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Poly(A):Poly(U); Poly(O8A):Poly(U); Poly(A):Poly(MeNH5U) and poly(O8A):Poly(MeNH5U) versus bida semliki forest virus in chick embryos
Toxicology Letters, 12 (1982) 41-52 Elsevier
Biomedical
47
Press
POLY(A):POLYW);
POLY(O*A):POLY(U);
POLY(O*A):POLY(MeNH%J) IN CHICK
JOSEPH
POLY(A):POLY(MeNH5U)
AND
VERSUS BIDA SEMLIKI FOREST VIRUS
EMBRYOS
OLOLADE
FOLAYAN
Biochemistry Department, Ife University, Ile-Ife, Oyo State (Nigeria) (Received
August
(Revision
received
(Accepted
2lst,
1981)
October
November
26th,
1981)
18th, 1981)
SUMMARY Poly(A):poly(U) acid:polyuridylic acid hybrid) hybrid) Nigerian
(polyadenylic acid:polyuridylic acid hybrid); poly(OsA):poly(U) (poly-8-oxy-adenylic acid hybrid); poly(A):poly(MeNHW) (polyadenylic acid:poly-5-methylamino uridylic
and poly(OsA):poly(MeNHsU)
were studied strain)
in chick embryos
AN 49809. The modified
than the unmodified
(poly-8-oxyadenylic
to compare polymers
the protection showed
acid:poly-5-methylamino offered
against
a higher activity
uridylic
acid
Semliki forest virus (the
index but were more toxic
analogue.
INTRODUCTION
The induction of cells to produce interferon [I] may be brought about by both synthetic [2-41 and natural polynucleotides [5]. The synthesis of polynucleotides is receiving much attention [6-91; some can be degraded by ribonucleases and many synthetically modified nucleotides are more resistant to ribonuclease hydrolysis than the unmodified analogues [lo]. These molecules must possess stable secondary structure, being double stranded hybrids with high melting temperatures [ 111; but this requirement also causes cytoxicity [ 1l- 121. We have studied the effects of these polynucleotides on local (Nigerian) viruses and investigated the effects of some polymers on Wesselsbron virus (H10964) in albino mice [13-l 51. We are also studying the effects of Bida (Nigerian) strain of Semliki Forest virus (AN49809) in albino mice as well as the effects of Wesselsbron virus in chick embryos. The present study has been undertaken to examine the effect of Bida Semliki forest virus in chick embryos. This virus causes brain damage in mice when injected 0378-4274/82/0000-0000/$02.75
0 Elsevier
Biomedical
Press
48
intracerebrally, and death usually within 24 h of administration [16]. Mice pretreated with synthetic polynucleotides may survive up to certain doses. This virus is abundant in mosquitoes from which it is extracted [ 161. As our birds (and other animals) are bred in atmospheres not completely free of mosquitoes, the possible commercial value of this study is obvious. MATERIALS
AND METHODS
The virus was obtained from the Virology Group of the University College Hospital (Ibadan), where isolation, purification and characterisation were carried out, having been isolated from mosquitoes Aedes (Neo) spp. and Mansonia africanus collected at Bida (in Niger State) [16]. Mice were injected with this virus and died when the concentration reached 1 . lo* units [ 161. The animals were then quickly frozen and kept at - 70 “C until required. After thawing under tap water, the brain was sucked out into a micro-mortar, ground and made up to the required volume with sterile distilled water. Nucleosides, nucleotides and other reagents were purchased from Sigma Chemical Co.; poly(08A) and poly(MeNH5U) were prepared as described [6, 151. The two appropriate polymers were mixed in equimolar solutions to make the required hybrids [6-91. Eggs were obtained from the University of Ife Farm after being fertilized and kept for 10 days. Pretreatment When the embryos were 10 days old, the eggs were candled to locate the airsacs. Injection of either the virus or the polymer was made at a point opposite the air sac care being taken to avoid the blood vessels. The eggs were stored with the point of inoculation uppermost. Solutions of polymers were made in sterile distilled water to which penstrep (penicillin/streptomycin) had been added. The volume injected into each egg was maintained at 0.1 ml and controls were given distilled sterile water only. Challenging The eggs were kept for 24 h after pretreatment to allow induction of interferon and were then given a counter injection of 0.1 ml virus at the desired concentration. The eggs were left for 5 days and then cracked. In water, eggs with dead embryos floated while those containing living embryos, sank. Percentage survival was calculated.
