_Archives
Vrology
Arch Virol (1992) 123:255-265
© Springer-Verlag 1992 Printed in Austria
Porcine retrovirus: optimal conditions for its biochemical detection L. Phan-Thanh,B. Kaeffer, and E. Bottreau Institut National de la Recherche Agronomique, Laboratoire de Pathologic Infccticuse et Immunologic, Centre de Recherche de Tours, Nouzilly, France Accepted September 13, 1991
Summary. The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn + + was indispensable, with an optimal concentration of 0.25mM. Monocations (K +, Na +) at 50mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells. Introduction Retroviruses have been found in various animal species associated or not with diseases. Porcine retroviruses have been detected in a cell line derived from swine malignant lymphomas (Shimozuma cells) [15], in blood of a miniature swine that had been exposed to strontium-90 radiation and that had developed haematopoietic disorders [4-] and in porcine cell lines after long-term propagation in vitro [11, 16]. Recently, we have found retroviruses in lymphoblastoid cell lines derived from pigs that had been inoculated with retroviruses [9]. Generally, retroviruses are identified by immunological methods using antibodies directed against one of the viral structural proteins, which, in most cases, cross-react with type C viruses of other species I-8, 11, 14-] or detected by biochemical assay of their reverse transcriptase (RT) activity. Electron microscopy is also helpful in identifying virus particles morphologically. The RT assay provides a convenient biochemical means for detecting the existence of
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retroviruses. W e have f o u n d however that the application to porcine systems o f p r o c e d u r e s that have been described for the estimation o f R T activity o f retroviruses in other species often lead to a m b i g u o u s or negative results where they should be positive. F o r the successful detection o f retroviruses in porcine systems, it is thus i m p o r t a n t to determine the optimal conditions for a sensitive and reliable R T assay. This paper reports these conditions, and the s p o n t a n e o u s reactivation o f retrovirus in porcine cell lines that have u n d e r g o n e a high n u m b e r o f passages.
Materials and methods
Reagents Synthetic primer oligo(dT)t2_18 and paired templates-primers poly(rA).(dT)l0, poly(rA).(dT)12_18, poly(dA).(dT)12_18, poly(rC).(dG)12_18 were purchased from Pharmacia (Bois d'Acry, France) and Boehringer Mannheim (Meylan, France), rabbit globin mRNA from Gibco-BRL (Cergy Pontoise, France), radioactive nucleotides and avian myeloblastosis virus RT from Amersham (Les Ulis, France). Glass microfibre filters (Whatman GF/ C) were punched into 14mm diameter discs from glass fibre sheets for cell harvest. The choice of glass fibre filters was important in lowering the background, since some trademarks of glass fibre were found to retain non-incorporated radioactive nucleotides. Bicinchoninic acid protein assay reagent was purchased from Pierce (Interchim, Mountlucon, France).
Cell lines Iowa swine testis (ST) cell line was a gift from Dr. Jean-Marie Aynaud (INRA, Nouzilly, France) and originated from a cell line selected by McClurkin and Norman [12], and cultured in our laboratory for more than 200 passages. Shimozuma cells derived from pig malignant lymphomas and known to contain porcine retroviruses [15] were a gift from Dr. Sonada (Tsukuba City of Science, Japan). One clone of these cells was called G 2, and propagated for more than 100 passages. RPJ (about 100 passages) and REB (50 passages) were pig kidney cell lines obtained by Jacqueline Gelfi (INRA, France) and by us, respectively, fiom a healthy animal. L 35 and L 52 were tymphoblastoid cell lines isolated in our laboratory from blood of a young inbred miniature boar which had been inoculated intraperitoneally with Shimozuma cells [9]. Also used for the RT assay were different cells freshly isolated from kidneys, spleen, muscle, skin and bone marrow of healthy pigs. All cells were rendered mycoplasma-free according to Chen [3] and propagated in Earle's minimum essential medium containing 10% fetal calf serum and 5mg/ml of penicillinstreptomycin. Fetal calf serum had been previously tested for the absence of bovine retroviruses. Porcine origin of cell lines was confirmed by karyotypic analysis as previously described [9].
Virus purification Virus particles were pelleted from clarified cell culture supernatants by centrifugation in a SW 28 Beckman swing rotor at 100 000 x g for 90 rain. The pellets were then resuspended in a minimum of Tris buffer saline (TBS) pH 7.6. A density gradient of 20-50% (w/v) sucrose in TBS was formed in 40 ml polycarbonate centrifuge tubes. Virus suspension (12 ml) was overlayed on the sucrose gradient and the centrifugation continued in a SW 28 Beckman rotor at 140000 x g for 16 h. After centrifugation the sucrose gradient was fractionated and the virus banding around density 1.16g/ml was collected, The pooled
Biochemical detection of porcine retrovirus
257
virus fractions were then diluted with TBS, pelleted by centrifugation at 100 000 x g for 1 h, resuspended in 50 mM Tris buffer pH 7.6 and stored at - 7 0 °C until used.
Electron-microscopy Purified virus particles were stained according to Nermut etal. [13]. Microscopic observations were done with a Philips electron microscope (60 kV).
Procedure for RT activity assay Assays of RT activity were performed on purified virus suspensions of concentrated (100200 fold) celt culture supernatants. The assay conditions were adopted from Hoffman and coworkers [7] with some modifications. Briefly, 10gl of virus preparation corresponding to 0.1-0.5 gg of viral proteins were mixed with 25 gt of a reagent cocktail so as to provide final concentrations of 50mM Tris pH 7.8, 5raM dithiotreitol, 1% Triton X-100, 5 mM KC1 and 2.5 gg (5 gl) of poly(rA).oligo(dT)12_18 and left at room temperature for 10 rain to allow the detergent to disrupt virus particles. After addition of 10 gCi (4 ~tM) of radioactive nucleotide the mixture was incubated at 37 °C for the duration of time chosen (normally 60 rain). The synthesized DNA was then precipitated by adding 1 ml of ice-cold 20% trichloracetic acid (TCA) and allowed to stand on ice for at least 15min. It was subsequently filtered through glass fibre discs that had been preimpregnated with 1 ml of 100mM sodium pyrophosphate solution in 1 M HC1. Filters were washed 10 times with 5ml of ice-cold 5% TCA and once with 5ml of ice-cold 95% ethanol, dried under an infrared lamp, placed in 2.5 ml of scintillation liquid and counted.
