV group could not, be detected in any of these ten fetal cat cell cultures. Viral RNA from one of the new isolates, from the CCC virus, and from RD114 virus each specifically anneal SI to an [3H]DNA transcript of the RD114 genome.
INTRODUCTION Domestic cats of various breeds have been found to contain t.ype C viruses which can cause leukemias and sarcomas (Jarrett et al., 1964; Kawakami et al., 1967; Rickard et al., 1969). These viruses have been isolated from the tissues of both tumor-bearing and normal cats and can be transmitted experimentally to various species, such as cats, dogs, rabbits, and monkeys (Gardner et al., 1970; Deinhardt et al., 1970; Rabin et al., 1972). The great majority of the type C feline leukemia virus (FeLV) isolates possess a common group-specific antigen (Hardy et al., 1969; Oroszlan et al., 1971) and a common reverse transcriptase antigen (Parks et al., 1972; Scolnick et al., 1972). Each of these common markers is distinctive enough to define a group of related viruses isolated from cat tissues. Subsequently, a type C virus, RD114, was isolated after passing the RD human rhabdomyosarcoma cell line through a fetal cat (McAllister et al., 1972). This virus has immunologic and host range properties different from previously isolated feline type C viruses (McAllister et al., 1972,1973; Oroszlan et al., 1972). 506 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
Recently, it was found that a type C virus could be induced from virus-free cell clones of the CCC continuous line of cat kidney cells (Livingston and Todaro, 1973; Fischinger et al., 1973). The type C virus from the CCC cells has antigenic properties, host-range properties, and viral-int’erference patterns virtually indistinguishable from those of RD114 (Livingston and Todaro, 1973; Fischinger et al., 1973). Since this virus can be induced from single cell clones of virusfree cat cells, it was suggested that the RD114 virus was a member of a group of endogenous type C viruses of the cat. To test whether viruses of this RD/CCC group could be found in diploid cat cells as well as in an established line and to test whether potentially infectious type C virus of this group was widespread in the domestic cat, we have examined cultures derived from a variety of feline tissues. MATERIALS
AND
METHODS
Cells. All cells were grown on plastic surfaces (Falcon Plastics, Ontario, CA) in Dulbecco’s modification of Eagle’s minimal essential medium containing 10 % calf serum
TYPE
C VIIZUSES
FROM
(Colorado Swum Co., Denver, CO) at 37°C. Cell cultures at either the third or the sixth passage were ohtaintbd as frozen ampoulcs from the Cell Culture Laboratory, Naval Biomrdical Research Laboratory, Oakland, CA. The fetal ccl1 strains includrd t\vo from kidwy (P‘Fc’LK and P’E’cSK), one from lung (I:I:&Lu), t,wo from thymus (IcE’c2Th and E’FcJTh), ow from tongue (FFc3Tg), thrw from whole c>mbryo (F’FcSWF; F’FcGOWF; and orw mixc>d spleen, thymus, liver, and boric marrow (FFcli3RES)). CCC is a clonal lirw of feline kidney fibroblasts isolated and charactNcrizedby Crandcll et al., CCC-%4 is a non-virus-producing subclonr of CCC kindly provided by Dr. Peter E’ischingcr (National Cancer Institutc). FEC is a strain of feline ~~mbryonic wlls (Lw et al., 1972). A’204 is a continuous lirw of human rhabdomyosarcoma cells dcvclopcld in this laborat’ory, and DBS-FRhL-1 (DBS-1) is a continuous line derived from fetal rhwus monkey lung fibroblasts and was kindly provided by Dr. Roslyn Wallace, Lederlc Laboratories, Pearl River, KY (Wallace et al., 1973). The various other cell cultures used in host range studies were obtainod from the> American Type Cult’ure Collcc+ion, Itockville, MD, c>xwpt,for a fetal caninn thymus liw, FCf2Th, which was obtaiwd in it,s 42nd passagefrom the Naval Biomndical Rwarch Laboratory, Oakland,
