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The In Vitro and In Vivo Effect of Chemotherapeutic Agents on the Marek’s Disease Herpesvirus of Chickensa,b
The In Vitro and In Vivo Effect of Chemotherapeutic Agents on the Marek's Disease Herpesvirus of Chickensab C . S. EIDSON, V . T . THAN AND S. H . K L E V E N
Poultry Disease Research Center, College of Veterinary Medicine, University of Georgia, Georgia 30601
Athens,
(Received for publication November 23, 1973)
POULTRY SCIENCE 53: 1533-1538, 1974
INTRODUCTION
M
AREK'S disease (MD) is a lymphoproliferative disease which occurs most frequently in young chickens. In vitro studies of infected materials in cell culture led to the discovery that the etiologic agent was a cell-associated group B herpesvirus (Churchill and Biggs, 1967; Nazerian et al., 1968; and Solomon et al., 1968). A highly pathogenic strain of this virus was attenuated by passage in cell culture, and when administered to chicks, it protected them against the development of MD (Churchill et al., 1969; Biggs et al., 1970; Eidson et al., 1971; and Eidson and Anderson, 1971). A herpesvirus of turkeys (HVT) has been shown to be antigenically related to the Marek's disease virus (MDV), and provides protection against a. University of Georgia, College of Veterinary Medicine and Institute of Comparative Medicine, Journal Series number 1114, Athens Georgia. b. Presented at the 73rd Annual Meeting of the American Society for Microbiology, Miami, Florida.
this disease when given parenterally to young chicks (Witter et al., 1970; and Okazaki et al., 1970). It has subsequently been used in the field as a heterotypic vaccine against MD (Purchase et al., 1970; and Eidson et al, 1971). Much effort has been expended to discover drugs for control of viral diseases. Despite such efforts, few antiviral drugs exist today which have clinical value, and these are active only against a relatively small spectrum of DNA or RNA viruses. Recent data indicates that l-p-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole), a highly soluble synthetic nucleoside, has activity against a broad spectrum of DNA and RNA viruses in vitro (Huffman et al., 1972; Witkowski et al., 1972; and Sidwell et al., 1973). The drug inhibited viral cytopathogenic effects in monolayers of cells infected with type 3 adenovirus, types 1 and 2 herpesvirus, myxoma virus, cytomegalovirus, vaccinia virus, infectious bovine rhinotracheitis virus, types 1A, 2, 8, 13, and 56 rhinoviruses, types 1 and 3 parainfluenza
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ABSTRACT Virazole (l-p-D-ribofuranosyl-l,2,4-triazole-3-carboxamide) has activity against a number of DNA and RNA viruses and AUS (P-amino-P'-ureidodiphyenyl sulfone) has been used for the prophylaxis of Marek's disease (MD). This report presents in vitro and in vivo data on the antiviral activity of virazole and AUS against the Marek's disease herpesvirus (MDV). In laboratory studies using 18-hour primary chick embryo fibroblasts, concentrations of Virazole and AUS ranging from 10 to 100 jig. /ml. were added to monolayers infected with the MDV. Concentrations of virazole as low as 10 |i.g./ml. had in vitro activity against the MDV; whereas, concentrations of AUS as high as 100 fig./ml. had no activity. White Leghorn chicks inoculated with MDV at 2 weeks of age were treated subcutaneously with 500 or 1000 pg. of virazole twice daily for 14 days beginning 24 hour post-virus exposure. The 500 ng., 1000 |xg. and untreated groups had 14%, 10% and 40% MD lesions respectively at 8 weeks of age. Virazole (0.002% in the feed) was ineffective in preventing the development of MD tumors; whereas, AUS (0.002% in the feed) was very effective in the reduction of MD lesions. Although this study indicates that virazole and AUS have activity against the MDV, neither appear to be as effective in preventing the development of MD tumors as the turkey herpesvirus vaccine.
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C. S. EIDSON, V. T. THAN AND S. H. KLEVEN
MATERIALS AND METHODS Origin of Herpesvirus. The Marek's disease herpesvirus designated as the GA isolate of MD was isolated by Eidson and Schmittle (1968). The virus preparation was a stock of cell-associated virus of the 10th passage in specific pathogen free (SPF) chick embryo fibroblasts with a titer of 9.5 x 105 PFU per ml. The turkey herpesvirus used throughout this study was a vaccine strain of HVT designated as FC126 (Witter et al, 1970), which was passed 11 times in duck embryo fibroblasts at the East Lansing Laboratory. The virus was supplied by Dr. William Okazaki, East Lansing, Michigan. The HVT vaccine was a stock of cell-associated virus of the 10th consecutive passage (after arrival at this laboratory) in SPF chick embryo fibroblasts and had a titer of 3150 plaque forming units (PFU) per bird dose (1000 bird doses/vial).
