Experimental Chemotherapy Chemotherapy 25: 162-169 (1979)
Effect of Disodium Phosphonoacetate and lododeoxyuridine on the Multiplication of African Swine Fever Virus in vitro Carmen Gil-Fernández, E. Páez, Pilar Vilas and A. Garcia Gancedo Departamento de Virología, Instituto Jaime Ferrán de Microbiología, Madrid
Key Words. Disodium phosphonoacetate • lododeoxyuridine ■African swine fever virus • Replication inhibition Abstract. Disodium phosphonoacetate (PAA) was found to inhibit the replication of African swine fever virus (ASFV). The action of this compound has been compared with the inhibitory capacity of iododeoxyuridine (IDU) upon ASFV growing in Vero cells. The study was done by the immunofluorescence technique in order to detect formations of cytoplasmic virus antigens and inclusion bodies; both were found to be inhibited by IDU and PAA. At 100 //g/ml, IDU blocked completely the multiplication of ASFV and with PAA, a few scattered cells showed positive fluorescence. The infectivity of the virus was reduced 1-5 log depending upon drug concentrations and time of exposure to the drugs. Inhibition of ASFV replication by PAA suggests that this virus, like other herpesviruses, involves a virus-specific DNA polymerase in its replication mechanism.
ported that PAA suppresses the replication of Epstein-Barr, varicella and cytomegalovi ruses [10, 17, 23, 25, 27]. We now report on the inhibition of ASFV replication with PAA.
Materials and Methods Cell Culture and Virus Vero cells were grown in medium 199 supple mented with 5% fetal bovine serum and sodium bicarbonate added to reach a pH of 7.4. All cell cultures in the experiments were carried out at 37 °C.
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The inhibitory effect of iododeoxyuri dine (IDU) on the multiplication of African swine fever (ASFV) has been reported by Haag et al. [6]. Phosphonoacetic acid (PAA) is an effective inhibitor of herpes simplex virus growth in tissue culture [3, 12, 19] and exerts antiviral activity in ani mals infected with herpes simplex type 1 [5, 7, 18, 24, 26] and type 2 [11, 22, 20]. In some cases, the inhibition is more effective than with IDU. PAA exerts a strong inhibi tory action toward DNA synthesis in nuclei isolated from vaccinia- and herpes simplexinfected HeLa cells [1]. It has also been re
Disodium Phosphonoacetate Action on African Swine Fever Virus
Chemicals PAA was kindly supplied by Abbott Laborato ries, Chicago, 111.; IDU was purchased from Sigma Chemical Co., St. Louis, Mo.; fetal bovine serum (mycoplasma- and virus-screened) and medium 199 modified with Hanks’ salts and glutamine were both purchased from Flow Laboratories, UK. Fluorescein-conjugated ASFV antiserum was kindly supplied by Prof. Sdnchez-Botija (Crida, Madrid). Antiviral Assays 3 days after seeding 5.0 X 104 Vero cells in Leighton tubes with coverslips, the cultures pre sent a confluent monolayer of cells. Growth medi um was discarded, the monolayer washed twice with Ca++ and Mg++ free phosphate-buffered sa line (PBS) and then inoculated at a multiplicity of infection (moi) of about 0.1. After 2 h adsorption of the virus to the monolayers, the unadsorbed vi rus was removed by two successive washings with PBS. Maintenance medium (199 + 2% fetal bo vine serum) was added, supplemented with the dif ferent doses of drugs. Untreated controls were performed in all experiments. At 24, 48, 72, 96 and 120 h after infection, the cultures were washed with PBS, fixed with ace tone for 15 min, stained with fluoresceinconjugated ASFV antiserum (diluted 1:50 in PBS) for 30 min, rinsed again with PBS and mounted in buffered glycerol. The cultures were examined by ultraviolet mi croscopy and the number of fluorescent foci was counted. Other experiments were performed with monolayers of Vero cells in test tubes inoculated with ASFV after washing with Ca*+ and Mg+t free PBS. After a 2-hour adsorption period, the monolayers were washed again to remove the unad
sorbed virus and maintenance medium with differ ent doses of drugs was added. At 24, 48 and 72 h after infection, the me dium was removed and maintenance medium free of drugs was added. After 3 days in drug-free me dium, four tubes for each dilution of drug, as well as the control without drug were frozen and thawed three times, cell debris were removed by centrifugation and afterwards total virus yield was determined by titration in Vero cells, four tubes per sample.