c.4. I’iw,ses. The> ICirsten strain of murine lruk(mia virus (Iii1JuLV) was grown in mouse NIH/BT3 cells (Jainchill et al., 1969). V-NRli is an cwdogenous rat typ(l C virus spontaneously wlcased from certain subclonts (Liebcr et al., 1973) of the NRIi lint (Due-Nguyen et al., 1966) of rat kidney fibroblasts and is continuously produced by thaw subclones. Thr woolly monkey type C virus (Wolf(, et al., 1972) was grown in this labortory in p1204 cells. Pig type C virus is cwntinually producrd by the porcine kidney c~pithcliul cell lint: (PI<-1.5) (Armstrong e( al., 1971; Licxber et al., 1973); this cell lint was obtained from the American Type Culture Collection (Rockville, MD). RD114 virus was grown in the RD linct of human Ihahdomyosarcorrla cells, while CCC virus \vas continually wcrt+>d by thcl Crandcll
FETAJ,
i-f,7
C:AT CKI,I,S
(CCC) fcliw kidncy ccl1 line. Vivian myeloblast’osis virus was provided by Dr. Jowph Brard t8hrough tht> Rcsourcw and Logistics Ofhw, Viral Caner Program, Sationdl Canwr Instit,utc. Thcl Gardner-Arnsbrin strain of f&w sarcoma virus (GA-P&V) was obtjairwd from El(~ct,ro-Sucl~onicv Laborat oriw, Bcthcsda, AID. Snyd~~r-Thc~ilcnstrain f&w kwlwmia virus was ohtaitwd from Universit,~ Laboratories, Highland Park, S.J. I’I’r7~s
tletecfifw
by superriafaul
wwrse
lmrr-
assay. This was pcrformcvt by the mc$thod of Ross ef. al. (1971). I:iftcw millilitws of mcsdium from each tissw caultjure flask was claritic~d by wntrifugation at 1‘2,OOO~q for 10 min. The supcrnatnilt was thaw tranxferwd to a wn- tubv and p(~lletc~d by wntrifugation through 20 “; gl~~ccrcllin 0.05 31 TrissHCl, pH 7.S; 0.10 JI KC1 at 10.5,OOOc/ for 90 miti at 4°C. Tlic, pcill(lt 11as rcsuspcndt>d in 0.1 ml of 0.0.5 .II Tris HCl, pH 7.S; 0.10 ilf SaCl; lo-” 11Ldithiothrc~itol (DTT) buffw wntainingO.l 5’;Triton X-100. Virus suspension (0.01 ml) \v:is :rdd(ld to a. O.Oli-ml rc~achionmixt,urcl containing 0.05 :I/ Tris-HCl, pH 73; 0.06 M IiCl; 2 X IO-:’ ;11 DTT; 5 X lo-? 111manganrw uwtatc~; 0.012 ‘4zs,,units poly(rA) (1lilrs Laboratoricu, 151khart, IS) ; 0.02 A2G0units oligo (dTp:-,,) (Collaborativcb Restawh, Waltham, ;\LZ 1; and 3 X 1O-6 :l[ (“H]TTI’ (40,000 (‘pm; p~nol~~i (KU\- I~:iigland Xwlwr Corporation, Best on. 11.4). Rcwults :w t~xprwwd as pic~omol(3of [aH]TXI I’ inc~orporatc~d illto radioact iw poly(dT) product in a GO-min iwubation at 37°C. 6-lotlot/eolryu;~jffi/~e (Id1 ‘) irdurfiotr f!i’ ljype C r+w. .~-Iodod(,ox~uridiI~(~ was ~nrrc~tlast~d from Calbiochr~m, Los Angelw, (‘.\. ‘I%~ iododtaoxyuriditwtreatcd culture UXYY’pwparrd by twating 6 X 10” wlls of ~(~1~st8rain lvith 100 pg/ml of drug in complt~tc~media for 2%hr at 37°C. Th(l mcxdinwas c~hangctit\z.iw \vith a 30.min intwval at 37Y’. and that culturw incubatc>d for 2 morr days. .1t that t)irncl,th(> wlls \vc:rcatrypsinizcd. mtl approximatc,ly .5 X IO” wlls \v(‘r(’ :tdd(~d to t’rwh indicator wll wlturw which had bwu sc~~icd thr day Irwfow at 3 X 10: wlls pc’t’ 7.1,cm2 flasli in cornpl(+cxmedia wmtailliiig pol!~twcwc~ (2 pg/ml).