In vitro reverse transcription of natural mRNA by porcine retrovirus In an Eppendoff tube 0.5 gg of rabbit globin mRNA and 0.5 gg of oligo(dT)15 were mixed and allowed to anneal by heating at 60 °C for 3 rain and immediately chilling on ice. 50 gCi (20 gM) of tritiated dTTP or dGTP and three other non-radioactive mononucleotide phosphates were added at the final concentration of 0.4 mM (for the final volume of 50 gl). Virus suspension and a reagent cocktail were added so as to provide 50raM Tris-HC1 pH7.8, 5raM dithiothreitol, 1% Triton X-t00, 50raM KC1, and 6raM MgC12 at final concentrations. The mixture was incubated at 37 °C for 3 h. In the experiment with destruction ofmRNA, ribonuclease A was added at a concentration of 10 gg/ml after virus particles were disrupted with detergent-containing buffer and the mixture was allowed to react for 30 min at room temperature before adding radioactive nucleotide.
Protein determination Viral protein was determined by the bicinchoninic acid micromethod according to the manufacturer's protocol.
Results Presence o f porcine retrovirus The presence o f t y p e - C retroviruses in porcine cell culture s u p e r n a t a n t s was evidenced b y electron-microscopic o b s e r v a t i o n a n d b y R T assay. The m o r p h o logical o b s e r v a t i o n o f porcine retrovirus particles has been previously described [9]. U n d e r o p t i m a l conditions o f the R T assay described hereunder, porcine retroviruses readily synthesized D N A f r o m b o t h n a t u r a l a n d synthetic R N A s .
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All tests were performed simultaneously with Shimozuma cell line serving as a positive reference.
Determination of optimal conditions for RT assay in porcine systems Requirement of templates-primers Table 1 shows the RT activity of porcine retrovirus in the presence of different synthetic templates-primers. The duplex poly(rA).oligo(dT) was found to be an absolute requirement for porcine retrovirus RT. Other templates-primers such as poly(rC).oligo(dG), poty(dA).oligo(dT), or primer oligo(dT) alone could not be used as substrates by porcine retrovirus RT. When coupled with the template poly(rA), the primer oligo(dT)12_18 was found to be three times more efficient than the shorter primer oligo(dT)10. Effect of p H In the presence of poly(rA).oligo(dT) and at optimal concentrations of monoand divalent cations (see later), the optimal RT activity of purified porcine retrovitus was found to be in the pH 7.6-8.6 range. However, when the assay was done with non-purified cell culture supernatants, the optimal pH range became much narrower: 7.6-8.0; at p H above 8.2 the RT activity was totally inhibited (Fig. 1). Effect of divalent cations In the presence of poly(rA).oligo(dT) porcine retrovirus RT required M n + + or Mg + + ions for activity, but had a marked preference for M n + + (Table 2). The activity in the presence of M n + + at an optimal concentration (0.25 mM) was some 60 times higher than that in the presence of Mg + + at its optimal
Table 1. Dependency of porcine retrovirus RT activity on synthetic templates-primers Template-primer
poly(rA).oligo(dT)t0 poly(rA).oligo(dT)12 18 poly(rC).oligo(dG)12_18 poly(dA).oligo(dT)1~18 oligo(dT) l~_l~
Incorporated radioactive dTTP or dGTP in cpm x 103 Porcine viral RT
Avian viral RT a
Mn + +
Mg+ +
Mn + +
Mg+ +
241 750 3 2 2
3 12 4 10 5
nd 376 112 2 nd
nd 95 850 45 nd
a For comparison, the activity of avian myeloblastosis virus RT (used at 0.001 enzyme unit per assay) is included. The value of negative control is 2 x 103 nd Not determined
Biochemical detection of porcine retrovirus
259
700 600 500 400 300
X E Q. v
200 [
100 6,5 400
7,0
I
I
,
7,5
!
.
8,0
I
.
8,5
I
i
9.0
!
9,5
o
8
E O.
300
200
100
0 6,5
"
. 7,0
7,5
8,0
8,5
I 9,0
~
I 9,5
pH Fig. 1. Effect of p H on porcine retrovirus RT. A Purified virus particles. B Concentrated cell culture supernatant containing virus
Table 2. Effect of divalent ions Mn + + and Mg + + on porcine retrovirus RT in the presence of different templates-primers Template-primer
Cell line
Mn + + (mM) 0 0.25
0.50
Mg + + (mM) 1
2
4
0 2 4
6
8 10
radioactive d T T P incorporated (in sample/control ratio) poly(rA).oligo(dT) ST 1 375 323 155 100 34 1 1 3 6 4 2 G2 1 325 291 284 185 95 1 1 5 8 7 3 AMV a 188 50 radioactive d G T P incorporated (in sample/control ratio) 1 2 2 1 1 1 122 2 1 1 poly(rC).oligo(dG) ST 1 2 2 1 1 1 1 1 1 1 1 1 G2 56 425 AMV a For comparison the activities of avian myeloblastosis virus RT used at 0.001 enzyme unit per assay at optimal concentrations of Mn + + and Mg + + are included
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concentration (6 raM). In the presence of poly(rC).oligo(dG), porcine retrovirus RT was inactive at any concentration of Mn + + or Mg + + Effect of monovalent cations Figure 2 represents the effect of varying concentrations of Na + and K + on the RT activity of porcine retrovirus. An optimal stimulatory effect was observed at 50 m M with both Na + and K + with a slightly superior efficiency for K +. The effective range of Na + was narrower than that of K + . At concentrations above 200raM, both Na + and K + became totally inhibitory for porcine retrovirus RT. Effect of non-ionic detergents Non-ionic detergents such as Triton X-100 and Nonidet P-40 increased the RT activity of porcine retrovirus particles. Triton X-100 and Nonidet P-40 had similar effects. At 1%, they enhanced several-fold the reaction. The permissible concentration range of Triton X-100 was broad, with optimal concentrations between 1 and 2%. Only very high concentrations (above 5%) of detergent appeared to inhibit porcine retrovirus RT activity. Effect of other compounds To increase the sensitivity of the RT assay various compounds have been tested: 13-mercaptoethanol, dithiothreitol, glycerol, ethylene glycol, polyethylene glycol, bovine serum albumin, ethylene diamine tetraacetic acid, ethylene glycol bis(13-aminoethyl ether) N,N'-tetraacetic acid, but few of them enhanced the RT activity of porcine retrovirus convincingly (results not shown). The most effective compound was dithiothreitol; the addition of 5 m M increased the RT activity by 75%.