Results Cytopathic Effects of ASFV in Vero Cells ASFV forms microscopic plaques in Vero cells starting 48 h after the inocula tion; these are very clear and distinct after 3 days. No formation of satellite plaques is visible until the 5th or 7th day. At this time, primary plaques have increased in size and develop a hollow in the center, formed by the dead cells which come off the glass. Sur rounding primary plaques, satellite plaques formed by clumps of 5-10 infected, round ed cells are visible. Due to this property of the virus, it was possible to count micro scopic plaques 3 days after the inoculation of the virus following Farnham’s [4] technique. A linear relationship was found between the dilution of virus inoculated and the number of microplaques counted. 48 h after infec tion, some cells in the stained cultures of Vero cells became rounded, margination of chromatin occurred, and inclusion bodies near the nucleus became frequent. Effect of PAA and IDU on the Replication of ASFV on Vero Cells Assayed by Immunofluorescence Three experiments were carried out in order to measure the inhibition of ASFV by
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ASFV adapted to grow in Vero cells was kind ly supplied by Dr. E. Viiiuela (Centro de Biologia Molecular, Madrid), and a stock virus was pre pared in these cells with a titer of 2.1 X 105 plaque forming units (PFU), and stored at -70 °C. Titration of virus was done according to Farnliam's [4] technique; this titration was performed in the absence of agar overlay and microscopic plaques were counted 3 days after the inoculation with ASFV. Groups of four tubes were used in each experiment.
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Gil-Fernândez/Pâcz/Vilas/Ganccdo
PAA and IDU. Vero cells were grown in Leighton tubes with coverslips. Cells were infected (see Materials and Methods) and af ter the adsorption period, maintenance me dium with 25, 50, 75 and 100 fig of PAA and IDU was added. Table I shows the inhibitory activity of IDU and PAA, expressed in percent loss of fluorescence foci with respect to the untreat ed controls. As can be seen, IDU has a greater inhibitory effect than PAA. The per centage of inhibition of fluorescent foci in creased with increasing drug concentration in the medium and also with increasing time of exposure to the drug. A 43% inhibition was reached at 24 h of treatment with PAA and 51% with IDU. Inhibition of cytopathic effects was complete at concentrations of 75 and 100 fig with both drugs, but in cultures stained with fluorescein-conjugated ASFV antiserum, it was possible to detect some scattered fluorescent cells.
In order to investigate whether the few cells which showed fluorescence contained virus which can replicate in a medium with out drug, another experiment was carried out in which the cultures were infected as before and kept with the above-mentioned different amounts of drug during 3 days; af ter this period of time, the medium was re moved and medium free of drug was added. Table II shows the results. The inhibition continues and increases at doses of 75 and 100 fig, with both drugs reaching 100% in hibition at 120 h after the inoculation with 75 and 100 fig of IDU and almost 100% with 100 fig of PAA and 50 fig of IDU. With 25 and 50 fig of PAA, treatment during 3 days slows down the infectious process. However, this process continues and the amount of virus increases a little, after removal of the drug. Figure 1 shows the fluorescent foci formed by the inocula tion of ASFV treated with 25 fig of PAA
Table I. Effect of PAA and IDU on the replication of ASFV on Vero cells expressed in percent loss of fluorescence foci with respect to untreated controls
Table II. Effect of PAA and IDU on the replication of ASFV on Vero cells expressed in percent loss of fluorescence foci with respect to untreated controls
Drug
Drug1
Time1 h
Loss of fluorescence foci according to drug doses G/g/ml), % 25
PAA
IDU
24 48 72 24 48 72
43 55 95 51 82 97
50 66 78 98 84 89 99
75 80 89 99 96 98 99,4
Time2 h
100 89 92 98 96 99 99
1 Every 24 h after infection, two coverslips with Vero cells ASFV-infected and drug-treated and two con trols (no drug) were removed stained and fluorescent foci counted.
PAA IDU
96 120 96 120
Loss of fluorescence foci according to drug doses fog/ml), % 25
50
75
100
91 91 96 98
95 95 98 99.9
99 98 99.9 100
99 99.9 99.9 100
1 Cultures were treated with drug during 3 days; the medium was then removed and drug-free medium was added. 2 Every 24 h after removal of the drug, two coverslips with Vero cells ASFV-infected and two controls (no drug) were removed, stained and fluorescence foci counted.