In Vitro Antiviral Activity of Virazole and AUS. For the assay of the MDV an ampule was thawed by immersion in cold tap water immediately after removal from liquid nitrogen storage. Serial tenfold dilutions of the virus were prepared in culture medium 199 containing 2% calf serum and 0.11% sodium bicarbonate. One ml. of each virus dilution (10"~' to 10"6) was distributed over the surface of three confluent chick embryo fibroblast monolayers from which the growth medium had been removed. An absorption period of 30 min. was allowed at 38° C. in an atmosphere of 5% C 0 2 . The results of the assay of MDV were read after 5 days incubation when discreet primary microplaques consisting of foci of cells exhibiting cytopathic effect were visible on microscopic examination. Counts were made and the mean value was taken from the 3 plates inoculated with the highest dilution demonstrating microplaques for the purpose of calculating the titer of MDV. To determine the in vitro effectiveness of Virazole and AUS against MDV, 0 to 1000 |xg./ml. of the compounds were added to cell culture medium 199. Three monolayers per virus dilution (10"' to 10"6) were pretreated for 24 hours with 100 jj,g./ml. of each compound. The compounds were removed and the virus dilutions added. Four ml. of cell culture medium 199 was added to each plate after the 30 min. adsorption period, and plaques were counted on the 5th day after the addition of the virus. Another 3 monolayers per virus dilution (10 _1 to 10"6) were treated with 10, 50 and 100 u-g./ml. of Virazole or AUS immediately after the 30 min. adsorption period. After a 24 hour incubation period the spent medium was removed and cell culture medium void of chemotherapeutic agents was added to each of the petri dishes. Another group of monolayers (3 per virus dilution) was treated with 100 jjLg./ml. of Virazole or AUS 24 hours after the addition of the virus dilutions. All
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virus, Newcastle disease virus and measles virus (Sidwell et al., 1972). A second compound, P-amino-P'ureidodiphenyl sulfone (AUS), has been shown to be effective against MD by suppressing visceral lesions and mortality of infected chickens at a concentration of 0.002% in the diet (Shen et al, 1971). The compounds of this group were reported to have low toxicity. They are nonviricidal, and they have only weak bacteriostatic and antiinflammatory activity. It has been suggested that the anti-MD activity has no relationship with antibiotic activity. The mechanism of action of these compounds is not understood, but it has been postulated that they may affect the membrane of virus-infected cells or have an effect on the immunological processes. This report describes the in vitro and in vivo effect of Virazole and AUS on the Marek's disease herpesvirus (MDV) and on the lesions and mortality induced by the virus.
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CHEMOTHERAPEUTIC TREATMENT OF MAKER'S DISEASE
of the in vitro tests to determine the effect Virazole and AUS have on MDV were repeated 4 times.
Experiment 2. The first 2 groups of birds, each consisting of 30 birds, were infected at 3 weeks of age subcutaneously with 0.2 ml. of MD-infective plasma and beginning one-day postinfection the birds were injected subcutaneously with either 500 or 1000 mg. of Virazole per bird twice daily for 2 weeks. The second two groups of 30 birds each were treated for the 8 week period with Virazole or AUS at a concentration of 1 gram per 100 lb. of feed. The birds in both groups were infected at 3 weeks of age with MD-infective plasma. The last two groups of birds of 30 birds each served as controls. The first groups served as vaccinal controls, with each bird receiving 1000 PFU of the HVT vaccine while
TABLE 1.—In Vitro antiviral activity of Virazole and AUS on MDV in chick embryo fribroblast monolayers 5 days 1 Drug concen. (M-g./ml.) 0 10 50 100 100 100 1
Visible cytotoxicity
Time cpd. added
— — — — — —
Control min. after virus 3 min. after virus 3 min. after virus 3 hr. before virus 4 hr. after virus 5
30 30 30 24 24
Virus titer (PFU) after mixing with: Virazole 2 9.0 x 105 7.0 x 105 5.1 x 105 8.0 x 104 8.5 x 105 9.5 x 105
AUS 2 9.5 9.2 8.9 9.5 9.3 9.1
x x x x x x
105 10= 105 105 105 105
Plaque forming units (PFU) were counted after 5 days incubation. Three monolayers per virus dilution (10_I to 10~6) were treated with either 0, 10, 50 or 100 p-g./ml. of Virazole on AUS. 'Virazole or AUS was incorporated in maintenance media and was left in contact with monolayers for 24 hr. 4 Virazole or AUS was in contact with monolayers for 24 hr. but was removed before addition of virus. 5 Virazole or AUS was added 24 hr. after virus and remained in contact with the monolayers for 24 hr. 2
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In Vivo Effectiveness of Virazole and AUS. Experiment 1. The first 4 groups of 10 chickens each were infected subcutaneously at 3 weeks of age with 0.2 ml. of MD-infective plasma. Beginning one-day post-infection 2 groups of birds were injected subcutaneously twice daily for 2 weeks with 25 mg. of Virazole, while the other 2 groups of birds received 50 mg. per bird twice daily for the 2 week period. The next 4 groups of 10 chickens each were medicated from one day of age until the termination of the trial at 8 weeks with either Virazole or AUS (2 groups each) in the feed at a concentration of 1 gram of the compound per 100 lb. of feed. At 3 weeks of age the 4 groups of birds were infected subcutaneously with 0.2 ml. of MD infective plasma. The last 4 groups served as controls to the groups treated with either Virazole or AUS. Two of the groups were vaccinated
at one day of age with 1000 PFU of the HVT vaccine and were infected at 3 weeks of age with MD-infective plasma. These birds served as vaccinal controls; whereas, the other two groups served as untreated controls and were infected with MD at 3 weeks of age.