soiptase
508
TODARO ET AL.
n&ant fluids from virus-producing cell cultures were filtered through 0.45 p Millipore filters (Millipore Co., Bedford, Mass.) and 1.5 ml was used to infect indicator cells plated the day previously at 3 X lo5 cells per flask in complete medium with polybrene (2 pg/ml). After a 1-hr adsorption period, complete media was added. Molecular hybridization technique. The production of reverse-transcriptase-catalyzed [3H]DNA transcripts of various types of viral RNA and the detection of their hybridization to purified viral 70s RNA have been reported elsewhere (Benveniste and Scolnick, 1973). The source of RD114 and CCC virus has been previously described (Livingston and Todaro, 1973). The virus from FFc2K cells was propagated in the human rhabdomyosarcoma cell line, A204; the supernatants were concentrated, and the virus isopycnically banded in sucrose. Polymeruse antibody-inhibition studies. Putative virus was concentrated, and standard reverse transcriptase reaction mixtures were used as described above. Antisera against viral polymerases were prepared as described previously (Parks et al., 1972). Reactions were initiated with a mixture of template, primer, and substrate. Detergent-disrupted virus (0.01 ml) diluted in 0.05 M Tris-HCl, pH 7.8; 0.10 M NaCl; lop3 M DTT was employed in each assay, and enzyme was added prior to IgG. In a standard reaction, a quantity of enzyme which led to incorporation of 0.184.20 pmoles of [3H]TMP (40,000 cpmjpmole) in 60 min was routinely added. Electron microscopy of cell mon.olayers.The cell monolayer was fixed in situ with buffered 2 % glutaraldehyde. The cells were then scraped off, pelleted, and post-fixed with 1% buffered osmium tetroxide. After incubation overnight in cold 0.5 % uranyl acetate: they were dehydrat,ed and embedded in Epon. Thin sections were doubly stained in uranyl acetate and lead citrate and examined with a SiemensElmiskop 1A electron microscope. RESULTS Virus production by primary cultures 4f feline cells. Ten fetal cat cell cultures were tested for spontaneous type C virus production by assaying their media for viral reverse
transcriptase activity. Two of the ten cultures, a fetal cat kidney strain (FFc2K) and a fetal cat strain from mixed spleen, thymus, liver, and bone marrow (FFc63RES), showed high levels of reverse transcriptase activity in their supernatant fluids and typical type C virus particles by electron microscopy. Figure 1 showselectron micrographs of budding and extracellular type C virus from the FFc2K cells. Immunologic relationship of the various feline viral polymerases. The reverse transcriptase activity in the supernatant fluids of these two cultures was not substantially inhibited by IgG from antiserum to the FeLV polymerase (Fig. 2). The enzymatic activity from both cell cult,ures, however, was markedly inhibited by antibody to the RD114 polymerase (> 50 % with 20 pg of anti-RD114 polymerase), suggesting that, like RD114 and CCC virus, these cells were producing a virus with properties quite different from FeLV. Host range properties of various feline type C viruses. The FFc2K and FFc63RES cells were cocultivated with a variety of cat cells (CCC-3A, FEC, FFc2Th, and FFcGOWF), a rhesus monkey cell line (DBS-FRhL-I), and a human rhabdomyosarcoma cell line (A204). As shown in Table 1, the virus grew readily in the two primate cell lines, but was unable to infect most of the feline cells. One exception is a cat cell line, FFcGOWF, derived from whole embryo tissue, which readily supports the growth of the FFc2K virus. Similar host range properties have been found for t,he CCC and RD114 viruses (Livingston and Todaro, 1973). Table 2 showsthat these viruses, whether growing in feline or in human cells, arc similarly inhibited by RD114 anti-polymerase IgG and not by control IgG or anti-FeLV polymerase IgG. Scolnick et al. (1972) have previously shown that there is no significant inhibition of FeLV polymerase by anti-RD114 polymerase IgG, or vice versa. Similarly, there is no detectable antigenic cross reaction between FeLV and CCC virus polymerase (Livingston and Todaro, 1973). Induction of infectious type C virus from nonvirus,producing feline cell strains. Lung cells (FFc2L) and thymus cells (FFc2Th) from the same cat fetus (FFc2) which had
510
ET AL.