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164
Disodium Phosphonoacetate Action on African Swine Fever Virus
165
V
rt? ** y
* 4 *
Effect of Disodium Phosphonoacetate and lododeoxyuridine on the Multiplication of African Swine Fever Virus in vitro Carmen Gil-Fernández, E. Páez, Pilar Vilas and A. Garcia Gancedo Departamento de Virología, Instituto Jaime Ferrán de Microbiología, Madrid
Key Words. Disodium phosphonoacetate • lododeoxyuridine ■African swine fever virus • Replication inhibition Abstract. Disodium phosphonoacetate (PAA) was found to inhibit the replication of African swine fever virus (ASFV). The action of this compound has been compared with the inhibitory capacity of iododeoxyuridine (IDU) upon ASFV growing in Vero cells. The study was done by the immunofluorescence technique in order to detect formations of cytoplasmic virus antigens and inclusion bodies; both were found to be inhibited by IDU and PAA. At 100 //g/ml, IDU blocked completely the multiplication of ASFV and with PAA, a few scattered cells showed positive fluorescence. The infectivity of the virus was reduced 1-5 log depending upon drug concentrations and time of exposure to the drugs. Inhibition of ASFV replication by PAA suggests that this virus, like other herpesviruses, involves a virus-specific DNA polymerase in its replication mechanism.
ported that PAA suppresses the replication of Epstein-Barr, varicella and cytomegalovi ruses [10, 17, 23, 25, 27]. We now report on the inhibition of ASFV replication with PAA.
Materials and Methods Cell Culture and Virus Vero cells were grown in medium 199 supple mented with 5% fetal bovine serum and sodium bicarbonate added to reach a pH of 7.4. All cell cultures in the experiments were carried out at 37 °C.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 6:50:43 PM
The inhibitory effect of iododeoxyuri dine (IDU) on the multiplication of African swine fever (ASFV) has been reported by Haag et al. [6]. Phosphonoacetic acid (PAA) is an effective inhibitor of herpes simplex virus growth in tissue culture [3, 12, 19] and exerts antiviral activity in ani mals infected with herpes simplex type 1 [5, 7, 18, 24, 26] and type 2 [11, 22, 20]. In some cases, the inhibition is more effective than with IDU. PAA exerts a strong inhibi tory action toward DNA synthesis in nuclei isolated from vaccinia- and herpes simplexinfected HeLa cells [1]. It has also been re
Disodium Phosphonoacetate Action on African Swine Fever Virus
Chemicals PAA was kindly supplied by Abbott Laborato ries, Chicago, 111.; IDU was purchased from Sigma Chemical Co., St. Louis, Mo.; fetal bovine serum (mycoplasma- and virus-screened) and medium 199 modified with Hanks’ salts and glutamine were both purchased from Flow Laboratories, UK. Fluorescein-conjugated ASFV antiserum was kindly supplied by Prof. Sdnchez-Botija (Crida, Madrid). Antiviral Assays 3 days after seeding 5.0 X 104 Vero cells in Leighton tubes with coverslips, the cultures pre sent a confluent monolayer of cells. Growth medi um was discarded, the monolayer washed twice with Ca++ and Mg++ free phosphate-buffered sa line (PBS) and then inoculated at a multiplicity of infection (moi) of about 0.1. After 2 h adsorption of the virus to the monolayers, the unadsorbed vi rus was removed by two successive washings with PBS. Maintenance medium (199 + 2% fetal bo vine serum) was added, supplemented with the dif ferent doses of drugs. Untreated controls were performed in all experiments. At 24, 48, 72, 96 and 120 h after infection, the cultures were washed with PBS, fixed with ace tone for 15 min, stained with fluoresceinconjugated ASFV antiserum (diluted 1:50 in PBS) for 30 min, rinsed again with PBS and mounted in buffered glycerol. The cultures were examined by ultraviolet mi croscopy and the number of fluorescent foci was counted. Other experiments were performed with monolayers of Vero cells in test tubes inoculated with ASFV after washing with Ca*+ and Mg+t free PBS. After a 2-hour adsorption period, the monolayers were washed again to remove the unad
sorbed virus and maintenance medium with differ ent doses of drugs was added. At 24, 48 and 72 h after infection, the me dium was removed and maintenance medium free of drugs was added. After 3 days in drug-free me dium, four tubes for each dilution of drug, as well as the control without drug were frozen and thawed three times, cell debris were removed by centrifugation and afterwards total virus yield was determined by titration in Vero cells, four tubes per sample.
