Comparison of Interferon Induction in Mice by Purified Penicillium chrysogenum Virus and Derived Double-stranded RNA

Transcription

1 J. gen. Virol. (1971), I2, Printed in Great Britain Comparison of Interferon Induction in Mice by Purified Penicillium chrysogenum Virus and Derived Double-stranded RNA By K. W. BUCK, E. B. CHAIN AND F. HIMMELWEIT Biochemistry Department, Imperial College of Science and Technology, London, S.W. 7 (Accepted 29 March I971 ) SUMMARY A virus preparation, obtained from the mycelium of Penicillium chrysogenum, strain QI76, was purified by isopycnic centrifugation on linear gradients of potassium tartrate. Double-stranded RNA, prepared from the virus particles, revealed three components on electrophoresis on polyacrylamide gels. The serum interferon levels in mice after intravenous injection of free virus RNA were higher and earlier than for purified virus particles, containing an equivalent amount of RNA. After reaching their maxima, the interferon activities declined at similar rates to very low levels after 45 hr for free RNA and 39 hr for virus particles. INTRODUCTION In recent years it has been shown that the active principle of Statolon, an antivirus agent isolated from Penicillium stoloniferum (Kleinschmidt & Probst, I962 ) is double-stranded RNA of virus origin (Banks et al. 1968, Kleinschmidt et al. I968 ). Both virus particles and derived RNA were found to exert their antivirus effect by inducing interferon production in the host animal. The active component of another fungal antivirus agent, Helenine, obtained from Penicilliumfuniculosum, was also shown to be double-stranded RNA (Lampson et al. I967), and considered to arise from a hypothetical virus infection. Later virus particles were detected in a different strain of P. funiculosum and a crude virus preparation was found to induce interferon production in mice (Banks et al. I968). Viruses have also been isolated from Penicillium cyaneo-fulvum (Banks et al. I969b) and Aspergillus foetidus (Banks et al. I97O) and in each case both virus particles and derived double-stranded RNA were potent stimulators of interferon production in animals. Following reports of the isolation of antivirus and interferon-stimulating agents from Penicillium chrysogenum (Belgian Patent, I968; D. E. Van Dijk et al. personal communication) we found virus particles in seven penicillin-producing strains of this fungus (Banks et al. I969a). We now report the isolation and purification of the P. chrysogenum QI76 virus, show that RNA extracted from the virus particles is double-stranded, and compare the production of interferon in mice induced by intact virus particles and extracted RNA. METHODS Preparation and Purification of virus. Penicillium chrysogenum strain Q 176 was grown in 6o 1. stainless steel fermenters using the medium and conditions described previously (Banks et al. I969a). A crude virus preparation, obtained from the mycelium, as described for the P. cyaneo-fulvum virus (Banks et al b), was further purified by isopyknic centri- I0-2

2 I32 K.W. BUCK, E. B. CHAIN AND F. HIMMELWEIT fugation on linear gradients of potassium tartrate. Crude virus preparation (I ml.) was layered onto a pre-formed linear gradient of potassium tartrate (50 ml., IO to 50 % w/w) and centrifuged for I8 hr at 20,000 rev./min, in a Beckman SW25"2 rotor. A band of precipitated virus which formed at a density of 1.27 g./ml, was collected and dialysed against 0"o3 M- sodium phosphate buffer, ph 7"6, until free from tartrate. Analytical centrifugation. Sedimentation coefficients were measured using a Beckman Model E analytical ultracentrifuge equipped with an ultraviolet scanner. A 2 aluminiumfilled Epon double sector centrepiece was used in the AN-H rotor at I6,OOO rev./min, for virus preparations and at 40,000 rev./min, for RNA preparations. Sucrose density gradient centrifugation. Virus preparation (I ml.), containing less than I mg. of protein, was layered on a IO to 50% w/v linear gradient of sucrose in 0"o3 M- sodium phosphate buffer, ph 7"6, and centrifuged for 3 hr at 24,ooo rev./min, in rotor SW25 of a Beckman L2 ultracentrifuge. Gradients were scanned at 265 nm. using an ISCO density gradient analyser. Preparation of antisera and gel diffusion analysis. These were as described by Banks et al. 0969b). Preparation of virus RNA. This was by the phenol + sodium dodecyl sulphate method, essentially as described by Franklin 0966). Polyacrylamide gel electrophoresis. 