%
5O0
4o0 --q
3oo
ID O
200
K+
O a00
I
0 0
100
200
Concentration
( rnM )
300
Fig.2. Effect of K + and Na + on porcine retrovirus RT activity in the presence of poly(rA).oligo(dT)
Biochemical detection of porcine retrovirus
261
Reaction duration Up to 4 h of reaction the incorporation of radioactive dTTP into acid-precipitable DNA synthesized by porcine retrovirus RT increased proportionally with the duration of incubation. Very extended incubation period (24h and over) decreased the radioactive incorporation.
In vitro reverse transcription of mRNA by porcine retrovirus Table 3 shows the amounts of acid-precipitable DNA synthesized by porcine retrovirus from rabbit globin mRNA under different conditions. To copy natural mRNAs porcine retrovirus required Mg + + instead of Mn + + as when copying synthetic RNAs (Table 3, cases a and b). Without primer, DNA synthesis did not occur (case i). The omission of one of the nucleotide triphosphates resulted in a marked decrease in the DNA synthesis (case e), proving that a heteropolymeric complementary DNA was being synthesized and not a product of a terminal deoxynucleotidyl transferase. When mRNA was destroyed by ribonuclease, no DNA synthesis occurred (cases h and j).
Screening of porcine cell tines for retroviruses Using the optimal conditions determined above, we performed the RT assay on concentrated culture supernatants of different porcine cell lines (Table4). Retrovirus-containing cell lines G 2, L 35, L 52 all showed RT activity. Cell lines with a high number of passages (over 100 passages) G2, ST, RPJ, even the ones that did not contain any retrovirus at the beginning of culture (ST, RPJ), showed high RT activity. That fact demonstrates that retrovirus could be spontaneously reactivated in porcine cells by long-term propagation in culture. As
T a b l e 3. I n v i t r o r e v e r s e t r a n s c r i p t i o n o f r a b b i t g l o b i n m R N A
by porcine retrovirus
Case Addition of globin Oligo(dT) mRNA
Dication
Virus
dNTW
RNase
Radioactive incorporation ( c p m x 10 3)
a b c d e f g h i j
Mn Mg Mg Mg Mg Mg Mg Mg Mg Mg
+ + + + + + + + +
4 4 4 4 3 dTTP 4 4 4 4
+ +
4 295 30 100 9 8 3 6 7 4
+ + + + + + + + -
+ + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + +
a Deoxynucleotide triphosphate. In case d radioactive dNTP was dGTP instead of dTTP as i n o t h e r c a s e s
262
L. Phan-Thanh et al. Table 4. RT activity found in concentrated culture supernatants of different porcine cell lines Cell line
RT activity (cpm x 103)
Shimozuma G 2 Testis ST Kidney RPJ Kidney REB Lymphoblastoid L 35 L 52 Skin cells Spleen cells Kidney RPCA RPNC Muscle cells Bone marrow Negative control
600 570 150 21 12 24 1.8 2 3.6 1.8 1.9 1.7 2
for cells newly prepared from normal healthy pig organs, we did not find any measurable RT activity. Those results were consistent with electron-microscopic observation.
Transmission of porcine retrovirus to pigs L 35 and L 52 were lymphoblastoid cell lines obtained from a pig that had been inoculated intraperitoneally with cells producing porcine retrovirus. The RT activity found in these cell lines showed that there was transmission of retrovirus to the pig. Details concerning the experimental infection and its consequences in the animal have been published elsewhere [9]. Discussion
The application to porcine systems of the assay conditions described for typeC retrovirus (oncovirus) of other species has not always lead to reliable results. The fact that porcine type-C retrovirus RT responded to poly(rA).oligo(dT) and not to poly(rC).oligo(dG) was unusual, since most animal type-C retrovirus RTs respond to both poly(rA).oligo(dT) and poly(rC).oligo(dG). It is known that some mammalian cells [5] and chick embryo cells [1] contained, besides a DNA-dependent D N A polymerase activity, a synthetic RNA-dependent D N A polymerase activity (they could synthesize D N A from a synthetic homopolymeric R N A but not from a natural heteropolymeric R N A served as template), and that cellular D N A polymerases can only utilize poly(rA).oligo(dT) and not poly(rC).oligo(dG) as substrate [17]. The same results we found with porcine retroviral RT initially suggested the suspicion that our samples possibly con-
Biochemical detection of porcine retrovirus
263
tained cellular enzymes and.not retroviruses, although Kiessling and Goulian [10] reported the same findings for simian sarcoma retrovirus. That doubt was then cleared, by the virus purification on sucrose gradient and by the capability of our virus preparations to reverse-transcribe a natural mRNA, which cellular enzymes could not do. It is noteworthy that in the reverse transcription of natural mRNAs porcine RT required Mg + + instead of Mn + + when copying synthetic RNAs. The reasons for this switch are unclear. Furthermore, unlike avian and some mammalian retroviruses, porcine retrovirus RT was found to contain no DNA-dependent DNA polymerase activity (it was inactive to poly(dA).oligo(dT)). This feature also distinguished porcine retrovirus RT from cellular DNA polymerases and allowed the use of poly(dA).oligo(dT) to check the presence of contaminating cellular DNA polymerase in samples. Furthermore, the inability of porcine retrovirus RT to use oligo(dT) as substrate in the absence of a template permitted the use of oligo(dT) to check the