TODARO
spontaneously yielded type C virus from its kidney cells were also tested for virus production by the supernatant reverse transcriptase assay. Neither produced detectable levels of type C virus spontaneously. Therefore, these cultures were treated with 5-iododeoxyuridine (IdTJ) (Lowy et al., 1971; Aaronson I
I
1
I 20
I
I
I 60
I
100 75 50 25
100 75 50 25 I 40 W
bd
FIG. 2. Inhibition of reverse transcriptase from supernatants of fetal cat cells by antisera IgG. Putative virus was concentrated, and standard reaction mixtures were used as described in Methods. Values given are percentage of [“H]TMP incorporation in the presence of IgG relative to that in its absence. (Top) FFc2K, (Bottom) FFc63RES. (0) Enzyme plus nonimmune IgG, (0) enzyme plus anti-FeLV polymerase IgG, (0) enzyme plus anti-R.Dll4 polymerase IgG.
et al., 1971; Klement et al., 1971) then cocultivated with various indicator cells. The IdU-treated feline lung cells and thymus cells produced an infectious, reverse-transcriptase-containing particle which grew readily in human rhabdomyosarcoma cells (A204) and in the rhesus monkey cell line, DBS-FRhL-1, but not in three of the four cat cells tested (Table 1). It, however, did grow well in FFc60WF. In each instance, the polymerase of the virus from the induced and cocultivated cultures was specifically inhibited by the anti-RD114 polymerase IgG (Table 2). The extent of polymerase inhibition of these four cat type C virus isolates grown in human cells, as tested with antiRD114 polymerase IgG, was comparable to that obtained with the induced or the spontaneously produced type C virus from the continuous line of cat kidney cells, CCC. Isolation of endogenousvirus by cocultivation with permissivecells. Although no significant reverse transcriptase activity was found in the culture supernatants from a number of fetal cat cell strains, this biochemical test is less sensitive than certain biological assays for detecting type C virus in other systems. To enhance the possibility of detecting lowlevel spontaneous producers of RD/CCC virus, a group of apparently type C virusnegative strains of cat cells were cocultivated with the permissive human host line, A204. Of the four strains tested, FFcSWF, FFcGOWF, FFcl04WF, and FFc3Tg, two
TABLE HOST Cell
RANGK
OF CAT TYPE
1 C VIRAL
Origin
Viruses FFc2Ka Supernatant
CCC-3A FEC FFc2Th FFcGOWF DBS-FRhL-1 A204 a Filtered supernatant b 5-Iododeoxyuridine c Not tested.
ISOLATES
Feline Feline Feline Feline Rhesus Human. fluids treated
monkey
<0.04 <0.04 <0.04 9.14 1.88 0.89
FFc2L* (IdU treated)
FFc63RES”
Reverse Transcriptase H3-TMP incorporated) <0.04 <0.04 <0.04 3.63 NT 1.20
used to infect as described in Materials and and cocultivated as described in Materials
Activity
<0.04 <0.04 NT NT 0.43 1.85 Methods. and Methods.