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C. S. EIDSON, V. T. THAN AND S. H. KLEVEN
the second group was not vaccinated or treated. Both groups were infected at 3 weeks of age with MD-infective plasma. All of the birds in experiments 1 and 2 were examined for the presence of MDV at the end of the 8-week-experimental period by culturing their kidneys as cell culture monolayers were examined for viral cytopathic effect. RESULTS
TABLE 2.—Effect
In Vivo Effectiveness of Virazole and AUS. In experiment 1 birds inoculated with 25 or 50 mg. of Virazole had fewer gross MD lesions when compared to the untreated controls. In the 2 groups of birds treated with 25 mg. of Virazole, only 1 of 20 birds had gross MD lesions. Only 1 of 18 birds had gross lesions in the 2 groups treated with 50 mg. The 2 groups of untreated controls were found to have gross lesion in 8 or 20 birds challenged with the virus. Virazole, when administered in the feed at the 0.002 percent level, was completely ineffective in reducing MD lesions. However, AUS administered in the feed at the 0.002 percent level was effective in reducing MD lesions with only 3 of 20 birds exhibiting gross lesions. None of the birds in the 2 groups vaccinated with the HVT vaccine developed MD lesions during the 8-week experimental period (Table 2). The results of experiment 2 (Table 3) were similar to those of experiment 1; however, increasing the dosage of Virazole to 500 or
of Virazole and AUS on the development of MD in chickens 8 weeks
Cpd. Virazole Virazole Virazole Virazole Virazole Virazole AUS AUS Control (HVT) 1 Control (HVT)' Control 2 Control 2
Method of medication Inj.« Inj. 2 3 Inj. 2 - 3 Inj.23 Feed 1 Feed 1 Feed 1 Feed 1 Control Control Control Control
Concn. of cpd. (mg.) 25 25 50 50 10004 10004 10004 10004 None None Control Control
MD +/no. started 1/10 0/10 0/8 1/10 5/10 6/10 1/10 2/10 0/10 0/10 5/10 4/10
% Pos. (10.0%) (0.0%) (0.0%) (10.0%) (50.0%) (60.0%) (10.0%) (20.0%) (0.0%) (0.0%) (50.0%) (40.0%)
•Birds not treated with virazole or AUS but were vaccinated at one day of age with 1000 PFU of 2the HVT vaccine. 3-Week-old chicks injected with MD-infected plasma. 3 Injected twice daily for 2 weeks with Virazole. 4 1 Gram per 100 lb. of feed for 8 weeks.
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In Vitro Antiviral Activity of Virazole and AUS. The results of an experiment to determine the viricidal effect of Virazole and AUS on MDV in cell culture indicated that 100 |xg./ml. Virazole added 30 min. after MDV was adsorbed to the chick embryo fibroblasts monolayers reduced the viral titer by approximately 90 percent (Table 1). Virazole at 10 or 50 jtg./ml. reduced the viral titer by approximately 22.2 percent and 43.2 percent, respectively (Table 1). Pretreatment of monolayers with 100 (ig./ml. of Virazole for 24 hr. before the addition of the virus did not reduce the viral titer, nor did 100 (jLg./ml. of Virazole when added 24 hours after the adsorption of the virus. Treat-
ment with AUS did not reduce viral titers when used at 100 fig./ml.
CHEMOTHERAPEUTIC TREATMENT OF MAREK'S DISEASE
TABLE 3.—Effect
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of Virazole and AUS on the development of MD in chickens
1000 mg./bird did not reduce the incidence of gross MD lesions below those which received 25 or 50 mg./bird in experiment 1. Virazole, when administered at the 0.002% level in the feed, was ineffective in reducing MD lesions; whereas, the AUS group had only 2 of 30 birds with gross lesions. There were no birds in the groups vaccinated with the HVT vaccine with gross lesions, and 12 of 30 of the untreated controls had MD lesions. The MDV was recovered from the kidneys of all birds in each of the 2 experiments regardless of the treatment or the presence of lesions. DISCUSSION The data presented indicate that the synthetic triazole nucleoside Virazole has significant antiviral activity against MDV in vitro as well as in the reduction of tumor development in chickens. Although it is quite significant that Virazole has the ability to protect chickens against MD, it is highly unlikely that Virazole would be used as a prophylactic agent against MD, because multiple injections are necessary to elicit the desired results. This would not be economically feasible for the poultry industry at the present time. Also, it was found that Virazole was ineffective against MD when added to the ration. Unlike Virazole, AUS added to the feed at a con-
centration of 0.002 percent protects chickens against the development of MD lesions. This means of protecting the chickens against MD would have an advantage over the present method of vaccinating the chicks with the HVT vaccine because the vaccination procedure requires that each bird be injected. Administration of AUS in the feed would eliminate the cost of injecting each bird. Although the poultry industry would find it highly desirable to eliminate the vaccination of each bird, it must be pointed out that the results of this study indicate that AUS was not as effective in preventing MD lesions as the HVT vaccine. Also of interest is the fact that neither the mechanism of protection of the HVT vaccine against MDV nor the mechanism of protection of AUS is not fully understood. Although both AUS and the HVT vaccine prevent tumor development, neither prevents the infection of the bird with MDV, since the virus was detected in all birds at the termination of the trial at 8 weeks. While neither Virazole nor AUS have been approved for use as a means to protect chickens against MD, the most important point for the poultry industry would be the cost of these compounds. Although, the authors do not have information as to the cost of treatment per bird medicated it is likely that the cost would be prohibitive since the cost of the vaccine (1000 PFU) for a broiler
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8 weeks Method of Concn. of MD+/no. Cpd. medication cpd. (mg.) started % Pos. Virazole Inj.2-3 500 4728 (14.3%) Virazole Inj.2-3 1000 3/30 (10.0%) Virazole Feed2 10004 9/30 (30.0%) AUS Feed2 10004 2/30 (6.7%) Control (HVT)1 Control None 0/30 (0.0%) Control2 Control None 12/30 (40.0%) 'Birds not treated with Virazole or AUS but were vaccinated at one day of age with 1000 PFU of 2the HVT vaccine. 3-Week-old chicks injected with MD-infected plasma. 3 Injected twice daily for 2 weeks with Virazole. 4 1 gram per 100 lb. of feed for 8 weeks.