49 RESULTS
The results are given in Tables I-IV.
TABLE I VIRUS DOSE RESPONSE Eggs were grouped and various doses of virus administered. They were cracked at 5 days and the embryos examined. CaIculations were made for percentage survival. Group 1
2 3 4 5 6 7 8 9 10
Virus dose (+10’ units)
Results after 5 days (70 survival)
0.00 0.01
100 f 62 + 46 rt 35 + 25 + 18 t 10 + 7f 5f 32
0.10
0.30 0.50 0.80 1.00 1so 2.00 2.50
2.0 2.0 4.0 5.0 5.0 5.0 6.0 7.0 7.0 7.0
LDso = 0.07.107 Units,
TABLE II POLYMER DOSE RESPONSE Eggs were pooled into 10 groups, given poly(A):poly(U), and were watched for 5 days before being cracked for examination and calculation of percentage survival. This was repeated for the other three analogues. Group
Polymer fig in 0.1 ml
1
0.00
2 3 4 5 6 7 8 9 10
0.01 0.10 1.00 10.00 30.00 50.00 70.00 85.00 100.00
LDso pg/O. 1 ml
% Survival for various polymers after 5 days Poly(A): poly(Uf
Poly(O*A) POlY(U)
Poly(A): poly(MeNHsU)
Poly(O*A): poly(MeNHsU)
lOOz!z 2 994 2 961t 4 921t 5 862 5 79f 7 68r 7 50+ 8 36+ 8 15rh: 10
loo+98+ 96+ 90+ 82+ 72+ 64t 48-+ 30+ 12 *
2 3 5 5 6 6 8 8 8 10
lOOk 96+ 90+ 86+ 77+ 6Oi 55+ 465 27 + 12*
2 4 6 6 7 7 8 8 10 10
100+ 96+ 90* 80& 72+ 58-t 52* 43* 25+ 10 f
2 4 6 6 8 8 8 8 10 12
68*
5
64*
6
6Ok
8
70*
5
50
TABLE III PROTECTION PROFILES Eggs were grouped and pretreated with different doses of polymers and later challenged by varying doses of virus. The following results were obtained from pretreatment with 10.0 pg polymer followed by challenges with 0.01, 0.10, 0.50 or 1.00.107 units of virus, respectively. Polymer (10.0 pg in 0.1 ml)
% Survival at various virus doses .I07 Units 0.10
0.01 Poly(A):poly(U) Poly(0sA):poly(U) Poly(A):poly(MeNHsU) Poly(08A):poly(MeNHsU)
85 t 82 + 76 + 72 +
5 5 7 7
82 + 15 f 70 f 66 +
0.50 6 6 8 8
1.00
70? 68+ 62& 58 &
8 8 8 10
66t 6Ot 57 t 52 +
8 8 10 10
TABLE IV ACTIVITY INDEX Several protection profiles (e.g. in Table III) were carried out with varying polymer concentrations. The optimum protections were found and the highest percentage protection also obtained as summarised below. Activity index was taken either as protection per gg (@/o/as)or as how much material was required to offer 1% protection &g/%). Polymer
Optimum concentration (pg in 0.1 ml)
Maximum protection (S)
Activity Index
18+3 14 + 3 12 zlz2 10 + 2
86 + 4 80 + 4 76 z!z4 70 * 5
209 175 158 143
(gg, %
,lO-3 Poly(A):poly(U) Poly(OsA):poly(U) Poly(A):poly(MeNHsU) Poly(OsA):poly(MeNHW)
. lO-2 477 571 633 700
DISCUSSION
All analogues of poly(A):poly(U) offered protection to chick embryos against Semliki forest virus. The results resemble those for poly(I):poly(C) and some of its analogues against Wesselsbron virus (H19964). The polymer which is not modified (poly(A):poly(U)) was found to be the least toxic. Next was one in which the poly(U) strand is unmodified (i.e. p~ly(O~A):poly(U)). The polymer in which both strands were modified had the highest toxicity followed by that in which the poly(A) strand alone was modified. The poly(U) strand may be the more critical of the two. Activity was found to be in the reverse order to toxicity. The most toxic is also the most active and the sequence follows that order (Table IV). It would appear that activity and toxicity are directly related as has been observed in other systems [ 11, 171.
