f. gen. Virol. (1975), 27, I73-r8o
I73
Printed in Great Britain
Type C Virus Production by a Continuous Line of
Pig Oviduct Cells (PFT)
By A. M. P. B O U I L L A N T , A. S. G R E I G ,
Animal Pathology Division, Health o f Animals Branch, Agriculture Canada,
Animal Diseases Research Institute (E), P.O. Box 113oo,
Postal Station 'H', Ottawa, Ontario, Canada. K2H 8P9
M. M. L I E B E R AND G. J. T O D A R O
Viral Leukemia and Lymphoma Branch, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
(Accepted 7 January 1975)
SUMMARY
Late passages of the PFT porcine oviduct cell line spontaneously release a typical
type C virus antigenically related to the type C virus released from the PK05)
porcine kidney cell line. The PFT virus was non-infective for a large variety of cell
lines. Type C virus can also be induced by BrdU treatment from earlier passages of
the PFT cell line.
INTRODUCTION
Mammalian cell lines derived from mouse (Hall et aL I967; Kindig & Kirsten, i967),
rat (Gazzolo, Simkovic & Martin-Berthelon, 1971; Bergs et al. I972), hamster (Freeman
et aL 1971 ; Lieber et al. I973a), and cat (Fischinger et aL 1973; Livingston & Todaro, I973)
tissues have been observed to begin producing type C virus particles spontaneously after
a period of in vitro cultivation. Cell lines derived from porcine tissues also appear to have
a high probability of spontaneously producing type C virus, since such virus particles have
frequently been observed by electron microscopy in most high passages of continuous pig
cell lines (Breese, 197o; Armstrong, Porterfield & de Madrid, I97I; Lieber et aL I973a ).
The type C virus released from the PK(I5) pig kidney celt line has been characterized biochemically: it has a density of I'I6 g/ml, contains 70 S virus RNA, RNA-dependent DNA
polymerase with a mol. wt. of 7oooo, and 'gs protein' of mol. wt. about 3oo0o with interspecies antigenic determinants detected by a 'gs-3' competitive radioimmunoassay (Woods
et al. 1973; Todaro et al. 1974a). Similar biochemical and serological results have also been
described for a type C virus (PLCP) released from a porcine lymphoma cell line (Moennig
et aL I974; Strandstr6m et aL 1974). DNA: RNA hybridization experiments have demonstrated that the PK(15) type C virus is distinctly different from the known murine, rat,
feline, hamster, and primate type C viruses, while D N A : D N A hybridization studies with
DNA from normal porcine tissues suggests that PK(IS) is an endogenous, vertically
transmitted virus (Todaro & Huebner, 1972; Todaro et aL 1974a; Benveniste & Todaro,
I975).
A new porcine cell line, PFT, has been established from the oviduct of a 2-year-old sow
(Bouillant, Genest & Greig, I973). Its growth characteristics and susceptibility to virus
infection have been previously described (Bouillant et aL I973; Bouillant et aL I975a;
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A. M. P. B O U 1 L L A N T
AND OTHERS
Bouillant, Genest & Greig, I975 b; Bouillant, Tabel & Greig, 1974)- Over a period of 41 years
the PFT culture has undergone an uninterrupted series of 250 subcultures. We now wish to
report that high passaged cultures of PFT also spontaneously produce type C viruses related
antigenically to the endogenous pig PK(I5) virus. Moreover, earlier passages of PFT can
be induced by chemical treatment to begin producing type C virus particles.
METHODS
Cells. All cells were grown at 37 °C in Eagle's minimal essential medium containing
Io ~o calf serum as previously described (Bouillant et al. I973)- Cell lines were obtained
from the following sources: PK(I5), E. Derm, MvlLu, MDBK, P1 I Ut, from the American
Type Culture Collection, Rockville, Md., U.S.A.; DBS-FRhL-I (Wallace et al. I973) was
a gift from Dr Roslyn Wallace and SC-I was a gift from Dr Janet Hartley, A2o4 was derived
in this laboratory (Giard et aL 1973) and N R K has been described previously (Duc-Nguyen,
Rosenblum & Ziegel, I966). The secondary pig kidney cell strain (PORC) was obtained
from Flow Laboratories, Rockville, Md., U.S.A.
Electron microscopy. At cell sheet confluency, cells of the I7th, 2ISt, 59th, 9oth, I3oth,
165th, 2o5th, 217th and 227th subculture were examined under the electron microscope.
Following trypsinization, they were centrifuged, rinsed in phosphate buffered saline solution,
then fixed in cold 2"5 ~ glutaraldehyde phosphate buffer and post-fixed in cold 2 ~ osmium
tetroxide. After dehydration in increasing concentrations of ethyl alcohol, the cells were
embedded in Spurr medium (Spurr, I963). Sections were post-stained in uranyl acetate and
lead citrate and examined at 6o kV in a Philips 2oi electron microscope.