4% polyacrylamide gels containing 0"04% bisacrylamide were prepared in glass tubes, 4 mm. internal diameter, essentially as described by Bishop, Claybrook & Spiegelman (i967). Running buffer was TAE (o'04 M-tris, 0"O5 M- acetate, o.ooi6 M-EDTA, ph 7"8) for virus preparations and twice this concentration of TAE for RNA preparations. Electrophoresis was carried out at 5 ma/tube for 2 to 4 hr. Extinction coefficient of virus RNA. A stock solution of virus RNA in SSC (o" I5 M-NaC1, o'oi5 M-Na citrate, ph 7"0; 12oo #g./ml.) was diluted from 0"5 ml. to 25 ml. with SSC and the extinction at 26o nm. was measured. From the same stock solution of RNA 0"5 ml. was mixed with 0"5 ml. of 0"5 M-sodium hydroxide; the mixture was incubated at 37 for 18 hr and then diluted to 25 ml. with water. The optical density at 260 rim. was measured and the extinction coefficient of the virus RNA was calculated from the published extinction coefficients of the four nucleotide products (Dunn & Hall, I968 ) and the measured nucleotide composition of the virus RNA. Base ratio analysis of virus RNA. This was as by Knight (I963). Determination of RNA content of virus and RNA preparations. The RNA content of virus preparations was determined by phosphorus estimation (Bartlett, I959) in conjunction with the measured base composition of the RNA. Concentrations of virus RNA preparations were determined spectrophotometrically using the measured extinction coefficient. Acid hydrolysis of virus RNA. Virus RNA preparations were hydrolysed with N-hydrochloric acid and the products were separated chromatographically (Smith & Markham, I95o). Thermal denaturation. The increase in optical density at 260 nm. of virus RNA preparations was measured using the heated cell compartment of a Beckman DK spectrophotometer. Thermal transition mid-points (Tm) were calculated as described by Marmur & Doty (I 962). lsopycnic centrifugation of virus RNA. Virus RNA (0"2 ml. of 15 #g./ml.) in SSC was mixed with a solution of caesium sulphate (9 ml., density 1"8 g./ml.) and SSC (8 ml. at Io concentration) and the density adjusted to I-6o g./ml, by addition of SSC. Samples of 2"5 ml. of this solution were overlaid with 2"5 ml. of liquid paraffin and centrifuged at 35,ooo rev./min, in a Beckman SW39L rotor for 6o hr at 4. The bottom of the tubes was pierced and 5 fractions of 5 drops each were collected using a Buchler density-gradient

3 P. chrysogenum virus and interferon induction I33 fractionater. The optical density and refractive index of each fraction was determined. Densities were estimated from refractive indices (Hearst & Vinograd, 196I ). Action of ribonuclease on virus RNA. A solution of I ml. of virus RNA (2o #g./ml.) in SSC or o.i SSC was mixed with Io/~1. of a solution of Boehringer pancreatic ribonuclease (2o #g./ml.) in SSC or o.x SSC and the optical density at 26o nm. was measured as a function of time at 23. Action ofdeoxyribonuclease on virus RNA. Virus RNA was incubated with Worthington deoxyribonuclease (ribonuclease-free) as described by Wood & Streissle (~97o). The optical density at a6onm, was measured as a function of time. After 2 hr the incubation mixture was examined by polyacrylamide gel electrophoresis. Interferon induction. Interferon was induced in 18 g. (_+ I g.) mice by intravenous or intraperitoneal injection. Interferon assays. These were made on pooled mouse sera by the dye uptake method (Finter, I969) and 5o% end-points were determined by probit analysis (Lindenmann & Gifford, I963). Protein determination. The protein content of virus RNA preparations was estimated using the method of Lowry et al. (I95I). Isolation and purification of virus particles RESULTS Virus preparations, obtained