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C. S. EIDSON, V. T. THAN AND S. H. KLEVEN
chick is approximately $1.60 to $2.50 per thousand chicks. Although, the data presented in this study indicate that AUS is not as effective as the HVT vaccine, it is nevertheless encouraging that a chemotherapeutic agent may soon be available that would be at least as effective as the HVT vaccine, thereby eliminating the necessity of treating each bird individually.
ACKNOWLEDGMENTS
ICN Nucleic Acid Research Institute, Irvine, California, for supplying the Virazole and to Mr. Stiles Lovern and to Mr. Lane Teet for technical assistance. REFERENCES Biggs, P. M., L. N. Payne, B. S. Miine, A. E. Churchill, R. C. Chubb, D. G. Powell and A. H. Harris, 1970. Field trials with an attenuated cell associated vaccine for Marek's disease. Vet. Rec. 87: 704-709. Churchill, A. E., and P. M. Biggs, 1967. Agent of Marek's disease in tissue culture. Nature, 215: 528-530. Churchill, A. E., R. C. Chubb and W. Baxendale, 1969. The attenuation, with loss of oncogenicity, of the herpestype virus of Marek's disease (strain HPRS-16) on passage in cell culture. J. Gen Virol. 4: 557-564. Eidson, C.S., and D.P.Anderson, 1971. Immunization against Marek's disease. Avian Dis. 15: 68-81. Eidson, C. S., D. P. Anderson, S. H. Kleven and J. Brown, 1971. Field trials of vaccines for Marek's disease. Avian Dis. 15: 312-322. Eidson, C. S., and S. C. Schmittle, 1968. Studies on acute Marek's disease. I. Characteristics of Isolate GA in chickens. Avian Dis. 12: 467-476. Eidson, C. S., and S. C. Schmittle, 1969. Studies on acute Marek's disease. XII. Detecting antibodies with a tannic acid indirect hemagglutinating test. Avian Dis. 13: 774-782. Huffman, J. H., R. W. Sidwell, G. P. Khane, J. T. Witkowski, L. B. Allen and R. K. Robins, 1973.
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The authors would like to express their sincere appreciation to Dr. R. W. Sidwell,
In vitro effect of l-p-D-ribofuranosyl-l,2,4-triazole3-carboxamide (virazole, ICN 1229) on deoxyribonucleic acid and ribonucleic acid viruses. Antimicrob. Ag. Chemother. 3: 235-241. Nazerian, K., J. J. Solomon, R. L. Witter and B. R. Burmester, 1968. Studies on the etiology of Marek's disease. II. Finding of a herpesvirus in cell culture. Proc. Soc. Exptl. Biol. Med., 127: 177-182. Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14: 413-429. Purchase, H. G., W. Okazaki and B. R. Burmester, 1970. Field trials with the herpes virus of turkeys (HVT) strain FC126 as a vaccine against Marek's disease. Poultry Sci. 50: 775-783. Shen, T. Y., D. B. R. Johnston, N. P. Jensen, W. V. Ruyle, J. J. Friedman, M. W. Fordice, J. F. McPherson, K. H. Boswell, T. A. Maag, R. W. Burg, R. M. Pellegrino, M. E. Jewell, C. A. Morris, H. L. Easterbrooks and B. J. Skelly, 1971. New chemoprophylactic agents for Marek's disease. Amer. Chem. Soc. Abstract NO. 44 in Section on Med. Chem. (No page no.) Sidwell, R. W., J. H. Huffman, G. P. Khare, L. B. Allen, J. T. Witkowski and R. K. Robins, 1972. Broad-spectrum antiviral activity of virazole: l-(3-Dribofuranosyl - 1, 2, 4 - triazole - 3 - carboxamide. Science, 177: 705-706. Sidwell, R. W., L. B. Allen, G. P. Khare, J. H. Huffman, J. T. Witkowski, L. N. Simon and R. K. Robins, 1973. Effect of 1-0-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole, ICN 1229) on herpes and vaccinia keratitis and encephatilis in laboratory animals. Antimcrob. Ag. Chemother. 3: 242-246. Sidwell, R. W., and L. N. Simon, 1972. Design synthesis, and broad spectrum antiviral activity of 1-0-D-ribofuranosyl- 1,2, 4-triazole-3-carboxamide and related nucleosides. J. Me. Chem. 15: 11501153. Solomon, J. J., R. L. Witter, K. Nazerian and B. R. Burmester, 1968. Studies on the etiology of Marek's disease. I. Propagation of the agent in cell culture. Proc. Soc. Exptl. Biol. Med. 127: 173-177. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 31: 525-538.