presence of terminal deoxynucleotidyl transferases, which would give false positive results to the RT assay. The difference in the optimal pH range of porcine RT in the case of purified retrovirus particles and the case of concentrated cell culture supernatants is probably due to some inhibiting factors existing in the cell culture fluids. Such factors had not been removed, as they had been by the purification process, and were active only at alkaline pHs. That is perhaps the reason why we often failed to detect retroviruses directly in porcine cell culture supernatants by assaying their RT activity at pHs as high as the values normally used (pH 8.3). Monovalent cations (Na +, K +) had an enhancing effect on porcine retrovirus RT activity at concentrations that had also been found for most other viral RTs (optimal at 50 mM), but for porcine retrovirus RT these monocations began to become inhibitory at relatively lower concentrations (150 mM as compared to 300raM). Na + and K + ions had a similar effect, but the enhancing range of K + concentrations was broader (Fig. 2). As a result, when cell culture supernatants or biological fluids are used as such in the assay, attention should be paid to monocation concentrations already present in samples and reactant solutions added so that the total amounts of Na + and K + should not exceed optimal doses and become inhibitory. In efforts to increase the sensitivity of the RT assay further, various substances have been used as additives by different authors [2, 6, 7, 10]. Several of these compounds have been tried by us, but only dithiothreitol had an unambiguous enhancing effect, although not to the extent reported by Hoffman and coworkers [7]. Furthermore, with other additives, the result reported by different authors are not uniform, sometimes even contradictory. Finally, the sensitivity of the assay could be enhanced by increasing the duration of incubation. Normally 1 h of incubation was sufficient to give significant signals for our virus-containing cell culture supernatants that had been concentrated to 50- to 100-fold. With scarcer amounts of virus a longer incubation was needed, but very extended incubation periods decreased the radioactive incorporation,
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probably because of the degradative action of c o n t a m i n a t i n g nucleases or a nuclease activity contained in the R T enzyme system itself as occurs with avian myeloblastosis virus RT. We did n o t count the minimal n u m b e r of retroviral particles u n a m b i g u o u s l y detectable by the assay, but 0.1 gg of retroviral protein regularly gave a signal of several h u n d r e d times when c o m p a r e d with the negative control background. Using the R T assay with optimal conditions determined above, we confirmed that: (1) procine retroviruses can be transmitted to the pig by intraperitoneal inoculation with virus-producting cells, and (2) retroviruses can be spontaneously p r o d u c e d by porcine cell lines after long-term propagation. The cause of this " s p o n t a n e o u s p r o d u c t i o n " of retrovirus is u n k n o w n .
Acknowledgements We thank Drs. Akiro Sonoda and Michi Kodama (National Institute of Animal Health, Tsukuba City of Science, Japan) for their kind gift of Shimozuma cells, and Dr. JeanMarie Aynaud (INRA, Nouzilly, France) for his gift of ST cells and helpful discussions.
References 1. Brun G, Rougeon F, Lauber M, Chapvitle F (I974) Purification and properties of DNA polymerases from chick embryo. Eur J Biochem 41:241-251 2. Chan EW, Dale PJ, Greco IL, Rob JG, O'Connor TE (1980) Effects of polyethylene glycol on reverse transcriptase and other polymerases activity. Biochim Biophys Acta 606:353-361 3. Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:255-262 4. Frazier ME (1985) Evidence for retrovirus in miniature swine with radiation-induced leukemia or metaplasia. Arch Virol 83:83 87 5. Fridlender B, Fry M, Bolden A, Weissbach A (1972) A new synthetic RNA-dependent DNA polymerase from human tissue culture cells (HeLa-fibrobtast-synthetic oligomtcteotides-template-purified enzymes). Proc Natl Acad Sci USA 69:452-455 6. Gregerson JP, Wege H, Preiss L, Jentsch KD (1988) Detection of human immunodeficient virus and other retroviruses in cell culture supernatants by a reverse transcriptase microassay. J Virol Methods 19:161-168 7. Hoffman AD, Banapour B, Levy JA (1985) Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 147:326-335 8. Huebner RJ, Igel HJ (1970) Immunological tolerance to gs antigens as evidence for vertical transmission of non-infectious C-type RNA virus genoms. Perspect Virol 7: 55-71 9. Kaeffer B, Bottreau E, Phan-Thanh L, Olivier M, Salmon H (1990) Histocompatible miniature boar model: selection of transformed cell lines of B and T lineages producing retrovirus. Int J Cancer 46:481-488 10. Kiessling AA, Goulian M (1976) A comparison of the enzymatic responses of the DNA polymerases from four RNA tumour viruses. Biochem Biophys Res Comm 71: 10691077 1t. Lieber MM, Sherr CJ, Benveniste RE, Todaro GJ (t975) Biologic and immunologic properties of porcine type C virus. Virology 66:616-619 12. McClurkin AW, Norman JO (1966) Studies on transmissible gastroenteritis of swine:
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13.
14. 15. 16. 17.