from the mycelium of Penicillium chrysogenum, were purified by isopyknic centrifugation on linear gradients, of potassium tartrate. This virus was unstable on the caesium chloride gradients used for purifying the viruses from P. cyaneofulvum (Banks et al. I969b) and Aspergillusfoetidus (Banks et al. I97o ). The yield of purified virus from a 6o 1. fermentation was approximately 45o mg. Preparations contained polyhedral virus particles approximately 35 nm. in diameter when examined by electron microscopy, as previously described (Banks et al. I969b) and had an ultraviolet spectrum typical of nucleoprotein with minimum at 244 nm. and maximum at 260 nm. The purity of virus preparations was shown by the following criteria: (a)in isopyknic centrifugation on linear gradients of potassium tartrate a single band of density I.z7 g./ml. was obtained; (b) in velocity centrifugation on linear gradients of Io to 5 % (w/v) sucrose a single band was formed; (c)in velocity centrifugation in o'o3 N-phosphate buffer, ph 7"6, containing o.i M-potassium chloride a single boundary was obtained with S~ 0 = I5o s; (d) in electrophoresis on 4 % polyacrylamide gels a single band was observed when the gels were stained with either amido black (Smith, I968) or acridine orange (Richards, Coll& Gratzer, I965); (e) in gel diffusion tests against rabbitantiserum to purified virus preparations only a single preeipitin line was obtained. There was no cross-reaction when virus preparations were tested against antisera to viruses obtained from P. stolon!ferum, P. funiculosum, P. cyaneo-fulvum or A. foetidus, or when antiserum to the P. chrysogenum virus was tested against virus preparations from the four fungi named above. Properties of virus RNA. Virus preparations were readily disrupted with 1% sodium dodecyl sulphate, and RNA, with less than 1% protein, could be obtained in yields of 70 to 8o % by phenol extraction. Although a single boundary (S~0 = I3 s in SSC) was observed when virus RNA preparations were centrifuged (zoo,ooo g), three clear bands of closely similar mobility were obtained when preparations were subjected to electrophoresis in 4 % polyacrylamide gels. The virus nucleic acid had a typical ultraviolet absorption spectrum with A maximum at

4 I34 K. W. BUCK, E. B. CHAIN AND F. HIMMELWEIT I I I I 1 '75 m ~ N d 1.ss e, 1.o f'~ "~, 1.so 0"5 T T v m O m Q ~ O ~ Q w O ~ Volume of gradient (ml.) Fig. I. Sedimentation of Penicillium chrysogenum virus RNA in caesium sulphate gradients E~60; m, density of Cs2SO4 (g./ml.). O m I I l I I I I I 1.4 o J ol I el ID.m..--~. i I I Temperature (o) Fig. 2. Thermal denaturation ofpenicillium chrysogenum virus RNA in o.i SSC.

5 P. chrysogenum virus and interferon induction nm., and ~ minimum at 231 nm. (E~/a. lem. (SSC) 203 ; E26o/E28 o 2" I7). The nucleic acid was RNA by the following criteria: (a) it was readily hydrolysed by dilute alkali to form, in 99 % yield, a mixture of GMP, CMP, AMP and UMP, which were characterized by their electrophoretic mobilities and ultraviolet spectra; (b) it was readily hydrolysed by dilute acid to form in high yield a mixture of guanine, adenine, CMP and UMP, which were characterized by their chromatographic mobilities and ultraviolet spectra; (c) it was readily hydrolysed, at low ionic strength, by pancreatic ribonuclease, but was unaffected by deoxyribonuclease; (d) on isopycnic centrifugation in self-generating gradients of caesium sulphate it formed an asymmetric band of density approximately 1.6o g./ml. (Fig. I), a value in the range for RNA, but not for DNA g ' v.