Reverse transcriptase and polymerase antibody inhibition assays. The techniques for the
supernatant reverse transcriptase assay (Todaro et al. I974a) and the polymerase antibody
inhibition assay (Aaronson et al. I97I; Parks et al. 1972; Scolnick et al. I972; Todaro
et al. I974a) were used exactly as previously described. Antisera directed against partially
purified reverse transcriptase of the porcine PK05) was prepared as follows: PK(15)virus
was concentrated from cells spontaneously releasing it (Todaro et al. 1974a), disrupted
in o'o5 M-tris-HC1 buffer, pH 7"8, containing o.6 M-KC1, I ~ Triton X-Ioo, and 2 mM-2mercaptoethanol, and chromatographed on Sephadex G-Ioo (9ox5.o cm column) as
described previously (Ross et al. I97I). Individual column fractions (I I ml) were assayed
for virus polymerase and the enzyme concentrated by pressure dialysis and used to immunize
rabbits (Parks et aI. I972). Polymerase inhibition studies were performed using purified
'anti-pol' IgG obtained by salt fractionation and DEAE cellulose chromatography (Parks
et al. 1972).
Virus induction. Virus induction was attempted by 5-bromodeoxyuridine (BrdU) treatment (Aaronson, Todaro & Scolnick, I971 ; Lowy et al. I971) at cell confluency with 60/zg[
ml BrdU (Nutritional Biochemicals Corp., Cleveland, Ohio, U.S.A.) in the I65th subculture
and 40 #g/ml in the 17th, 59th, 9oth and I3oth subcultures. After a 3-day treatment, supernatant fluid was discarded and replaced by fresh medium for another 3 days. Untreated
and BrdU-treated cells were processed for electron microscopy six days after starting virus
induction.
RESULTS
The porcine cell line (PFT), derived from oviduct tissue, underwent an uninterrupted
series of 25o subcultures. The general morphology of the cell line was epithelium-like until
approx, the 5oth subculture, when a morphological alteration occurred with the gradual
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Type C virus in pig oviduct cell line
Fig. I. Type C virus spontaneous release in the 2zvth (a) and 2IVth (b, e) subcultures:
complete virus particles (a, b) and budding particle (c).
I00
B
50
w
v
I
lfl
I
l
I
20
30
40
IgG concentration (ug/ml)
I
50
Fig. 2. Effect of anti-PK(I5) polymerase and non-immune lgG on virus polymerase activity.
Assays were performed as described by Parks et al. (I 972) and Todaro et aL (1974 a). Basal activity in
the absence of added IgG was about 25ooo ct/min [3H]-TMP incorporated. O - - O , PFT virus
polymerase incubated with non-immune IgG. Enzymes incubated with anti-PK(I5) polymerase:
0 - - 0 , PFT; ~,__A, PK([5); ~ - - A , feline leukaemia virus (Gardner-Arnstein); v1--D, simian
sarcoma associated virus.
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:t75
I76
A. M. P. BOUILLANT AND OTHERS
Table r. Non-permissive cell lines for replication of the PFT virus*
Species
Cell line
Tissue origin
Human
Rhesus monkey
Horse
Mink
Pig
Cow
Mouse
Rat
Raccoon
A204
DBS-FRhL-I
E. Derm
MvlLu
PORC
MDBK
SC-I
NRK
PI I Ut
Rhabdomyosarcoma
Lung
Skin
Lung
Kidney
Kidney
Foetus
Kidney
Uterus
* Supernatant media from cultures of PFT were filtered through Millipore filters and then used to infect
cultures of the following cell lines set up z4 h previously in complete media containing Polybrene (2 #g/ml).
Additionally, IO8 PFT cells were co-cultivated with Ios host ceils. After I week, supernatant fluids from the
co-cultivated cultures were filtered through Millipore filters and used to infect the respective host cell line set
up 24 h before in media containing Polybrene. The cultures were tested for type C virus replication at Iand 2-month periods after infection; no significant reverse transcriptase activity was present in the supernatant fluid from any of these host cell lines.
emergence of fibroblast-like cells (Bouillant et al. I973). N o type C virus particles (Dalton,
I971) were observed by electron microscopy in cultures of the P F T cell line in I7th, 2ISt,
59th, 9oth, 13oth, I65th and 2o5th subcultures. However, type C virus particles were readily
observed by electron microscopy in the cells of the 217th and 227th subcultures (Fig. I).
Subcultures from the 2o5th and 225th subcultures were also tested for type C virus production by supernatant reverse transcriptase assay. The 2o5th subculture was 'virus-negative'
by this assay, but the 225th and subsequent subcultures were found to produce high levels
of supernatant reverse transcriptase activity. Fig. 2 presents the results of a polymerase
inhibition assay performed using antisera developed against partially purified reverse
transcriptase from the P K U 5 ) virus. Both PK(15) and P F T enzymes are markedly inhibited
( > 80 ~ ) by Io #g/ml of anti-PK(I 5) polymerase IgG; the polymerases from feline leukaemia
virus, simian sarcoma associated virus (Fig. 2), Rauscher murine leukaemia virus, and RDI 14 virus (data not shown) are much less inhibited by this concentration of anti-polymerase
IgG. However, all virus enzymes tested are somewhat inhibited by l g G developed against
the PK(I5) enzyme. This result suggests that the two pig viruses share common antigenic
determinants on their polymerases which are not present on the polymerases of the other
viruses tested, but that there are also common antigens shared by the P K 0 5 ) polymerase
and the polymerases from other mammalian type C virus enzymes. Thus, by these
morphological, biochemical, and serological criteria, the P F T cell line is releasing a typical
mammalian type C virus.