FFc2Thb (IdU treated (pmoles,
<0.04 NT” NT NT NT 1.52
TYPE TABLK
<: VIE:UHES
FROM
FETAL
CAT
CELLS
2
TABLI1:
C~CULTIVATION
OF
A204 Cat cell strain a? Inhibition of [3H]TMP incorporation in supernatant reverse transcriptase assay” Type
of IgG Added
Control
Virrls from cat (aells FPc2K 67
FFcYWF FFcGOWF FFcl04WF FFc3Tg
51 1 3
C.\T CELL CULTDKI:S KITEI THE HUM-IN CELL 1,1x1.; Supernatant polymerase activity (pmoles [zH]‘I’MP incorporated)* I.7
___u Assayed at 3 weeks and after two subcultures of t,he cocultivnted cells. By this time, t,he culture consists almost entirely of the more rapidly growing human rhabdomyosarroma cells, .420-t.
sarcoma ccl1line, ,4204, \vas uscldsince it had previously been shown to ho a pcrmissivr: host for the type C viruses from the CCC cat cell lincx (Livingston and Todnro, 1973) and from thtl spontaneously type C virusreleasing fetal cat rcllls (FFc2Ii and FI~cMRES). Ability of emlogel~,ous virus of the cat to ~/ww in clistady related cell hosts.Table 5 shows a more extensive study of the host range of the virus being spontaneously produced by the fetal cat kidney strain, I~I’c2Ii. It is compared with the host) rang{’ of two related viruses, RD114 and CCC. Xonc~ of the viruses successfully infect, cat CCC-SA cells, but all can grow in the fetal cast strain, FI’cGOWI~‘. The mouse, rat, and raccoon cells uscbdin this study did not support virus replicat,ion, but, the viruses all rradily repliyirldrld infec*tious polymctrasc:-cont,aining cated in fetal dog thymus cells (ICf2Th) and particlcs after 3 weeks and two suhcultivain bat lung cells (TblLu). Thexcl cell strains tions of tht> mixed cell cultures (Table 3). In appear comparable in their ability- to support both inst’ances, tho enzymes were inhibited virus replication to the primate (rhesus by anti-RD114 polymerasc IgG, but not monkey and human) cells that wcrc prclI&(; directc>d against the I~cLV enzyme viously described as permissive hosts (Liv(Table 2). ingston and Todaro, 1973). Thr two cndogTablr> 4 summarizes the stud& used to cnous type C viruses growing in human detect’ type‘ C viruses released by fetal feline cells, IJFc21< virus in A204 cells and RD114 cc&. Six of the ten lines test,ed produce a virus, arc’ also able to grow in mink and t\.pc‘ C virus related to CCC and RD114. buffalo lung cells, \vhile the endogcnous virus TR.Oof these>strains product enough virus to from CCC cells appears unable to grow. This bc dr:tc>ctcd directly in t’he supcrnatant fluids may rrflect a diffcrcnco in the three isolates, of tho cultures themselves by the reverse or, perhaps, R host’ modificat,ion resulting transcriptasr> assay; four others rcyuire co- from the replication by RD114 and FFc2K cultivation with a permissive host cell. In in human cells. The viruses in the info&d thcxsct ctxpfriments t,hc human rhabdomyo- buffalo lung ~~11sand dog thymus ~~11s havtl
512
TODARO
ET
a polymerase with the antigenic properties of those of the RD/ ‘CCC group. The four fetal ck cell strains that are apparently virus-negative have not been exhaustively tested by induction and cocultivation with each of the potentially permissive host cells shown in Table 5. Nucleic acid homology of RNA from various
AL.