Poultry Disease Research Center, College of Veterinary Medicine, University of Georgia, Georgia 30601
Athens,
(Received for publication November 23, 1973)
POULTRY SCIENCE 53: 1533-1538, 1974
INTRODUCTION
M
AREK'S disease (MD) is a lymphoproliferative disease which occurs most frequently in young chickens. In vitro studies of infected materials in cell culture led to the discovery that the etiologic agent was a cell-associated group B herpesvirus (Churchill and Biggs, 1967; Nazerian et al., 1968; and Solomon et al., 1968). A highly pathogenic strain of this virus was attenuated by passage in cell culture, and when administered to chicks, it protected them against the development of MD (Churchill et al., 1969; Biggs et al., 1970; Eidson et al., 1971; and Eidson and Anderson, 1971). A herpesvirus of turkeys (HVT) has been shown to be antigenically related to the Marek's disease virus (MDV), and provides protection against a. University of Georgia, College of Veterinary Medicine and Institute of Comparative Medicine, Journal Series number 1114, Athens Georgia. b. Presented at the 73rd Annual Meeting of the American Society for Microbiology, Miami, Florida.
this disease when given parenterally to young chicks (Witter et al., 1970; and Okazaki et al., 1970). It has subsequently been used in the field as a heterotypic vaccine against MD (Purchase et al., 1970; and Eidson et al, 1971). Much effort has been expended to discover drugs for control of viral diseases. Despite such efforts, few antiviral drugs exist today which have clinical value, and these are active only against a relatively small spectrum of DNA or RNA viruses. Recent data indicates that l-p-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole), a highly soluble synthetic nucleoside, has activity against a broad spectrum of DNA and RNA viruses in vitro (Huffman et al., 1972; Witkowski et al., 1972; and Sidwell et al., 1973). The drug inhibited viral cytopathogenic effects in monolayers of cells infected with type 3 adenovirus, types 1 and 2 herpesvirus, myxoma virus, cytomegalovirus, vaccinia virus, infectious bovine rhinotracheitis virus, types 1A, 2, 8, 13, and 56 rhinoviruses, types 1 and 3 parainfluenza
1533
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ABSTRACT Virazole (l-p-D-ribofuranosyl-l,2,4-triazole-3-carboxamide) has activity against a number of DNA and RNA viruses and AUS (P-amino-P'-ureidodiphyenyl sulfone) has been used for the prophylaxis of Marek's disease (MD). This report presents in vitro and in vivo data on the antiviral activity of virazole and AUS against the Marek's disease herpesvirus (MDV). In laboratory studies using 18-hour primary chick embryo fibroblasts, concentrations of Virazole and AUS ranging from 10 to 100 jig. /ml. were added to monolayers infected with the MDV. Concentrations of virazole as low as 10 |i.g./ml. had in vitro activity against the MDV; whereas, concentrations of AUS as high as 100 fig./ml. had no activity. White Leghorn chicks inoculated with MDV at 2 weeks of age were treated subcutaneously with 500 or 1000 pg. of virazole twice daily for 14 days beginning 24 hour post-virus exposure. The 500 ng., 1000 |xg. and untreated groups had 14%, 10% and 40% MD lesions respectively at 8 weeks of age. Virazole (0.002% in the feed) was ineffective in preventing the development of MD tumors; whereas, AUS (0.002% in the feed) was very effective in the reduction of MD lesions. Although this study indicates that virazole and AUS have activity against the MDV, neither appear to be as effective in preventing the development of MD tumors as the turkey herpesvirus vaccine.
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C. S. EIDSON, V. T. THAN AND S. H. KLEVEN
MATERIALS AND METHODS Origin of Herpesvirus. The Marek's disease herpesvirus designated as the GA isolate of MD was isolated by Eidson and Schmittle (1968). The virus preparation was a stock of cell-associated virus of the 10th passage in specific pathogen free (SPF) chick embryo fibroblasts with a titer of 9.5 x 105 PFU per ml. The turkey herpesvirus used throughout this study was a vaccine strain of HVT designated as FC126 (Witter et al, 1970), which was passed 11 times in duck embryo fibroblasts at the East Lansing Laboratory. The virus was supplied by Dr. William Okazaki, East Lansing, Michigan. The HVT vaccine was a stock of cell-associated virus of the 10th consecutive passage (after arrival at this laboratory) in SPF chick embryo fibroblasts and had a titer of 3150 plaque forming units (PFU) per bird dose (1000 bird doses/vial).