51
Toxicity was estimated by taking the reciprocal of the LDso, since toxicity and LDso values are inversely related. Similarly, the activity index was taken either as the amount of polymer that would offer 1% protection under optimum concentrations or the amount of protection offered by 1 pg material at optimum concentrations. In the series of synthetic polymers studied, there was little difference between the LDso values, indicating that modification has not drastically increased toxicity. However, as reported [lo], the modifications have led to increased stability of secondary structure, resistance to ribonucleases and increased M.P. values. These new analogues would therefore appear to have potential for interferon induction in chick embryos. CONCLUSION
These polymers in chick embryo cells induce the production of interferon which is active against Semliki forest virus. This bears some similarity to the activity of poly(I):poly(C) analogues in chick embryos against Wesselsbron virus. The modified polymers have some advantage over the unmodified analogue as they are more stable.
The author acknowledges the University of Ife, Ile-Ife, Oyo State of Nigeria for the award of the research grant with which this laboratory was financed. The assistance of other colleagues, Dr. E.O. Ngaha, Mr. Doyin Okusanya, Mr. A.A. Sijuade is also appreciated, as is the secretarial assistance of Mr. Ademola Ibraheem. Grateful acknowledgement is made to the Virology Group, University College Hospital, Ibadan, for donations of the virus. REFERENCES 1 A. Isaacs and J. Lindenmann, Virus interference, I. The interferon, Proc. Roy. Sot. B., 147 (1957) 258. 2 G. Colby and M.J. Chamberlin, The specificity of interferon induction in chick embryo cells by helical RNA, Proc. Natl. Acad. Sci. USA, 63 (1969) 160. 3 A.K. Field, A.A. Tytell, G.P. Lampson and M.R. Hilleman, inducers of interferon and host resistance, II. Multistrand~ synthetic polynucl~tide complexes, Proc. Natl. Acad. Sci. USA, 58 (1967) 1004. 4 P.M. Pitha, L.W. Marshall and W.A. Cater, interferon induction: Rate of cellular attachment to poly(I):C, J. Gen. Virol., 15 (1972) 89. 5 G.P. Lampson, A.A. Tytell, A.K. Field, M.M. Nemes and M.R. Hilleman, Inducers of interferon and host resistance, I. Double-stranded RNA from extracts of Penicifhm funiculosum. Proc. Natl. Acad. Sci. USA, 58 (1967) 782. 6 J.O. Folayan and D.W. Hutchinson, Poly-8-oxy-adenylic acid, Biochim. Biophys. Acta, 474 (1977) 329.
52
7 J.O. Folayan
and D.W. Hutchinson,
Poly-Sfluorocytidylic
acid, Biochim.
Biophys.
Acta, 340 (1974)
194. 8 J.O. Folayan, polyriboinosinic 9 J.O.
Folayan
D.W. Hutchinson acid), Interferon and D.W.
and M.D. Johnston, Antiviral Sci. Memoranda (1973) Memo
Hutchinson,
activity l-906.
Poly-5-dimethylamino-cytidylic
of poly(5-fluorocytidylic
acid.
Tetrahedron
Lett.,
51
(1973) 5077. 10 J.O. Folayan
and E.O. Ngaha,
Hydrolysis
of synthetic
nucleotides
by ribonucleases,
Nig. J. Sci., 12
(1978) 405. 11 M. Absher
and W.R.
Stinebring,
Toxic properties
of a synthetic
double-stranded
RNA, Nature,
223
(1969) 715. 12 H.L.
Lindsay,
Nature, 13 J.O. 14 J.O. 15 J.O.
P.W.
Trown,
J. Brandt
and M. Forbes,
Progenicity
of Poly(l):Poly(C)
in rabbits,
223 (1969) 717.
Folayan,
Poly-8-azidoadenylic
Folayan, Folayan,
Poly-8-dimethylamino adenylic acid, Drugs of the Future, 4 (1979) 824. Poly-5-methylaminocytidylic acid, Drugs of the Future, 6 (1981) 155.
acid,
Drugs of the Future,
4 (1979) 73 1.