type C viruses with the DNA transcript of RDll4 RNA. Viral RNA extracted from the RD114, CCC, and FFc2lX viruses was annealed to the [3H]DNA product of an “endogenous” reverse transcriptase reaction with RD114 RNA as template. Using a singlestrand specific nuclease digestion method to generate specific RNA *DNA hybrids, sub-
TABLE
4
SUMMARY OF CAT CELL CULTURES RELEASING TYPE C VIRUSES OF THE RD/CCC Tissue
Strain
Infectious Spontaneously
FFc2K FFc3K FFc2Lu FFc2Th FFc3Th FFc3Tg FFc9WF FFcBOWF FFcl04WF FFc63RES
a Not
Kidney Kidney Lung Thymus Thymus Tongue Whole fetus Whole fetus Whole fetus Mixed spleen-thymusliver-bone marrow
type
C virus
GROUP release
Cocultivation
NTa -
f -
NT NT + + -
NT NT -
f
I
Inductioncocultivation
+ -
NT NT -
+ NT
NT NT
tested. TABLE
COMPARISON OF HOST RANGE OF RD114, Cell line
CCC-3A FFcGOWF MDCK FCf2Th MDBK NRK MHlG NIH/3T3 SIRC Bu (IMR-41) Mv 1 Lu Tb 1 Lu Pl 1 ut IgH-2 DBS-FRhL-1 A204
Species
Cat Cat Dog Dog cow Kat Rat Mouse Rabbit Buff alo Mink Bat Raccoon Iguana Monkey Human
Tissue
5 CCC, type
AND THE VIRUS FROM FFc2K Supernatant (pmoles
transcriptase activity incorporated)*
FFc2Kb
ccc
RD114
<0.04 1.2 0.2 7.3 0.4 <0.04 <0.04 <0.04 <0.04 0.2 1.1 5.5 <0.04 <0.04 7.5 2.6
<0.04 1.4 0.1 3.4 0.2 <0.04 <0.04 <0.04 0.7 <0.04 <0.04 8.3 <0.04 (0.04 5.6 0.6
<0.04 0.9 1.2 5.6 1.3 <0.04 <0.04 <0.04 0.6 0.9 3.6 2.4 <0.04 <0.04 4.9 1.7
Kidney Fetus Kidney Thymus Kidney Kidney Hepatoma Fetus Cornea Lung Lung Lung Uterus Heart Lung Sarcoma
Q Culture fluids were assayed 10 days after infection. b Virus was obtained from human A204 cell cultures lated from the cat FFc2K cells.
reverse [!HlTMP
CELLS
chronically
producing
the virus
originally
iso-
TYPE
C VIRUSES
FROM
stantial homology (> 60 %) was detected between the grnomes of RD114, CCC, and FFc2K virus, as shown in Table 6. Less than 2 % of the RD114 transcript anneals to RNA from mouse, rat, woolly monkey, pig, and avian type C viruses. Previous studies have shown that RD114 and FeLV are not significantly related (< 5 %) by DNA-RNA hybridization (Bctnveniste and Todaro, 1973). Sincr the CCC and FFc2Ii RNA is only able to sat#urate 60-X0? of the RD114[3H]DNA product’ as compared to the RD114 RNA, it would appear t,hat these viruses, while clearly relatt>d to one another, may not be identiral.
Of t,hc t,rn frtal cat cell cultures studied, six have> been found to produce type C viruses of the RD/CCC group. Two strains spontaneously produce high levels, and the virus can bc visualized by electron microscopy. Four other lines yield infectious virus upon cocult,ivation with a permissive host ~~11. Trt>atmcant of t,he cells with IdU prior to coc>ultivation facilitates t’he detection of typcx C virus rclcaac>. Infectious type C viruscbs have> b(L(>n found in caat crll cultures d(brivcld from kidney, thymus, and lung, as TABLH HTIIMDIZ.ITION
TO THE FROM
Viral
RI)114
6
DNA VIRAL
PKODUCT RNA
ICXA
It 1111-l CCC FFc2K FeLV (Snyder-Theilen Ki -RZuLV Rat (V-NIZK) Woolly monkey Pig (PK.15) Avian myeloblastosis
PREP.IRED
‘2 Hybridizationa
strain)
100 75 66 4.5 1.0 1.4 1.6 1.4 0.3
0 The percent hybridization values have been normalized with respect to the final percent hybridization obt,ained with RI1114 (7OG/;,). The values shown represent average final saturating percentage values obtained after adding varying amount,s of viral R.NA (up to 5 pg) to the RD114 [“H]I>XA product (Benveniste and Scolnick, 1073; Benveniste and Todaro, 1973).