In Vitro Antiviral Activity of Virazole and AUS. For the assay of the MDV an ampule was thawed by immersion in cold tap water immediately after removal from liquid nitrogen storage. Serial tenfold dilutions of the virus were prepared in culture medium 199 containing 2% calf serum and 0.11% sodium bicarbonate. One ml. of each virus dilution (10"~' to 10"6) was distributed over the surface of three confluent chick embryo fibroblast monolayers from which the growth medium had been removed. An absorption period of 30 min. was allowed at 38° C. in an atmosphere of 5% C 0 2 . The results of the assay of MDV were read after 5 days incubation when discreet primary microplaques consisting of foci of cells exhibiting cytopathic effect were visible on microscopic examination. Counts were made and the mean value was taken from the 3 plates inoculated with the highest dilution demonstrating microplaques for the purpose of calculating the titer of MDV. To determine the in vitro effectiveness of Virazole and AUS against MDV, 0 to 1000 |xg./ml. of the compounds were added to cell culture medium 199. Three monolayers per virus dilution (10"' to 10"6) were pretreated for 24 hours with 100 jj,g./ml. of each compound. The compounds were removed and the virus dilutions added. Four ml. of cell culture medium 199 was added to each plate after the 30 min. adsorption period, and plaques were counted on the 5th day after the addition of the virus. Another 3 monolayers per virus dilution (10 _1 to 10"6) were treated with 10, 50 and 100 u-g./ml. of Virazole or AUS immediately after the 30 min. adsorption period. After a 24 hour incubation period the spent medium was removed and cell culture medium void of chemotherapeutic agents was added to each of the petri dishes. Another group of monolayers (3 per virus dilution) was treated with 100 jjLg./ml. of Virazole or AUS 24 hours after the addition of the virus dilutions. All
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virus, Newcastle disease virus and measles virus (Sidwell et al., 1972). A second compound, P-amino-P'ureidodiphenyl sulfone (AUS), has been shown to be effective against MD by suppressing visceral lesions and mortality of infected chickens at a concentration of 0.002% in the diet (Shen et al, 1971). The compounds of this group were reported to have low toxicity. They are nonviricidal, and they have only weak bacteriostatic and antiinflammatory activity. It has been suggested that the anti-MD activity has no relationship with antibiotic activity. The mechanism of action of these compounds is not understood, but it has been postulated that they may affect the membrane of virus-infected cells or have an effect on the immunological processes. This report describes the in vitro and in vivo effect of Virazole and AUS on the Marek's disease herpesvirus (MDV) and on the lesions and mortality induced by the virus.
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CHEMOTHERAPEUTIC TREATMENT OF MAKER'S DISEASE
of the in vitro tests to determine the effect Virazole and AUS have on MDV were repeated 4 times.
Experiment 2. The first 2 groups of birds, each consisting of 30 birds, were infected at 3 weeks of age subcutaneously with 0.2 ml. of MD-infective plasma and beginning one-day postinfection the birds were injected subcutaneously with either 500 or 1000 mg. of Virazole per bird twice daily for 2 weeks. The second two groups of 30 birds each were treated for the 8 week period with Virazole or AUS at a concentration of 1 gram per 100 lb. of feed. The birds in both groups were infected at 3 weeks of age with MD-infective plasma. The last two groups of birds of 30 birds each served as controls. The first groups served as vaccinal controls, with each bird receiving 1000 PFU of the HVT vaccine while
TABLE 1.—In Vitro antiviral activity of Virazole and AUS on MDV in chick embryo fribroblast monolayers 5 days 1 Drug concen. (M-g./ml.) 0 10 50 100 100 100 1
Visible cytotoxicity
Time cpd. added
— — — — — —
Control min. after virus 3 min. after virus 3 min. after virus 3 hr. before virus 4 hr. after virus 5
30 30 30 24 24
Virus titer (PFU) after mixing with: Virazole 2 9.0 x 105 7.0 x 105 5.1 x 105 8.0 x 104 8.5 x 105 9.5 x 105
AUS 2 9.5 9.2 8.9 9.5 9.3 9.1
x x x x x x
105 10= 105 105 105 105
Plaque forming units (PFU) were counted after 5 days incubation. Three monolayers per virus dilution (10_I to 10~6) were treated with either 0, 10, 50 or 100 p-g./ml. of Virazole on AUS. 'Virazole or AUS was incorporated in maintenance media and was left in contact with monolayers for 24 hr. 4 Virazole or AUS was in contact with monolayers for 24 hr. but was removed before addition of virus. 5 Virazole or AUS was added 24 hr. after virus and remained in contact with the monolayers for 24 hr. 2
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In Vivo Effectiveness of Virazole and AUS. Experiment 1. The first 4 groups of 10 chickens each were infected subcutaneously at 3 weeks of age with 0.2 ml. of MD-infective plasma. Beginning one-day post-infection 2 groups of birds were injected subcutaneously twice daily for 2 weeks with 25 mg. of Virazole, while the other 2 groups of birds received 50 mg. per bird twice daily for the 2 week period. The next 4 groups of 10 chickens each were medicated from one day of age until the termination of the trial at 8 weeks with either Virazole or AUS (2 groups each) in the feed at a concentration of 1 gram of the compound per 100 lb. of feed. At 3 weeks of age the 4 groups of birds were infected subcutaneously with 0.2 ml. of MD infective plasma. The last 4 groups served as controls to the groups treated with either Virazole or AUS. Two of the groups were vaccinated
at one day of age with 1000 PFU of the HVT vaccine and were infected at 3 weeks of age with MD-infective plasma. These birds served as vaccinal controls; whereas, the other two groups served as untreated controls and were infected with MD at 3 weeks of age.