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II. Selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis. Can J Comp Med Vet Sci 30:190-198 Nermut MV, Frank H, Schaeffer W (1972) Properties of mouse leukemia viruses. III. Electron-microscopic appearance as revealed after conventional preparation techniques as well as freeze-drying and freeze-etching. Virology 49:345-358 Nowinski RC, Peters ED (1973) Cell surface antigens associated with murine leukemia virus: definition of GL and GT antigenic systems. J Virol 12:1104-1117 Suzuka I, Seikiguchi K, Kodama M (1985) Some characteristics of a porcine retrovirus from a cell line derived from swine malignant lymphomas. FEBS Lett 183:124-128 Todaro GJ (1972) Detection and characterization of RNA tumour viruses in normal and transformed cells. Perspect Virol 8:81-101 Weissbach A, Bolden A, Mueller R, Hanafusa H, Hanafusa T (1972) Deoxyribonucleic acid polymerase activities in normal and leukovirus-infected chicken embryo cells. J Virology 10:321-327
Authors' address: Dr. L. Phan-Thanh, Pathologie Infectieuse et Immunologie, INRA, F-37380 Nouzilly, France. Received June 20, 1991
Vrology
Arch Virol (1992) 123:255-265
© Springer-Verlag 1992 Printed in Austria
Porcine retrovirus: optimal conditions for its biochemical detection L. Phan-Thanh,B. Kaeffer, and E. Bottreau Institut National de la Recherche Agronomique, Laboratoire de Pathologic Infccticuse et Immunologic, Centre de Recherche de Tours, Nouzilly, France Accepted September 13, 1991
Summary. The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn + + was indispensable, with an optimal concentration of 0.25mM. Monocations (K +, Na +) at 50mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells. Introduction Retroviruses have been found in various animal species associated or not with diseases. Porcine retroviruses have been detected in a cell line derived from swine malignant lymphomas (Shimozuma cells) [15], in blood of a miniature swine that had been exposed to strontium-90 radiation and that had developed haematopoietic disorders [4-] and in porcine cell lines after long-term propagation in vitro [11, 16]. Recently, we have found retroviruses in lymphoblastoid cell lines derived from pigs that had been inoculated with retroviruses [9]. Generally, retroviruses are identified by immunological methods using antibodies directed against one of the viral structural proteins, which, in most cases, cross-react with type C viruses of other species I-8, 11, 14-] or detected by biochemical assay of their reverse transcriptase (RT) activity. Electron microscopy is also helpful in identifying virus particles morphologically. The RT assay provides a convenient biochemical means for detecting the existence of
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L. Phan-Thanh et al.
retroviruses. W e have f o u n d however that the application to porcine systems o f p r o c e d u r e s that have been described for the estimation o f R T activity o f retroviruses in other species often lead to a m b i g u o u s or negative results where they should be positive. F o r the successful detection o f retroviruses in porcine systems, it is thus i m p o r t a n t to determine the optimal conditions for a sensitive and reliable R T assay. This paper reports these conditions, and the s p o n t a n e o u s reactivation o f retrovirus in porcine cell lines that have u n d e r g o n e a high n u m b e r o f passages.
Materials and methods
Reagents Synthetic primer oligo(dT)t2_18 and paired templates-primers poly(rA).(dT)l0, poly(rA).(dT)12_18, poly(dA).(dT)12_18, poly(rC).(dG)12_18 were purchased from Pharmacia (Bois d'Acry, France) and Boehringer Mannheim (Meylan, France), rabbit globin mRNA from Gibco-BRL (Cergy Pontoise, France), radioactive nucleotides and avian myeloblastosis virus RT from Amersham (Les Ulis, France). Glass microfibre filters (Whatman GF/ C) were punched into 14mm diameter discs from glass fibre sheets for cell harvest. The choice of glass fibre filters was important in lowering the background, since some trademarks of glass fibre were found to retain non-incorporated radioactive nucleotides. Bicinchoninic acid protein assay reagent was purchased from Pierce (Interchim, Mountlucon, France).
Cell lines Iowa swine testis (ST) cell line was a gift from Dr. Jean-Marie Aynaud (INRA, Nouzilly, France) and originated from a cell line selected by McClurkin and Norman [12], and cultured in our laboratory for more than 200 passages. Shimozuma cells derived from pig malignant lymphomas and known to contain porcine retroviruses [15] were a gift from Dr. Sonada (Tsukuba City of Science, Japan). One clone of these cells was called G 2, and propagated for more than 100 passages. RPJ (about 100 passages) and REB (50 passages) were pig kidney cell lines obtained by Jacqueline Gelfi (INRA, France) and by us, respectively, fiom a healthy animal. L 35 and L 52 were tymphoblastoid cell lines isolated in our laboratory from blood of a young inbred miniature boar which had been inoculated intraperitoneally with Shimozuma cells [9]. Also used for the RT assay were different cells freshly isolated from kidneys, spleen, muscle, skin and bone marrow of healthy pigs. All cells were rendered mycoplasma-free according to Chen [3] and propagated in Earle's minimum essential medium containing 10% fetal calf serum and 5mg/ml of penicillinstreptomycin. Fetal calf serum had been previously tested for the absence of bovine retroviruses. Porcine origin of cell lines was confirmed by karyotypic analysis as previously described [9].
Virus purification Virus particles were pelleted from clarified cell culture supernatants by centrifugation in a SW 28 Beckman swing rotor at 100 000 x g for 90 rain. The pellets were then resuspended in a minimum of Tris buffer saline (TBS) pH 7.6. A density gradient of 20-50% (w/v) sucrose in TBS was formed in 40 ml polycarbonate centrifuge tubes. Virus suspension (12 ml) was overlayed on the sucrose gradient and the centrifugation continued in a SW 28 Beckman rotor at 140000 x g for 16 h. After centrifugation the sucrose gradient was fractionated and the virus banding around density 1.16g/ml was collected, The pooled
Biochemical detection of porcine retrovirus
257
virus fractions were then diluted with TBS, pelleted by centrifugation at 100 000 x g for 1 h, resuspended in 50 mM Tris buffer pH 7.6 and stored at - 7 0 °C until used.
Electron-microscopy Purified virus particles were stained according to Nermut etal. [13]. Microscopic observations were done with a Philips electron microscope (60 kV).
Procedure for RT activity assay Assays of RT activity were performed on purified virus suspensions of concentrated (100200 fold) celt culture supernatants. The assay conditions were adopted from Hoffman and coworkers [7] with some modifications. Briefly, 10gl of virus preparation corresponding to 0.1-0.5 gg of viral proteins were mixed with 25 gt of a reagent cocktail so as to provide final concentrations of 50mM Tris pH 7.8, 5raM dithiotreitol, 1% Triton X-100, 5 mM KC1 and 2.5 gg (5 gl) of poly(rA).oligo(dT)12_18 and left at room temperature for 10 rain to allow the detergent to disrupt virus particles. After addition of 10 gCi (4 ~tM) of radioactive nucleotide the mixture was incubated at 37 °C for the duration of time chosen (normally 60 rain). The synthesized DNA was then precipitated by adding 1 ml of ice-cold 20% trichloracetic acid (TCA) and allowed to stand on ice for at least 15min. It was subsequently filtered through glass fibre discs that had been preimpregnated with 1 ml of 100mM sodium pyrophosphate solution in 1 M HC1. Filters were washed 10 times with 5ml of ice-cold 5% TCA and once with 5ml of ice-cold 95% ethanol, dried under an infrared lamp, placed in 2.5 ml of scintillation liquid and counted.