FETAL
CAT
CELLS
5 13
well as from whole embryo cultures. It is of interest that three tissues from one cat fetus (FFc2) yielded virus; one from the kidncly was spontaneously producing virus, while the other two, from lung and thymus, produced tvpe C virus after induction and cocultiva&. On the other hand, thrccl cell strains from another cat fetus (FFc3) \v(bre apparently negative both spontaneously and after cocultivation. Whether still more sensitive methods or the use of diffrrtant pcbrmissive hosts will allow dctcction of typcl C viruses from thcscl cat cells as well as thtl others remains to be determined. The other cat ccl1 culture (l~l~cGOW1~) from which virus has so far not’ bt>cln dctc>ctrld even after induction and cocultivation is unusual among cat cells in being a permissive host for the replication of typt> C virus produced by the fetal cat cells. This cat cell lint: permits viral replication at least as ~~(111as any of the permissive primatcx ccl1 hosts \vc havcl so far tcstvd. This data suggclsts that, the rbndogctnous cat type C viruses may be analogous to cndogcnous mouscb viruscls where, generally, virus from one moustt strain is able to replicate much bc>ttrlr in (~11s from other mouse strains (Hartley pf rrl.. 1970; Aaronson et al., 1969). As in the, mouse’t!pct C virus systems, th(l host restrict,ion is not absolute; with high titrrs of endogcnous virus from th(l CCC cell lint, w(’ have>found that there is some viral production aftclr infection of other feline cells. How-cvr>r, itsis difficult to be certain, at this timch,that this is due to replication of the input virus rathclr than activation of th(l ondog~~noustype, C virus in the indicator cells. 1Iorcovc~r, the Sit0 of CT1iUhr rChktiOri t0 CWi~JgNlOUs type C virus growth in that (aat ~~11is not yclt und(lrstood. From the fntal ccl1 strains testcld \v(t have not bcm abl(t to obtain, cithrr spontanctously or by induction and cocultivation, pvidtqlce for thr production of a type C’ virus of the E’eLV group. A cell lint, ho\\-cvclr, tlcbrivcd from the boncbmarrow of an adult cat Gth lymphoma (E’lb), productbd high titchrs of type‘ C virus wit’h antigcnic and host- range properties similar to that of th(i l”clLV group (unpublished experiments). Th(> results dcscrihed here demonstrate that domestic cat’ cell culturc>s can spontja-
514
TODARO
neously produce type C virus with an immunologically related reverse transcriptase, host range for growth, and, at least in part, the nucleic acid sequence of RD114 or CCC virus. In each instance, these viral properties are distinct from those of the FeLV group of type C feline viruses, although it now appears that certain feline cells are permissive for the RD/CCC virus group. As has been shown, RD114/CCC-like virus can be activated either spontaneously or by chemical induction from several non-virus-producing feline cells, whether single cell clones or diploid strains. Importantly, as shox-n by the results of the A204 cocultivation experiments, certain feline cells spontaneously produce low levels of virus, which, except for the permissive cell amplification system, might have gone otherwise undetected. Thus, the presence of six additional isolates of RD114/ CCC like type C virus indicates the presence of a definable class of viruses that can be frequently expressed by normal cat cells. Since members of the FeLV and RD/CCC groups can readily be distinguished from one another, it will be of interest to clarify their relationship to normal feline physiology and to the diseases affecting cats. Whether the type C viruses of the RD/CCC group play a role in the naturally occurring leukemias, lymphomas, and other spontaneous tumors in cats is not yet resolved. The suggestion that FeLV is an endogenous virus of another species which is now endemic in cats (Todaro, 1973), spreading readily from cell to cell and from animal to animal in the manner of a more conventional infectious virus (Hardy, et al., 1973), should also be considered. The use of cat cells harboring a latent type C virus that readily grows in primate and human cells may constitute a potential risk for those working with such cells. Similarly, the use and handling of live virus vaccines prepared in cat cells which are spontaneously producing RD group viruses could also constitute a potential risk to exposed humans. ACKNOWLEDGMENTS We thank Charlote Meyer, Celeste Meade, Lou Fedele, and Don Stuart for their excellent assistance. The research was supported in part by a contract from the Special Virus Cancer Program
ET
AL.