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C. S. EIDSON, V. T. THAN AND S. H. KLEVEN
the second group was not vaccinated or treated. Both groups were infected at 3 weeks of age with MD-infective plasma. All of the birds in experiments 1 and 2 were examined for the presence of MDV at the end of the 8-week-experimental period by culturing their kidneys as cell culture monolayers were examined for viral cytopathic effect. RESULTS
TABLE 2.—Effect
In Vivo Effectiveness of Virazole and AUS. In experiment 1 birds inoculated with 25 or 50 mg. of Virazole had fewer gross MD lesions when compared to the untreated controls. In the 2 groups of birds treated with 25 mg. of Virazole, only 1 of 20 birds had gross MD lesions. Only 1 of 18 birds had gross lesions in the 2 groups treated with 50 mg. The 2 groups of untreated controls were found to have gross lesion in 8 or 20 birds challenged with the virus. Virazole, when administered in the feed at the 0.002 percent level, was completely ineffective in reducing MD lesions. However, AUS administered in the feed at the 0.002 percent level was effective in reducing MD lesions with only 3 of 20 birds exhibiting gross lesions. None of the birds in the 2 groups vaccinated with the HVT vaccine developed MD lesions during the 8-week experimental period (Table 2). The results of experiment 2 (Table 3) were similar to those of experiment 1; however, increasing the dosage of Virazole to 500 or
of Virazole and AUS on the development of MD in chickens 8 weeks
Cpd. Virazole Virazole Virazole Virazole Virazole Virazole AUS AUS Control (HVT) 1 Control (HVT)' Control 2 Control 2
Method of medication Inj.« Inj. 2 3 Inj. 2 - 3 Inj.23 Feed 1 Feed 1 Feed 1 Feed 1 Control Control Control Control
Concn. of cpd. (mg.) 25 25 50 50 10004 10004 10004 10004 None None Control Control
MD +/no. started 1/10 0/10 0/8 1/10 5/10 6/10 1/10 2/10 0/10 0/10 5/10 4/10
% Pos. (10.0%) (0.0%) (0.0%) (10.0%) (50.0%) (60.0%) (10.0%) (20.0%) (0.0%) (0.0%) (50.0%) (40.0%)
•Birds not treated with virazole or AUS but were vaccinated at one day of age with 1000 PFU of 2the HVT vaccine. 3-Week-old chicks injected with MD-infected plasma. 3 Injected twice daily for 2 weeks with Virazole. 4 1 Gram per 100 lb. of feed for 8 weeks.
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In Vitro Antiviral Activity of Virazole and AUS. The results of an experiment to determine the viricidal effect of Virazole and AUS on MDV in cell culture indicated that 100 |xg./ml. Virazole added 30 min. after MDV was adsorbed to the chick embryo fibroblasts monolayers reduced the viral titer by approximately 90 percent (Table 1). Virazole at 10 or 50 jtg./ml. reduced the viral titer by approximately 22.2 percent and 43.2 percent, respectively (Table 1). Pretreatment of monolayers with 100 (ig./ml. of Virazole for 24 hr. before the addition of the virus did not reduce the viral titer, nor did 100 (jLg./ml. of Virazole when added 24 hours after the adsorption of the virus. Treat-
ment with AUS did not reduce viral titers when used at 100 fig./ml.
CHEMOTHERAPEUTIC TREATMENT OF MAREK'S DISEASE
TABLE 3.—Effect
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of Virazole and AUS on the development of MD in chickens
1000 mg./bird did not reduce the incidence of gross MD lesions below those which received 25 or 50 mg./bird in experiment 1. Virazole, when administered at the 0.002% level in the feed, was ineffective in reducing MD lesions; whereas, the AUS group had only 2 of 30 birds with gross lesions. There were no birds in the groups vaccinated with the HVT vaccine with gross lesions, and 12 of 30 of the untreated controls had MD lesions. The MDV was recovered from the kidneys of all birds in each of the 2 experiments regardless of the treatment or the presence of lesions. DISCUSSION The data presented indicate that the synthetic triazole nucleoside Virazole has significant antiviral activity against MDV in vitro as well as in the reduction of tumor development in chickens. Although it is quite significant that Virazole has the ability to protect chickens against MD, it is highly unlikely that Virazole would be used as a prophylactic agent against MD, because multiple injections are necessary to elicit the desired results. This would not be economically feasible for the poultry industry at the present time. Also, it was found that Virazole was ineffective against MD when added to the ration. Unlike Virazole, AUS added to the feed at a con-
centration of 0.002 percent protects chickens against the development of MD lesions. This means of protecting the chickens against MD would have an advantage over the present method of vaccinating the chicks with the HVT vaccine because the vaccination procedure requires that each bird be injected. Administration of AUS in the feed would eliminate the cost of injecting each bird. Although the poultry industry would find it highly desirable to eliminate the vaccination of each bird, it must be pointed out that the results of this study indicate that AUS was not as effective in preventing MD lesions as the HVT vaccine. Also of interest is the fact that neither the mechanism of protection of the HVT vaccine against MDV nor the mechanism of protection of AUS is not fully understood. Although both AUS and the HVT vaccine prevent tumor development, neither prevents the infection of the bird with MDV, since the virus was detected in all birds at the termination of the trial at 8 weeks. While neither Virazole nor AUS have been approved for use as a means to protect chickens against MD, the most important point for the poultry industry would be the cost of these compounds. Although, the authors do not have information as to the cost of treatment per bird medicated it is likely that the cost would be prohibitive since the cost of the vaccine (1000 PFU) for a broiler
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8 weeks Method of Concn. of MD+/no. Cpd. medication cpd. (mg.) started % Pos. Virazole Inj.2-3 500 4728 (14.3%) Virazole Inj.2-3 1000 3/30 (10.0%) Virazole Feed2 10004 9/30 (30.0%) AUS Feed2 10004 2/30 (6.7%) Control (HVT)1 Control None 0/30 (0.0%) Control2 Control None 12/30 (40.0%) 'Birds not treated with Virazole or AUS but were vaccinated at one day of age with 1000 PFU of 2the HVT vaccine. 3-Week-old chicks injected with MD-infected plasma. 3 Injected twice daily for 2 weeks with Virazole. 4 1 gram per 100 lb. of feed for 8 weeks.