8th Report, Jan. 1971-Dec. 1972. 16 University of Ibadan, Virus Research Laboratory, 17 W.A. Carter, P.M. Pitha and L.W. Marshall, Structural Requirements of the r I,: rC, complex induction 18 D.H.
of interferon,
J. Mol. Biol., 70 (1972) 567.
Metz, The mechanism
of action
of interferon,
Cell, 6 (1975) 629.
for
Biomedical
47
Press
POLY(A):POLYW);
POLY(O*A):POLY(U);
POLY(O*A):POLY(MeNH%J) IN CHICK
JOSEPH
POLY(A):POLY(MeNH5U)
AND
VERSUS BIDA SEMLIKI FOREST VIRUS
EMBRYOS
OLOLADE
FOLAYAN
Biochemistry Department, Ife University, Ile-Ife, Oyo State (Nigeria) (Received
August
(Revision
received
(Accepted
2lst,
1981)
October
November
26th,
1981)
18th, 1981)
SUMMARY Poly(A):poly(U) acid:polyuridylic acid hybrid) hybrid) Nigerian
(polyadenylic acid:polyuridylic acid hybrid); poly(OsA):poly(U) (poly-8-oxy-adenylic acid hybrid); poly(A):poly(MeNHW) (polyadenylic acid:poly-5-methylamino uridylic
and poly(OsA):poly(MeNHsU)
were studied strain)
in chick embryos
AN 49809. The modified
than the unmodified
(poly-8-oxyadenylic
to compare polymers
the protection showed
acid:poly-5-methylamino offered
against
a higher activity
uridylic
acid
Semliki forest virus (the
index but were more toxic
analogue.
INTRODUCTION
The induction of cells to produce interferon [I] may be brought about by both synthetic [2-41 and natural polynucleotides [5]. The synthesis of polynucleotides is receiving much attention [6-91; some can be degraded by ribonucleases and many synthetically modified nucleotides are more resistant to ribonuclease hydrolysis than the unmodified analogues [lo]. These molecules must possess stable secondary structure, being double stranded hybrids with high melting temperatures [ 111; but this requirement also causes cytoxicity [ 1l- 121. We have studied the effects of these polynucleotides on local (Nigerian) viruses and investigated the effects of some polymers on Wesselsbron virus (H10964) in albino mice [13-l 51. We are also studying the effects of Bida (Nigerian) strain of Semliki Forest virus (AN49809) in albino mice as well as the effects of Wesselsbron virus in chick embryos. The present study has been undertaken to examine the effect of Bida Semliki forest virus in chick embryos. This virus causes brain damage in mice when injected 0378-4274/82/0000-0000/$02.75
0 Elsevier
Biomedical
Press
48
intracerebrally, and death usually within 24 h of administration [16]. Mice pretreated with synthetic polynucleotides may survive up to certain doses. This virus is abundant in mosquitoes from which it is extracted [ 161. As our birds (and other animals) are bred in atmospheres not completely free of mosquitoes, the possible commercial value of this study is obvious. MATERIALS
AND METHODS
The virus was obtained from the Virology Group of the University College Hospital (Ibadan), where isolation, purification and characterisation were carried out, having been isolated from mosquitoes Aedes (Neo) spp. and Mansonia africanus collected at Bida (in Niger State) [16]. Mice were injected with this virus and died when the concentration reached 1 . lo* units [ 161. The animals were then quickly frozen and kept at - 70 “C until required. After thawing under tap water, the brain was sucked out into a micro-mortar, ground and made up to the required volume with sterile distilled water. Nucleosides, nucleotides and other reagents were purchased from Sigma Chemical Co.; poly(08A) and poly(MeNH5U) were prepared as described [6, 151. The two appropriate polymers were mixed in equimolar solutions to make the required hybrids [6-91. Eggs were obtained from the University of Ife Farm after being fertilized and kept for 10 days. Pretreatment When the embryos were 10 days old, the eggs were candled to locate the airsacs. Injection of either the virus or the polymer was made at a point opposite the air sac care being taken to avoid the blood vessels. The eggs were stored with the point of inoculation uppermost. Solutions of polymers were made in sterile distilled water to which penstrep (penicillin/streptomycin) had been added. The volume injected into each egg was maintained at 0.1 ml and controls were given distilled sterile water only. Challenging The eggs were kept for 24 h after pretreatment to allow induction of interferon and were then given a counter injection of 0.1 ml virus at the desired concentration. The eggs were left for 5 days and then cracked. In water, eggs with dead embryos floated while those containing living embryos, sank. Percentage survival was calculated.
49 RESULTS
The results are given in Tables I-IV.