of the National Cancer Institute tories, Springfield, Virginia.
to Meloy
Labora-
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TYPE
C VIRUSES
FROM
I:. J. (1970). Host range restrictions of murine leukemia viruses in mouse embryo cell cultures. J. Viral. 5, 221-225. J \TKCIITLL, J. I,., A.~RONSON, S. A., and TOD~RO, (;. J. (1969). Murine sarcoma and leukemia viruses: assay using clonal lines of cont,act inhibited cells. J. Viral. 4, 549. JIILRKTT, W. F., CUWFORD, E. M., MARTIN, W. B., and I)I\vIF:, F. (1964). A virus-like particle associated with leukemia (lymphosarcoma). Salure (Londott) 202, 567-569. K.\\v.~K.uvII, T. (+., THISILKN, (+. H., DUNCV-ORTH, I). 1,. , MUXS, li. J., and BEALL, S. G. (1967). “(:“-t,ype viral particles in plasma of cats with lerlkemia. Scie~rcr 1049, 158-159. KLK~~I.:NT, V., N~co~,so,u, M. O., HUETINER, Ii. J., (~ILDICN, 1~. V., 01toszL~4~, S., b~M.4, P. Fi., l~os~r:~, R. W., and (GARDNER, M. B. (1971). Rat C-type virus indured in rat sarcoma cells by 5.bromodeoxyuridine. .Vafrrre (Londo?z) 12, 234237. Lr,:b,, Ii. I,., XOMUR.\, S., BASSIN, It. H., and FISC’HIKGKI<, P. J. (1972). Use of an established cat rell line for investigation and quantitation of feline oncornaviruses. J. ~Vul. Cancer Ivat. $9, 5560. LIE~KI~, M. M., BENVENISTP:, R. E., LIVINGSTON, 1). M., and T~TMRO, G. J. (1973). Mammalian cells in cultsure frequently release type C viruses. Scieuce, in press. LIUSGSTON, 1). M., and TOD.\RO, Cr. J. (1973). k;ndogenous type C virus from a cat cell clone with properties distinct from previously described feline type C viruses. Virology 53, 142151. Low>-, 1). R., Kowlc, W. P., TEICH, N., and HURTLKY, J. W. (1971). Murine leukemia virus: High frequency activation i,/ vitro by 5.iododeoxyuridine and 5-bromodeoxyuridine. Science 174, 155-156. Mc~I,I,IsTI~:~~, 1~. M., NICHOLSON, M. O., (:ARDKER, M. B., RONGKY, R. W., RSHEID, S., S.II~M.~, 1’. S., HUEANKH, R. J., HATAN~KA, M., OaoszL\S, s., (;lLI)~N, I<. V., K3BIGTING, A. and VFXWON, L. (1972). C-type virus released from cldhlred human rhabdomyosarcoma cells. Naftrre (London) 23.5, 3-6. MCALLISTER, Ii. ill., NICHOLSON, M., GSRDNER, M. B., I~.ZSHPXI), S., RONGEY, 1~. W., HARDY, W. I)., and C~ILD~:N, I<. V. (1973). RD-114 virus compared with feline and murine type C viruses
FETAL
CAT
CELLS
released 75-78.
from
1~11 cells.
5 15 .Yafwp
(Ltm~toor~‘~
242,
S., H~KHNKR, li. J., and (TILDES, R. V. (1971). Species-specific and interspecies nntigenie determinants associated with the strnctural protein of feline C-type virrrs. Prrv, .Yat. Acad. Sci. USA 68, 901-904. OROSZL.\N, S., Bov.\, l)., M.\RTIN-WHITE, RI. IT., TONI, lt., FOIZF:M.\N, C., and (;ILDF:N, I(. V. (1972). Purifiration and immunologica characterixat,ion of t.he major internal prot,ein of the KD-114 virus. Prof. A1-n/. dco((. Sri. 7-R.f 69, 1211-1215. P.\Rss, w. P., 8C’0LNICK, E. M., I
OROSZL,\N,