-~ I I I I Time (min.) Fig. 3. Action of ribonuclease on Penicillium chrysogenum virus RNA. O, in o.i SSC; V--V, in SSC. The virus RNA was shown to be double-stranded as follows: (1) Its melting curves in SSC (Tm lo3 ) and o.i SSC (Tm 88, Fig. 2) and behaviour on treatment with ribonuclease in SSC and o.i SSC (Fig. 3, Table I) were very similar to those described for the doublestranded RNAs from the P. cyaneo-fulvum virus (Banks et al. I969b ) and the A.foetidus virus (Banks et al. I97o). (2) The base ratio analysis (Table 2) showed approximately equimolar amounts of G and C, and A and U, and was consistent with, although not proof of, a predominantly double-stranded structure. Induction of interferon by P. chrysogenum virus and virus RNA Both the virus and virus RNA were good inducers of interferon in mice. Comparative activities of serum interferon 18 hr after an intraperitoneal injection of virus or virus RNA in mice are given in Table I. To obtain a further comparison of the interferon-inducing capacities of the virus and virus RNA the levels of serum interferon were measured as a function of time after intravenous injection of equivalent amounts of RNA as virus particles or as virus RNA. The free virus RNA induced an earlier peak activity of serum interferon (Fig. 4) than did the virus particles. Of greater contrast was the fourfold higher maximum

6 136 K. W. BUCK~ E. B. CHAIN AND F. HIMMELWEIT activity, induced by the free RNA. Both interferon activities then declined exponentially at similar rates to reach very low levels at about 45 hr for the free RNA and at 39 hr for the particles. Table I. Production of interferon by P. chrysogenum virus and virus RNA preparations Preparation Purified intact virus preparation 80 in SSC Virus RNA in SSC Virus RNA in SSC treated with 80 ribonuclease (o.z/~g./ml.) for z hr at z5 Virus RNA in o'i SSC treated with ribonuclease (o'z #g./ml.) for z hr at 25 Weight of RNA injected/mouse (#g.) 8o 8o Relative interferon activity of pooled sera from 15 mice (I8 g.) Mice were injected intraperitoneally with P. chrysogenum virus or RNA preparations and were bled after t6 hr. IOO o IO "~ _~ 40 2O 10 < Time (hrl Fig. 4. Kinetics of interferon production in the mouse in response to the intravenous injection at time zero of (a) 8o #g. double-stranded RNA, in o.z ml SSC, extracted from Penicillium chrysogenum virus particles ( -- ), and (b) a o.2 ml. suspension in SSC of such particles containing the equivalent of 8o #g. virus double-stranded RNA (0 Q). Interferon assays for each point were made on pooled sera from 15 mice (18 g.). A standard serum was included for reference in each assay. Table 2. Base composition of P. chrysogenum virus RNA Molar ratio (%) and standard deviation in five hydrolyses Adenine Uracil o-7 Guanine 26. I + o.6 Cytosine 24"5 + o.6 DISCUSSION All viruses so far isolated from the Penicillia and Aspergilli appear to contain a genome of double-stranded RNA. The virus found in Penicillium chrysogenum strain Q I76 conforms to this pattern. Viruses from P. stoloniferum and Aspergillus foetidus appear to be multi-

7 P. chrysogenum virus and interferon induction I37 component virus systems and virus RNA gave multiple bands on gel electrophoresis (Buck & Kempson-Jones, I97o; Banks et al. I97O; K. W. Buck and G. Ratti, unpublished results). For P. chrysogenum virus there was no evidence of more than one type of virus particle, although isolated RNA showed three components of closely similar electrophoretic mobility on polyacrylamide gels. The presence of multiple bands of double-stranded RNA has been reported for many viruses (reovirus, Shatkin, Sipe & Loh, 1968; wound turnout virus, Wood & Streissle, 197o; cytoplasmic polyhedrosis virus, Kalmakoff, Lewandowski & Black, I969; rice dwarf virus, Fuji-Kawata, Miura & Fuke, I97O; blue tongue virus, Verwoerd, Louw & Oellermann, 197o; African horse sickness virus, Oellermann, Els & Erasmus, I97O). Fuji-Kawata et al. (I97O) have suggested that this multiple bands property may be characteristic of all such double-stranded RNAs. After this work was submitted for publication, Cox, Kanagalingam & Sutherland (197o) described the isolation and properties of two species of double-stranded RNA from the mycelium ofp. chrysogenum. The larger of these two species, with S 0. w = s, appears to have similar properties to the virus RNA described here; however, we could not detect the smaller species, with S 0,,~ = 7"3 s, in RNA isolated from purified virus preparations. Double-stranded RNA of diverse origins have been shown to act as powerful inducers of interferon production