In vitro reverse transcription of natural mRNA by porcine retrovirus In an Eppendoff tube 0.5 gg of rabbit globin mRNA and 0.5 gg of oligo(dT)15 were mixed and allowed to anneal by heating at 60 °C for 3 rain and immediately chilling on ice. 50 gCi (20 gM) of tritiated dTTP or dGTP and three other non-radioactive mononucleotide phosphates were added at the final concentration of 0.4 mM (for the final volume of 50 gl). Virus suspension and a reagent cocktail were added so as to provide 50raM Tris-HC1 pH7.8, 5raM dithiothreitol, 1% Triton X-t00, 50raM KC1, and 6raM MgC12 at final concentrations. The mixture was incubated at 37 °C for 3 h. In the experiment with destruction ofmRNA, ribonuclease A was added at a concentration of 10 gg/ml after virus particles were disrupted with detergent-containing buffer and the mixture was allowed to react for 30 min at room temperature before adding radioactive nucleotide.
Protein determination Viral protein was determined by the bicinchoninic acid micromethod according to the manufacturer's protocol.
Results Presence o f porcine retrovirus The presence o f t y p e - C retroviruses in porcine cell culture s u p e r n a t a n t s was evidenced b y electron-microscopic o b s e r v a t i o n a n d b y R T assay. The m o r p h o logical o b s e r v a t i o n o f porcine retrovirus particles has been previously described [9]. U n d e r o p t i m a l conditions o f the R T assay described hereunder, porcine retroviruses readily synthesized D N A f r o m b o t h n a t u r a l a n d synthetic R N A s .
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All tests were performed simultaneously with Shimozuma cell line serving as a positive reference.
Determination of optimal conditions for RT assay in porcine systems Requirement of templates-primers Table 1 shows the RT activity of porcine retrovirus in the presence of different synthetic templates-primers. The duplex poly(rA).oligo(dT) was found to be an absolute requirement for porcine retrovirus RT. Other templates-primers such as poly(rC).oligo(dG), poty(dA).oligo(dT), or primer oligo(dT) alone could not be used as substrates by porcine retrovirus RT. When coupled with the template poly(rA), the primer oligo(dT)12_18 was found to be three times more efficient than the shorter primer oligo(dT)10. Effect of p H In the presence of poly(rA).oligo(dT) and at optimal concentrations of monoand divalent cations (see later), the optimal RT activity of purified porcine retrovitus was found to be in the pH 7.6-8.6 range. However, when the assay was done with non-purified cell culture supernatants, the optimal pH range became much narrower: 7.6-8.0; at p H above 8.2 the RT activity was totally inhibited (Fig. 1). Effect of divalent cations In the presence of poly(rA).oligo(dT) porcine retrovirus RT required M n + + or Mg + + ions for activity, but had a marked preference for M n + + (Table 2). The activity in the presence of M n + + at an optimal concentration (0.25 mM) was some 60 times higher than that in the presence of Mg + + at its optimal
Table 1. Dependency of porcine retrovirus RT activity on synthetic templates-primers Template-primer
poly(rA).oligo(dT)t0 poly(rA).oligo(dT)12 18 poly(rC).oligo(dG)12_18 poly(dA).oligo(dT)1~18 oligo(dT) l~_l~
Incorporated radioactive dTTP or dGTP in cpm x 103 Porcine viral RT
Avian viral RT a
Mn + +
Mg+ +
Mn + +
Mg+ +
241 750 3 2 2
3 12 4 10 5
nd 376 112 2 nd
nd 95 850 45 nd
a For comparison, the activity of avian myeloblastosis virus RT (used at 0.001 enzyme unit per assay) is included. The value of negative control is 2 x 103 nd Not determined
Biochemical detection of porcine retrovirus
259
700 600 500 400 300
X E Q. v
200 [
100 6,5 400
7,0
I
I
,
7,5
!
.
8,0
I
.
8,5
I
i
9.0
!
9,5
o
8
E O.
300
200
100
0 6,5
"
. 7,0
7,5
8,0
8,5
I 9,0
~
I 9,5
pH Fig. 1. Effect of p H on porcine retrovirus RT. A Purified virus particles. B Concentrated cell culture supernatant containing virus
Table 2. Effect of divalent ions Mn + + and Mg + + on porcine retrovirus RT in the presence of different templates-primers Template-primer
Cell line
Mn + + (mM) 0 0.25
0.50
Mg + + (mM) 1
2
4
0 2 4
6
8 10
radioactive d T T P incorporated (in sample/control ratio) poly(rA).oligo(dT) ST 1 375 323 155 100 34 1 1 3 6 4 2 G2 1 325 291 284 185 95 1 1 5 8 7 3 AMV a 188 50 radioactive d G T P incorporated (in sample/control ratio) 1 2 2 1 1 1 122 2 1 1 poly(rC).oligo(dG) ST 1 2 2 1 1 1 1 1 1 1 1 1 G2 56 425 AMV a For comparison the activities of avian myeloblastosis virus RT used at 0.001 enzyme unit per assay at optimal concentrations of Mn + + and Mg + + are included
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concentration (6 raM). In the presence of poly(rC).oligo(dG), porcine retrovirus RT was inactive at any concentration of Mn + + or Mg + + Effect of monovalent cations Figure 2 represents the effect of varying concentrations of Na + and K + on the RT activity of porcine retrovirus. An optimal stimulatory effect was observed at 50 m M with both Na + and K + with a slightly superior efficiency for K +. The effective range of Na + was narrower than that of K + . At concentrations above 200raM, both Na + and K + became totally inhibitory for porcine retrovirus RT. Effect of non-ionic detergents Non-ionic detergents such as Triton X-100 and Nonidet P-40 increased the RT activity of porcine retrovirus particles. Triton X-100 and Nonidet P-40 had similar effects. At 1%, they enhanced several-fold the reaction. The permissible concentration range of Triton X-100 was broad, with optimal concentrations between 1 and 2%. Only very high concentrations (above 5%) of detergent appeared to inhibit porcine retrovirus RT activity. Effect of other compounds To increase the sensitivity of the RT assay various compounds have been tested: 13-mercaptoethanol, dithiothreitol, glycerol, ethylene glycol, polyethylene glycol, bovine serum albumin, ethylene diamine tetraacetic acid, ethylene glycol bis(13-aminoethyl ether) N,N'-tetraacetic acid, but few of them enhanced the RT activity of porcine retrovirus convincingly (results not shown). The most effective compound was dithiothreitol; the addition of 5 m M increased the RT activity by 75%.