TABLE I VIRUS DOSE RESPONSE Eggs were grouped and various doses of virus administered. They were cracked at 5 days and the embryos examined. CaIculations were made for percentage survival. Group 1
2 3 4 5 6 7 8 9 10
Virus dose (+10’ units)
Results after 5 days (70 survival)
0.00 0.01
100 f 62 + 46 rt 35 + 25 + 18 t 10 + 7f 5f 32
0.10
0.30 0.50 0.80 1.00 1so 2.00 2.50
2.0 2.0 4.0 5.0 5.0 5.0 6.0 7.0 7.0 7.0
LDso = 0.07.107 Units,
TABLE II POLYMER DOSE RESPONSE Eggs were pooled into 10 groups, given poly(A):poly(U), and were watched for 5 days before being cracked for examination and calculation of percentage survival. This was repeated for the other three analogues. Group
Polymer fig in 0.1 ml
1
0.00
2 3 4 5 6 7 8 9 10
0.01 0.10 1.00 10.00 30.00 50.00 70.00 85.00 100.00
LDso pg/O. 1 ml
% Survival for various polymers after 5 days Poly(A): poly(Uf
Poly(O*A) POlY(U)
Poly(A): poly(MeNHsU)
Poly(O*A): poly(MeNHsU)
lOOz!z 2 994 2 961t 4 921t 5 862 5 79f 7 68r 7 50+ 8 36+ 8 15rh: 10
loo+98+ 96+ 90+ 82+ 72+ 64t 48-+ 30+ 12 *
2 3 5 5 6 6 8 8 8 10
lOOk 96+ 90+ 86+ 77+ 6Oi 55+ 465 27 + 12*
2 4 6 6 7 7 8 8 10 10
100+ 96+ 90* 80& 72+ 58-t 52* 43* 25+ 10 f
2 4 6 6 8 8 8 8 10 12
68*
5
64*
6
6Ok
8
70*
5
50
TABLE III PROTECTION PROFILES Eggs were grouped and pretreated with different doses of polymers and later challenged by varying doses of virus. The following results were obtained from pretreatment with 10.0 pg polymer followed by challenges with 0.01, 0.10, 0.50 or 1.00.107 units of virus, respectively. Polymer (10.0 pg in 0.1 ml)
% Survival at various virus doses .I07 Units 0.10
0.01 Poly(A):poly(U) Poly(0sA):poly(U) Poly(A):poly(MeNHsU) Poly(08A):poly(MeNHsU)
85 t 82 + 76 + 72 +
5 5 7 7
82 + 15 f 70 f 66 +
0.50 6 6 8 8
1.00
70? 68+ 62& 58 &
8 8 8 10
66t 6Ot 57 t 52 +
8 8 10 10
TABLE IV ACTIVITY INDEX Several protection profiles (e.g. in Table III) were carried out with varying polymer concentrations. The optimum protections were found and the highest percentage protection also obtained as summarised below. Activity index was taken either as protection per gg (@/o/as)or as how much material was required to offer 1% protection &g/%). Polymer
Optimum concentration (pg in 0.1 ml)
Maximum protection (S)
Activity Index
18+3 14 + 3 12 zlz2 10 + 2
86 + 4 80 + 4 76 z!z4 70 * 5
209 175 158 143
(gg, %
,lO-3 Poly(A):poly(U) Poly(OsA):poly(U) Poly(A):poly(MeNHsU) Poly(OsA):poly(MeNHW)
. lO-2 477 571 633 700
DISCUSSION
All analogues of poly(A):poly(U) offered protection to chick embryos against Semliki forest virus. The results resemble those for poly(I):poly(C) and some of its analogues against Wesselsbron virus (H19964). The polymer which is not modified (poly(A):poly(U)) was found to be the least toxic. Next was one in which the poly(U) strand is unmodified (i.e. p~ly(O~A):poly(U)). The polymer in which both strands were modified had the highest toxicity followed by that in which the poly(A) strand alone was modified. The poly(U) strand may be the more critical of the two. Activity was found to be in the reverse order to toxicity. The most toxic is also the most active and the sequence follows that order (Table IV). It would appear that activity and toxicity are directly related as has been observed in other systems [ 11, 171.