in animals (Nemes et al. 1969). It is probable that interferon induction by both P. chrysogenum virus and isolated RNA is due to double-stranded RNA. The slower action of the virus particles in stimulating interferon production is probably a reflexion of the slow release of RNA from the virus particles. More surprising is the lower yield of interferon induced by virus than by an equivalent amount of virus RNA. A possible explanation is that different cells are stimulated in the two cases. Spleen and liver cells of the reticulo-endothelial system (Kono & Ho, 1965), circulating lymphocytes (Jullien & De Maeyer, I966; J. De Maeyer-Guigrard and E. De Maeyer, personal communication), and non-phagocytic mononuclear leucocytes (Lee & Ozere, 1965), have been invoked in the production of circulating interferon by different inducers. These possibilities have not yet been distinguished but it is hoped to obtain further information by studying the fate of labelled P. chrysogenum virus and virus RNA after intravenous injection into mice. Essentially similar results were obtained by Planterose et al. (197o), who dosed mice intraperitoneally with double-stranded RNA, obtained from virus-like particles from P. stolon# ferum, and obtained a peak of serum interferon activity about 4 hr later. Lower levels of serum interferon were produced after intraperitoneal injection of the virus-like particles, containing an equivalent amount of double-stranded RNA; in this case, however, serum interferon levels were still rising 24 hr after injection. The difference between these results and those obtained with P. chrysogenum virus and virus RNA is probably due to a much slower release of virus RNA from the P. stoloniferum virus than from the P. chrysogenum virus. This could be due to the different routes of injection, intraperitoneal or intravenous, or to differences in stability of the two types of virus particle. We wish to thank Mr G. T. Banks and his colleagues for pilot plant fermentations, Mrs J. E. Darbyshire for gel diffusion analysis and electron microscopy, Mr I. P. Blench and Mr R. Hay for technical assistance and the Medical Research Council for a grant. The work reported in this paper forms part of a collaborative study on antivirus agents with Dr D.N. Planterose and his colleagues at Beecham Research Laboratories, Betchworth, Surrey.

8 138 K.W. BUCK, E. B. CHAIN AND F. HIMMELWEIT REFERENCES BANKS, G. T., BUCK, K. W., CHAIN, E. B., DARBYSHIRE, J. E. & HIMMELWEIT, F. (I969a). Virus-Iike particles in penicillin producing strains of Penicillium chrysogenum. Nature, London 222, 89. BANKS, G. T., BUCK, K. W., CHAIN, E. B., DARBYSHIRE, J. E. & HIMMELWEIT, F. (19693). Penicillium cyaneo-fulvum virus and interferon stimulation. Nature, London 223, 155. BANKS, G. T., BUCK, K. W., CHAIN, E. B., DARBYSHIRE, J. E., HIMMELWEIT, F., RATTI, 0. 9 SHARPE, T. J. & PLANTEROSE D. N. (1970). Antiviral activity of double-stranded RNA derived from a virus isolated from Aspergillus foetidus. Nature, London 227, 5o5. BANKS, G. T., BUCK, K. W., CHAIN, E. B., HIMMELWEIT, E.~ MARKS, J. E., TYLER, J. M., HILLINGS, M., LAST, F. T. & STONE, O. M. (1968). Viruses in fungi and interferon stimulation. Nature, London 218, 542. BARTLETT, G. R. (I959). Phosphorus assay in column chromatography. Journal of Biological Chemistry 234, 466. BELGIAN PATENT (I968). NO. 691,88I, Glaxo Laboratories Ltd. BISHOP, D. W. L., CLAYBROOK, J. R. & SPIEGELMAN, S. (1967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. Journal of Molecular Biology 26, 373. BUCK, K. W. & KEMPSON-JONES, G. F. (I970). Three types of virus particle in Penicillium stoloniferum. Nature, London 225, 945. cox, R. A., KANAGALINGAM, K. & SUTHERLAND, E. S. (1970). Double-helical character of ribonucleic acid from virus-like particles found in Penicillium chrysogenum. Biochemical Journal 12o, 549. DUNN, D. B. & HALL, R. H. (r968). Purines, pyrimidines, nucleosides and nucleotides: physical constants and spectral properties. In Handbook of Biochemistry: Selected Data for Molecular Biology, p. G 3- Ed. by H. A. Sober: The Chemical