%
5O0
4o0 --q
3oo
ID O
200
K+
O a00
I
0 0
100
200
Concentration
( rnM )
300
Fig.2. Effect of K + and Na + on porcine retrovirus RT activity in the presence of poly(rA).oligo(dT)
Biochemical detection of porcine retrovirus
261
Reaction duration Up to 4 h of reaction the incorporation of radioactive dTTP into acid-precipitable DNA synthesized by porcine retrovirus RT increased proportionally with the duration of incubation. Very extended incubation period (24h and over) decreased the radioactive incorporation.
In vitro reverse transcription of mRNA by porcine retrovirus Table 3 shows the amounts of acid-precipitable DNA synthesized by porcine retrovirus from rabbit globin mRNA under different conditions. To copy natural mRNAs porcine retrovirus required Mg + + instead of Mn + + as when copying synthetic RNAs (Table 3, cases a and b). Without primer, DNA synthesis did not occur (case i). The omission of one of the nucleotide triphosphates resulted in a marked decrease in the DNA synthesis (case e), proving that a heteropolymeric complementary DNA was being synthesized and not a product of a terminal deoxynucleotidyl transferase. When mRNA was destroyed by ribonuclease, no DNA synthesis occurred (cases h and j).
Screening of porcine cell tines for retroviruses Using the optimal conditions determined above, we performed the RT assay on concentrated culture supernatants of different porcine cell lines (Table4). Retrovirus-containing cell lines G 2, L 35, L 52 all showed RT activity. Cell lines with a high number of passages (over 100 passages) G2, ST, RPJ, even the ones that did not contain any retrovirus at the beginning of culture (ST, RPJ), showed high RT activity. That fact demonstrates that retrovirus could be spontaneously reactivated in porcine cells by long-term propagation in culture. As
T a b l e 3. I n v i t r o r e v e r s e t r a n s c r i p t i o n o f r a b b i t g l o b i n m R N A
by porcine retrovirus
Case Addition of globin Oligo(dT) mRNA
Dication
Virus
dNTW
RNase
Radioactive incorporation ( c p m x 10 3)
a b c d e f g h i j
Mn Mg Mg Mg Mg Mg Mg Mg Mg Mg
+ + + + + + + + +
4 4 4 4 3 dTTP 4 4 4 4
+ +
4 295 30 100 9 8 3 6 7 4
+ + + + + + + + -
+ + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + +
a Deoxynucleotide triphosphate. In case d radioactive dNTP was dGTP instead of dTTP as i n o t h e r c a s e s
262
L. Phan-Thanh et al. Table 4. RT activity found in concentrated culture supernatants of different porcine cell lines Cell line
RT activity (cpm x 103)
Shimozuma G 2 Testis ST Kidney RPJ Kidney REB Lymphoblastoid L 35 L 52 Skin cells Spleen cells Kidney RPCA RPNC Muscle cells Bone marrow Negative control
600 570 150 21 12 24 1.8 2 3.6 1.8 1.9 1.7 2
for cells newly prepared from normal healthy pig organs, we did not find any measurable RT activity. Those results were consistent with electron-microscopic observation.
Transmission of porcine retrovirus to pigs L 35 and L 52 were lymphoblastoid cell lines obtained from a pig that had been inoculated intraperitoneally with cells producing porcine retrovirus. The RT activity found in these cell lines showed that there was transmission of retrovirus to the pig. Details concerning the experimental infection and its consequences in the animal have been published elsewhere [9]. Discussion
The application to porcine systems of the assay conditions described for typeC retrovirus (oncovirus) of other species has not always lead to reliable results. The fact that porcine type-C retrovirus RT responded to poly(rA).oligo(dT) and not to poly(rC).oligo(dG) was unusual, since most animal type-C retrovirus RTs respond to both poly(rA).oligo(dT) and poly(rC).oligo(dG). It is known that some mammalian cells [5] and chick embryo cells [1] contained, besides a DNA-dependent D N A polymerase activity, a synthetic RNA-dependent D N A polymerase activity (they could synthesize D N A from a synthetic homopolymeric R N A but not from a natural heteropolymeric R N A served as template), and that cellular D N A polymerases can only utilize poly(rA).oligo(dT) and not poly(rC).oligo(dG) as substrate [17]. The same results we found with porcine retroviral RT initially suggested the suspicion that our samples possibly con-
Biochemical detection of porcine retrovirus
263
tained cellular enzymes and.not retroviruses, although Kiessling and Goulian [10] reported the same findings for simian sarcoma retrovirus. That doubt was then cleared, by the virus purification on sucrose gradient and by the capability of our virus preparations to reverse-transcribe a natural mRNA, which cellular enzymes could not do. It is noteworthy that in the reverse transcription of natural mRNAs porcine RT required Mg + + instead of Mn + + when copying synthetic RNAs. The reasons for this switch are unclear. Furthermore, unlike avian and some mammalian retroviruses, porcine retrovirus RT was found to contain no DNA-dependent DNA polymerase activity (it was inactive to poly(dA).oligo(dT)). This feature also distinguished porcine retrovirus RT from cellular DNA polymerases and allowed the use of poly(dA).oligo(dT) to check the presence of contaminating cellular DNA polymerase in samples. Furthermore, the inability of porcine retrovirus RT to use oligo(dT) as substrate in the absence of a template permitted the use of oligo(dT) to check the presence of terminal deoxynucleotidyl transferases, which would give false positive results to the RT assay. The difference in the optimal pH range of porcine RT in the case of purified retrovirus particles and the case of concentrated cell culture supernatants is probably due to some inhibiting factors existing in the cell culture fluids. Such factors had not been removed, as they had been by the purification process, and were active only at alkaline pHs. That is perhaps the reason why we often failed to detect retroviruses directly in porcine cell culture supernatants by assaying