Results Cytopathic Effects of ASFV in Vero Cells ASFV forms microscopic plaques in Vero cells starting 48 h after the inocula tion; these are very clear and distinct after 3 days. No formation of satellite plaques is visible until the 5th or 7th day. At this time, primary plaques have increased in size and develop a hollow in the center, formed by the dead cells which come off the glass. Sur rounding primary plaques, satellite plaques formed by clumps of 5-10 infected, round ed cells are visible. Due to this property of the virus, it was possible to count micro scopic plaques 3 days after the inoculation of the virus following Farnham’s [4] technique. A linear relationship was found between the dilution of virus inoculated and the number of microplaques counted. 48 h after infec tion, some cells in the stained cultures of Vero cells became rounded, margination of chromatin occurred, and inclusion bodies near the nucleus became frequent. Effect of PAA and IDU on the Replication of ASFV on Vero Cells Assayed by Immunofluorescence Three experiments were carried out in order to measure the inhibition of ASFV by
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 6:50:43 PM
ASFV adapted to grow in Vero cells was kind ly supplied by Dr. E. Viiiuela (Centro de Biologia Molecular, Madrid), and a stock virus was pre pared in these cells with a titer of 2.1 X 105 plaque forming units (PFU), and stored at -70 °C. Titration of virus was done according to Farnliam's [4] technique; this titration was performed in the absence of agar overlay and microscopic plaques were counted 3 days after the inoculation with ASFV. Groups of four tubes were used in each experiment.
163
Gil-Fernândez/Pâcz/Vilas/Ganccdo
PAA and IDU. Vero cells were grown in Leighton tubes with coverslips. Cells were infected (see Materials and Methods) and af ter the adsorption period, maintenance me dium with 25, 50, 75 and 100 fig of PAA and IDU was added. Table I shows the inhibitory activity of IDU and PAA, expressed in percent loss of fluorescence foci with respect to the untreat ed controls. As can be seen, IDU has a greater inhibitory effect than PAA. The per centage of inhibition of fluorescent foci in creased with increasing drug concentration in the medium and also with increasing time of exposure to the drug. A 43% inhibition was reached at 24 h of treatment with PAA and 51% with IDU. Inhibition of cytopathic effects was complete at concentrations of 75 and 100 fig with both drugs, but in cultures stained with fluorescein-conjugated ASFV antiserum, it was possible to detect some scattered fluorescent cells.
In order to investigate whether the few cells which showed fluorescence contained virus which can replicate in a medium with out drug, another experiment was carried out in which the cultures were infected as before and kept with the above-mentioned different amounts of drug during 3 days; af ter this period of time, the medium was re moved and medium free of drug was added. Table II shows the results. The inhibition continues and increases at doses of 75 and 100 fig, with both drugs reaching 100% in hibition at 120 h after the inoculation with 75 and 100 fig of IDU and almost 100% with 100 fig of PAA and 50 fig of IDU. With 25 and 50 fig of PAA, treatment during 3 days slows down the infectious process. However, this process continues and the amount of virus increases a little, after removal of the drug. Figure 1 shows the fluorescent foci formed by the inocula tion of ASFV treated with 25 fig of PAA
Table I. Effect of PAA and IDU on the replication of ASFV on Vero cells expressed in percent loss of fluorescence foci with respect to untreated controls
Table II. Effect of PAA and IDU on the replication of ASFV on Vero cells expressed in percent loss of fluorescence foci with respect to untreated controls
Drug
Drug1
Time1 h
Loss of fluorescence foci according to drug doses G/g/ml), % 25
PAA
IDU
24 48 72 24 48 72
43 55 95 51 82 97
50 66 78 98 84 89 99
75 80 89 99 96 98 99,4
Time2 h
100 89 92 98 96 99 99
1 Every 24 h after infection, two coverslips with Vero cells ASFV-infected and drug-treated and two con trols (no drug) were removed stained and fluorescent foci counted.
PAA IDU
96 120 96 120
Loss of fluorescence foci according to drug doses fog/ml), % 25
50
75
100
91 91 96 98
95 95 98 99.9
99 98 99.9 100
99 99.9 99.9 100
1 Cultures were treated with drug during 3 days; the medium was then removed and drug-free medium was added. 2 Every 24 h after removal of the drug, two coverslips with Vero cells ASFV-infected and two controls (no drug) were removed, stained and fluorescence foci counted.
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164
Disodium Phosphonoacetate Action on African Swine Fever Virus
165
V
rt? ** y
* 4 *