51
Toxicity was estimated by taking the reciprocal of the LDso, since toxicity and LDso values are inversely related. Similarly, the activity index was taken either as the amount of polymer that would offer 1% protection under optimum concentrations or the amount of protection offered by 1 pg material at optimum concentrations. In the series of synthetic polymers studied, there was little difference between the LDso values, indicating that modification has not drastically increased toxicity. However, as reported [lo], the modifications have led to increased stability of secondary structure, resistance to ribonucleases and increased M.P. values. These new analogues would therefore appear to have potential for interferon induction in chick embryos. CONCLUSION
These polymers in chick embryo cells induce the production of interferon which is active against Semliki forest virus. This bears some similarity to the activity of poly(I):poly(C) analogues in chick embryos against Wesselsbron virus. The modified polymers have some advantage over the unmodified analogue as they are more stable.
The author acknowledges the University of Ife, Ile-Ife, Oyo State of Nigeria for the award of the research grant with which this laboratory was financed. The assistance of other colleagues, Dr. E.O. Ngaha, Mr. Doyin Okusanya, Mr. A.A. Sijuade is also appreciated, as is the secretarial assistance of Mr. Ademola Ibraheem. Grateful acknowledgement is made to the Virology Group, University College Hospital, Ibadan, for donations of the virus. REFERENCES 1 A. Isaacs and J. Lindenmann, Virus interference, I. The interferon, Proc. Roy. Sot. B., 147 (1957) 258. 2 G. Colby and M.J. Chamberlin, The specificity of interferon induction in chick embryo cells by helical RNA, Proc. Natl. Acad. Sci. USA, 63 (1969) 160. 3 A.K. Field, A.A. Tytell, G.P. Lampson and M.R. Hilleman, inducers of interferon and host resistance, II. Multistrand~ synthetic polynucl~tide complexes, Proc. Natl. Acad. Sci. USA, 58 (1967) 1004. 4 P.M. Pitha, L.W. Marshall and W.A. Cater, interferon induction: Rate of cellular attachment to poly(I):C, J. Gen. Virol., 15 (1972) 89. 5 G.P. Lampson, A.A. Tytell, A.K. Field, M.M. Nemes and M.R. Hilleman, Inducers of interferon and host resistance, I. Double-stranded RNA from extracts of Penicifhm funiculosum. Proc. Natl. Acad. Sci. USA, 58 (1967) 782. 6 J.O. Folayan and D.W. Hutchinson, Poly-8-oxy-adenylic acid, Biochim. Biophys. Acta, 474 (1977) 329.
52
7 J.O. Folayan
and D.W. Hutchinson,
Poly-Sfluorocytidylic
acid, Biochim.
Biophys.
Acta, 340 (1974)
194. 8 J.O. Folayan, polyriboinosinic 9 J.O.
Folayan
D.W. Hutchinson acid), Interferon and D.W.
and M.D. Johnston, Antiviral Sci. Memoranda (1973) Memo
Hutchinson,
activity l-906.
Poly-5-dimethylamino-cytidylic
of poly(5-fluorocytidylic
acid.
Tetrahedron
Lett.,
51
(1973) 5077. 10 J.O. Folayan
and E.O. Ngaha,
Hydrolysis
of synthetic
nucleotides
by ribonucleases,
Nig. J. Sci., 12
(1978) 405. 11 M. Absher
and W.R.
Stinebring,
Toxic properties
of a synthetic
double-stranded
RNA, Nature,
223
(1969) 715. 12 H.L.
Lindsay,
Nature, 13 J.O. 14 J.O. 15 J.O.
P.W.
Trown,
J. Brandt
and M. Forbes,
Progenicity
of Poly(l):Poly(C)
in rabbits,
223 (1969) 717.
Folayan,
Poly-8-azidoadenylic
Folayan, Folayan,
Poly-8-dimethylamino adenylic acid, Drugs of the Future, 4 (1979) 824. Poly-5-methylaminocytidylic acid, Drugs of the Future, 6 (1981) 155.
acid,
Drugs of the Future,
4 (1979) 73 1.
8th Report, Jan. 1971-Dec. 1972. 16 University of Ibadan, Virus Research Laboratory, 17 W.A. Carter, P.M. Pitha and L.W. Marshall, Structural Requirements of the r I,: rC, complex induction 18 D.H.
of interferon,
J. Mol. Biol., 70 (1972) 567.
Metz, The mechanism
of action
of interferon,
Cell, 6 (1975) 629.
for