their RT activity at pHs as high as the values normally used (pH 8.3). Monovalent cations (Na +, K +) had an enhancing effect on porcine retrovirus RT activity at concentrations that had also been found for most other viral RTs (optimal at 50 mM), but for porcine retrovirus RT these monocations began to become inhibitory at relatively lower concentrations (150 mM as compared to 300raM). Na + and K + ions had a similar effect, but the enhancing range of K + concentrations was broader (Fig. 2). As a result, when cell culture supernatants or biological fluids are used as such in the assay, attention should be paid to monocation concentrations already present in samples and reactant solutions added so that the total amounts of Na + and K + should not exceed optimal doses and become inhibitory. In efforts to increase the sensitivity of the RT assay further, various substances have been used as additives by different authors [2, 6, 7, 10]. Several of these compounds have been tried by us, but only dithiothreitol had an unambiguous enhancing effect, although not to the extent reported by Hoffman and coworkers [7]. Furthermore, with other additives, the result reported by different authors are not uniform, sometimes even contradictory. Finally, the sensitivity of the assay could be enhanced by increasing the duration of incubation. Normally 1 h of incubation was sufficient to give significant signals for our virus-containing cell culture supernatants that had been concentrated to 50- to 100-fold. With scarcer amounts of virus a longer incubation was needed, but very extended incubation periods decreased the radioactive incorporation,
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probably because of the degradative action of c o n t a m i n a t i n g nucleases or a nuclease activity contained in the R T enzyme system itself as occurs with avian myeloblastosis virus RT. We did n o t count the minimal n u m b e r of retroviral particles u n a m b i g u o u s l y detectable by the assay, but 0.1 gg of retroviral protein regularly gave a signal of several h u n d r e d times when c o m p a r e d with the negative control background. Using the R T assay with optimal conditions determined above, we confirmed that: (1) procine retroviruses can be transmitted to the pig by intraperitoneal inoculation with virus-producting cells, and (2) retroviruses can be spontaneously p r o d u c e d by porcine cell lines after long-term propagation. The cause of this " s p o n t a n e o u s p r o d u c t i o n " of retrovirus is u n k n o w n .
Acknowledgements We thank Drs. Akiro Sonoda and Michi Kodama (National Institute of Animal Health, Tsukuba City of Science, Japan) for their kind gift of Shimozuma cells, and Dr. JeanMarie Aynaud (INRA, Nouzilly, France) for his gift of ST cells and helpful discussions.
References 1. Brun G, Rougeon F, Lauber M, Chapvitle F (I974) Purification and properties of DNA polymerases from chick embryo. Eur J Biochem 41:241-251 2. Chan EW, Dale PJ, Greco IL, Rob JG, O'Connor TE (1980) Effects of polyethylene glycol on reverse transcriptase and other polymerases activity. Biochim Biophys Acta 606:353-361 3. Chen TR (1977) In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:255-262 4. Frazier ME (1985) Evidence for retrovirus in miniature swine with radiation-induced leukemia or metaplasia. Arch Virol 83:83 87 5. Fridlender B, Fry M, Bolden A, Weissbach A (1972) A new synthetic RNA-dependent DNA polymerase from human tissue culture cells (HeLa-fibrobtast-synthetic oligomtcteotides-template-purified enzymes). Proc Natl Acad Sci USA 69:452-455 6. Gregerson JP, Wege H, Preiss L, Jentsch KD (1988) Detection of human immunodeficient virus and other retroviruses in cell culture supernatants by a reverse transcriptase microassay. J Virol Methods 19:161-168 7. Hoffman AD, Banapour B, Levy JA (1985) Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 147:326-335 8. Huebner RJ, Igel HJ (1970) Immunological tolerance to gs antigens as evidence for vertical transmission of non-infectious C-type RNA virus genoms. Perspect Virol 7: 55-71 9. Kaeffer B, Bottreau E, Phan-Thanh L, Olivier M, Salmon H (1990) Histocompatible miniature boar model: selection of transformed cell lines of B and T lineages producing retrovirus. Int J Cancer 46:481-488 10. Kiessling AA, Goulian M (1976) A comparison of the enzymatic responses of the DNA polymerases from four RNA tumour viruses. Biochem Biophys Res Comm 71: 10691077 1t. Lieber MM, Sherr CJ, Benveniste RE, Todaro GJ (t975) Biologic and immunologic properties of porcine type C virus. Virology 66:616-619 12. McClurkin AW, Norman JO (1966) Studies on transmissible gastroenteritis of swine:
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13.
14. 15. 16. 17.
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II. Selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis. Can J Comp Med Vet Sci 30:190-198 Nermut MV, Frank H, Schaeffer W (1972) Properties of mouse leukemia viruses. III. Electron-microscopic appearance as revealed after conventional preparation techniques as well as freeze-drying and freeze-etching. Virology 49:345-358 Nowinski RC, Peters ED (1973) Cell surface antigens associated with murine leukemia virus: definition of GL and GT antigenic systems. J Virol 12:1104-1117 Suzuka I, Seikiguchi K, Kodama M (1985) Some characteristics of a porcine retrovirus from a cell line derived from swine malignant lymphomas. FEBS Lett 183:124-128 Todaro GJ (1972) Detection and characterization of RNA tumour viruses in normal and transformed cells. Perspect Virol 8:81-101 Weissbach A, Bolden A, Mueller R, Hanafusa H, Hanafusa T (1972) Deoxyribonucleic acid polymerase activities in normal and leukovirus-infected chicken embryo cells. J Virology 10:321-327
Authors' address: Dr. L. Phan-Thanh, Pathologie Infectieuse et Immunologie, INRA, F-37380 Nouzilly, France. Received June 20, 1991