Rubber Co: Cleveland Ohio. FINTER, i",1. B. (I969). Dye uptake methods for assessing viral cytopathogenicity and their application to interferon assays. Journal of General Virology 5, 419. FRANKLIN, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage R 17. Proceedings of the National Academy of Sciences of the United States of America 58, 782. FUJI-KAWATA, I., MIURA, K. & FUKE, i. (I970). Segments of genome of viruses containing double-stranded ribonucleic acid. Journal of Molecular Biology 51, 247. HEARST, J. L. & VINOGRAD, J. (I961). The net hydration of T-4 bacteriophage deoxyribonucleic acid and the effect of hydration on buoyant density behaviour in a density gradient at equilibrium in the ultracentrifuge. Proceedings of the National Academy of Sciences of the United States of America 47, loo5. JULLIEN, P. & DE MAEYER, E. (I966). Interferon synthesis in X-irradiated animals. I. Depression of circulating interferon in CDH mice after total body irradiation. International Journal of Radiation Biology 11, 567. KALMAKOFF, J., LEWANDOWSKI, L. J., & BLACK, n. R. 0969). Comparison of the ribonucleic subunits of reovirus, cytoplasmic polyhedrosis virus and wound tumour virus. Journal of Virology 4, 851. KLEINSCHMIDT, W. J., ELLIS, L. E., VAN FRANK, R. M. & MURPHY, E. B. (I968). Interferon stimulation by a doublestranded RNA from a mycophage in statolon preparations. Nature, London 22o, 157. KLEINSCHMIDT, W. J. & PROBST, G. W. (1962). The nature of statolon, an antiviral agent. Antibiotics and Chemotherapy 12, 298. KNIGHT, C. A. (1963). The Chemistry of Viruses, p. 82. Wien: Springer-Verlag. KONO, Y. & HO, M. (1965). The role of the reticuloendothelial system in interferon formation in the rabbit. Virology 25, I62. LAMPSON, G. P., TYTELL, A. A., FIELD, A. K., NEMES, H. M. & HILLEMAN, M. R. (1967). Inducers of interferon and host resistance. I. Double-stranded RNA from extracts of Penicillium funiculosum. Proceedings of the National Academy of Sciences of the United States of America 58, 782. LEE, S. H. S. & OZORE, R. L. (1965). Production of interferon by human mononuclear leukocytes. Proceedings of the Society for Experimental Biology and Medicine 118, I9O. LINDENMANN, J. & GIFFORD, G. E. (I963). Studies on vaccinia virus plaque formation and its inhibition by interferon. Virology I9, 3o2. LOWRY, U. H., ROSEBROUGH, N. J., EARR, A. L. & RANDALL, R. J. (I95I). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 165. MARMIIR, J. & DUTY, P. (I962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. Journal of Molecular Biology 5, IO9. NEMES, M. M., TYTELL, A. A., LAMPSON, G. P., FIELD, A. K. & HILLEMAN, M. R. (1969)-Inducers of interferon and host resistance. VII. Antiviral efficacy of double-stranded RNA of natural origin. Proceedings of the Society for Experimental Biology and Medicine 132, 784. OELLERMANN, R. A., ELS, H. J. & ERASMUS, B. J. (1970). Characterization of African horsesickness virus. Archly fiir die gesamte Virusforschung 29, 163. PLANTEROSE, D. N., BIRCH, P. J., PILCH, D. J. P. & SHARPE, T. J. (I970)- Antiviralactivityof double-strandedrna and virus-like particles from Penicillium stoloniferum. Nature, London 227, 5o4. RICHARDS, E.G., CULL, J.A. & GRATZER, W.B. (I965). Disc electrophoresis of ribonucleic acid in polyacrylamide gels. Analytical Biochemistry 12, 452.

9 P. chrysogenum virus and interferon induction I39 SrtATKIN, A. J., SIPE, S. D. & LOH, P. (1968). Separation of ten reovirus genome segments by polyacrylamide gel electrophoresis. Journal of Virology 2, 986. SMITH, L (I968). Chromatographic and Electrophoretic Techniques, Vol. n, p London: Heinemann. SMITH, J. D. & MARKHAM, R. (I950). Chromatographic studies of nucleic acids. ]L The quantitative analysis of ribonucleic acids. Biochemical Journal 46, 5o9. VERWOERD, D. W., LOUW, H. & OELLERMANN, R. A. (1970). Characterization of blue tongue virus ribonucleic acid. Journal of Virology 5, I. wooo, H. A. & STReISSLE, G. 0 97O). Wound tumour virus; purification and fractionation of the double-stranded ribonucleic acid. Virology 40, 319. (Received I7 November r97o)