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C. S. EIDSON, V. T. THAN AND S. H. KLEVEN
chick is approximately $1.60 to $2.50 per thousand chicks. Although, the data presented in this study indicate that AUS is not as effective as the HVT vaccine, it is nevertheless encouraging that a chemotherapeutic agent may soon be available that would be at least as effective as the HVT vaccine, thereby eliminating the necessity of treating each bird individually.
ACKNOWLEDGMENTS
ICN Nucleic Acid Research Institute, Irvine, California, for supplying the Virazole and to Mr. Stiles Lovern and to Mr. Lane Teet for technical assistance. REFERENCES Biggs, P. M., L. N. Payne, B. S. Miine, A. E. Churchill, R. C. Chubb, D. G. Powell and A. H. Harris, 1970. Field trials with an attenuated cell associated vaccine for Marek's disease. Vet. Rec. 87: 704-709. Churchill, A. E., and P. M. Biggs, 1967. Agent of Marek's disease in tissue culture. Nature, 215: 528-530. Churchill, A. E., R. C. Chubb and W. Baxendale, 1969. The attenuation, with loss of oncogenicity, of the herpestype virus of Marek's disease (strain HPRS-16) on passage in cell culture. J. Gen Virol. 4: 557-564. Eidson, C.S., and D.P.Anderson, 1971. Immunization against Marek's disease. Avian Dis. 15: 68-81. Eidson, C. S., D. P. Anderson, S. H. Kleven and J. Brown, 1971. Field trials of vaccines for Marek's disease. Avian Dis. 15: 312-322. Eidson, C. S., and S. C. Schmittle, 1968. Studies on acute Marek's disease. I. Characteristics of Isolate GA in chickens. Avian Dis. 12: 467-476. Eidson, C. S., and S. C. Schmittle, 1969. Studies on acute Marek's disease. XII. Detecting antibodies with a tannic acid indirect hemagglutinating test. Avian Dis. 13: 774-782. Huffman, J. H., R. W. Sidwell, G. P. Khane, J. T. Witkowski, L. B. Allen and R. K. Robins, 1973.
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The authors would like to express their sincere appreciation to Dr. R. W. Sidwell,
In vitro effect of l-p-D-ribofuranosyl-l,2,4-triazole3-carboxamide (virazole, ICN 1229) on deoxyribonucleic acid and ribonucleic acid viruses. Antimicrob. Ag. Chemother. 3: 235-241. Nazerian, K., J. J. Solomon, R. L. Witter and B. R. Burmester, 1968. Studies on the etiology of Marek's disease. II. Finding of a herpesvirus in cell culture. Proc. Soc. Exptl. Biol. Med., 127: 177-182. Okazaki, W., H. G. Purchase and B. R. Burmester, 1970. Protection against Marek's disease by vaccination with a herpesvirus of turkeys. Avian Dis. 14: 413-429. Purchase, H. G., W. Okazaki and B. R. Burmester, 1970. Field trials with the herpes virus of turkeys (HVT) strain FC126 as a vaccine against Marek's disease. Poultry Sci. 50: 775-783. Shen, T. Y., D. B. R. Johnston, N. P. Jensen, W. V. Ruyle, J. J. Friedman, M. W. Fordice, J. F. McPherson, K. H. Boswell, T. A. Maag, R. W. Burg, R. M. Pellegrino, M. E. Jewell, C. A. Morris, H. L. Easterbrooks and B. J. Skelly, 1971. New chemoprophylactic agents for Marek's disease. Amer. Chem. Soc. Abstract NO. 44 in Section on Med. Chem. (No page no.) Sidwell, R. W., J. H. Huffman, G. P. Khare, L. B. Allen, J. T. Witkowski and R. K. Robins, 1972. Broad-spectrum antiviral activity of virazole: l-(3-Dribofuranosyl - 1, 2, 4 - triazole - 3 - carboxamide. Science, 177: 705-706. Sidwell, R. W., L. B. Allen, G. P. Khare, J. H. Huffman, J. T. Witkowski, L. N. Simon and R. K. Robins, 1973. Effect of 1-0-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole, ICN 1229) on herpes and vaccinia keratitis and encephatilis in laboratory animals. Antimcrob. Ag. Chemother. 3: 242-246. Sidwell, R. W., and L. N. Simon, 1972. Design synthesis, and broad spectrum antiviral activity of 1-0-D-ribofuranosyl- 1,2, 4-triazole-3-carboxamide and related nucleosides. J. Me. Chem. 15: 11501153. Solomon, J. J., R. L. Witter, K. Nazerian and B. R. Burmester, 1968. Studies on the etiology of Marek's disease. I. Propagation of the agent in cell culture. Proc. Soc. Exptl. Biol. Med. 127: 173-177. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 31: 525-538.