Repeated attempts have been made to find cell lines permissive for replication of the virus
released from PFT. Both infectivity assays using filtered supernatant fluids of P F T media
as well as co-cultivation of P F T cells with various cell lines have failed to identify any host
cell lines permissive for replication from among a large variety of mammalian cell lines
tested (Table I). We have also been unable to demonstrate 'rescue' by the P F T virus of the
Kirsten murine sarcoma virus (MSV) genome from Ki-MSV-transformed nonproducer
cell lines derived from the rat N R K , mouse BALB/3T3, and mink CCL 64 cell lines
(Aaronson & Weaver, I97t; Henderson, Lieber & Todaro, I974). In these experiments,
P F T cells were co-cultivated with the nonproducer cells of mouse, rat and mink for I week;
supernatant fluids filtered and tested on N R K and CCL 64 cells showed no focus formation. The same procedures can readily detect the infectivity of endogenous type C viruses
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I77
Type C virus in pig oviduct cell line
Fig. 3. BrdU induction of type C virus in the I65th (a, b) and 9oth (c) subcultures:
complete virus particles (a) and budding particles (b, c).
in mouse (Aaronson & Stephenson, 1973; Benveniste, Lieber & Todaro, I974), cat (Fischinger et aL I973; Todaro et aL I973), and baboon (Benveniste et al. I974; Todaro et al.
1974 b) cells.
Treatment with BrdU induced readily observable (by electron microscopy) type C virus
particles in the 9oth, I3oth and I65th subcultures (Fig. 3). However, no particles were seen
after similar BrdU treatment of the I7th and 59th subcultures.
DISCUSSION
The PFT pig oviduct cell line has been cultivated for 4½ years without any known contact
with the other pig cell lines. The appearance of typical type C virus antigenically related to
the PK(I5) virus in the high passaged subcultures of the PFT cell line suggests that this
culture also has the potential to spontaneously produce an endogenous type C virus genetically transmitted in its genome (Todaro & Huebner, I972; Todaro et al. I974a ). The lack of
infectivity of this PFT virus for other cell lines argues against its being a laboratory contaminant. The ability to induce type C virus particles in the PFT cell line by BrdU treatment also
argues in favour of the PFT cell line producing an endogenous virus.
Our results demonstrate that induction with BrdU of PFT virus can occur at the 9oth
subculture, several passages before any karyotypic changes can be observed in the cell'~
(Bouillant et aL I973). A pericentric inversion and a translocation in two different chromosomes occurred at the Iooth and Ia9th subcultures respectively (Bouillant et al. I975 b) and
a high diploidy ratio (up to 80 ~) still exist in these cultures. The spontaneous release of
type C virus occurred after a substantial drop of the diploid ratio from 64 ~ to 24 ~o in the
2o5th and 226th subcultures respectively and at that time a third new chromosomic marker
was observed in the 225th subculture (P. Genest, unpublished data).
I2
VIR 27
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I78
A. M. P. B O U I L L A N T
AND
OTHERS
Thus, PFT, a fallopian tube culture, can be added to the growing list of continuous porcine cell lines which release type C viruses after prolonged subculturing (Breese, I97o;
Armstrong et al. I97I ; Todaro et al. I973). The inability of the porcine PK(t5) virus to
cause productive infections or to rescue the sarcoma genome is shared by the PFT virus.
Whether such behaviour will be generally typical of porcine type C viruses remains to be
determined; the porcine lymphoma PLCP virus is reportedly infectious for pig kidney cells
(Strandstr6m et al. I974). Among the species groups of mammalian type C viruses studied
so far, hamster and rat endogenous type C viruses share a similar inability to cause productive infections, although viruses from this group will rescue and pseudotype the MSV genome
(Aaronson, i97I ; Freeman et al. I97I ; Klement, Nicolson & Huebner, I97 0.
This data supports the broad generalization that normal pig cells, along with cells from
the mouse, rat and cat species have a high probability of both spontaneous transformation
and release of endogenous type C virus during serial subcultivation in vitro. The facilitated
expression of previously unexpressed endogenous type C 'virogenes' in these circumstances
suggests that the cellular regulatory mechanisms of these transformed cells become progressively more disturbed during serial in vitro passage (Aaronson et al. I969; Todaro,
I972; Lieber, Livingston & Todaro, I973b; Lieber & Todaro, I973).
The existence of continuous cell lines producing porcine type C viruses in high titre
PK(I5), PFT, and the porcine lymphoma line 38A-~ (Strandstr6m et al. I974 ) should
permit the development of species specific serologic reagents which will, in turn, allow
further investigation of the function of porcine type C viruses in vivo and the possible role
on these viruses in porcine disease.
We thank Dr Charles Sherr for preparation of the anti-polymerase sera and Miss S.
Becker, Mr Robert Begin and Mr Louis Fedele for their excellent technical assistance.
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