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Inhibition of semliki forest virus multiplication in L-cells by combinations of interferon and ribavirin as measured by plaque titration and direct enzyme immunoassay
Zbl. Bakt. 280, 386-391 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Inhibition of Semliki Forest Virus Multiplication in L-Cells by Combinations of Interferon and Ribavirin as Measured by Plaque Titration and Direct Enzyme Immunoassay THEO HARMSEN, DIDY VAN VEENENDAAL, and CORNELIS A. KRAAIJEVELD" From the Eijkman-Winkler Laboratory for Medical Microbiology, Utrecht University, Utrecht, The Netherlands
Received July 13, 1992 . Revision received April 26, 1993 . Accepted June 4, 1993
Summary Inhibition of Semliki Forest virus (SFV) multiplication in L-cell monolayers by combinations of mouse interferon (IFN) and ribavirin was measured by plaque titration and by direct enzyme immunoassay of SFV in L-cells. When critically inhibitory quantities of IFN and ribavirin were combined, an additive inhibitory effect was observed in either assay. Zusammenfassung Die Hemmung der Vermehrung von Semliki-Forest-Virus (SFV) in L-Zellen-Monolayers durch Kombination von murinem Interferon (IFN) und Ribavirin wurde mittels PlaqueTitration und directem Enzym-Immunoassay von SFV in L-Zellen gemessen. Bei einer Kombination von hemmenden Mengen von IFN und Ribavirin wurde in beiden Priifungen eine additive Hemmwirkung beobachtet. Introduction Combinations of antiviral agents might have either an antagonistic, additive or a synergistic effect on virus multiplication. The inhibitory effects of antiviral agents are mostly measured by yield reduction assays, e.g. by plaque titration of culture fluids. Earlier, we have developed enzyme immunoassays (EIAs) for detection of Semliki Forest virus (SFV) in glutaraldehyde-fixed L-cell monolayers using horseradish peroxidase-labelled SFV-specific monoclonal antibodies (MAbs) (6). These EIAs proved to * Corresponding author
Inhibition of Semliki Forest Virus Multiplication
387
be suitable for the measurement of the antiviral effect of IFN and ribavirin (7, 8). In the present study, we investigated whether direct EIA of SFV could be used to measure the inhibitory effect of combinations of IFN and ribavirin by comparing it with the aid of plaque titration. EIA measures cell-associated virus at the end of the infection period while plaque titration of supernatant fluids measures the production of infectious virus (minus thermo-inactivation) over the entire period.
Materials and Methods Semliki Forest virus (SFV). The prototype strain of SFV was obtained from H. GaTOff (1). After one passage over L-cells, the culture fluid contained 109 plaque forming units (PFU) per ml and was stored in small portions at -70 0e. Plaque titration of SFV and other general virological methods have been described earlier (2). Cells and media. L-cells, a continuous line of mouse fibroblasts, were maintained in Dulbecco's minimal essential medium (DMEM), buffered with 0.01 M N-2-hydroxyethylpiperazine-N'-ethane sulphonic acid, and supplemented with 10% calf serum, 0.2% tryptose (Difco Laboratories, Detroit, USA) and antibiotics. Cells, SFV, interferon and ribavirin were diluted to the required concentrations in the same medium with 5% calf serum. Interferon (IFN). IFN was induced in L-cell monolayers with ultraviolet light-inactivated Newcastle disease virus as described previously (3). Culture medium containing L-cell (mouse) IFN was divided in to small portions and stored at -20°e. Ribavirin. Ribavirin was obtained from Servo Feinbiochemica, Heidelberg, Germany. Monoclonal antibody (MAb). SFV in cell culture was detected with horseradish peroxidase (HRPO)-Iabelled MAb UM 1.4. This SFV-neutralizing MAb (IgG2a) is specific for the E2 membrane protein of SFV (5). Horseradish peroxidase was conjugated to MAb UM 1.4 (ascitic fluid) by the periodate method (4). The conjugate was diluted 1: 10 in phosphate buffered saline (PBS) of pH 7.2 with 1 % bovine serum albumin and stored in small portions in sterile siliconized plastic tubes at 4°C. Immediately before use, the conjugate was diluted to a final dilution of 1: 40000 in PBS with 0.5% Tween 20. Determination of the combined antiviral activity of IFN and ribavirin by direct enzyme immunoassay of SFV in cell culture. To each well of 96-well plates (catalogue No. 3596, Costar Plastics, Cambridge, MA, USA), 15000 L-cells (0.1 ml) were seeded to form monolayers. After 3 h incubation at 37"C, the medium was discarded. Dilutions (0.05 ml per well) of IFN were added to the monolayers followed by incubation for 24 h at 37°e. Then dilutions (0.05 ml) of ribavirin and SFV (0.05 ml) were added to the wells. Suitable infectious doses of SFV proved to be 3000 to 12000 PFU per well equalling to multiplicities of infection (MOIs) of 0.2 to 0.8. After an infection period of 19 h at 37°C, the supernatant fluids (0.15 ml) were transfered individually to wells of other plates, stored at -20°C and used later for plaque titration (pools of 4 identical wells). The monolayers were fixed with 0.05% glutaraldehyde (E. Merck AG, Darmstadt, Germany) for 10 min at room temperature. Thereafter, these mono layers were washed with tap water, rinsed with PBS and shaken until dry. Subsequently 0.1 ml aliquots of SFV-specific HRPO-Iabelled MAb UM 1.4 (1: 40000) were pipetted into the wells and incubated for 1 h at 37°e. The plates were washed again with PBS, shaken until dry, and then 0.1 ml volumes of a solution of 3'3'5'5' tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO, USA) and ureaperoxide (Organon, Teknika, Boxtel, The Netherlands) were added. After 30 min incubation at room temperature, the enzyme reaction was stopped with 0.1 ml of 0.19 M H 2 S0 4 per well. The colour reaction was measured as optical density at 450 nm with a multichannel photometer (Flow Laboratories, Irvine, Scotland, U.K.). Inhibition of virus multiplication was calculated as percentage of control: % of inhibition = 100% - [(~50 of inhibitor - ~50 of cell control) /(~50 of virus control - A450 of cell control)] X 100%. Titres were arbitrarily defined as dilutions causing 50% inhibition.
388
T. Harmsen, D. van Veenendaal, and C. A. Kraaijeveld
Results Predetermined critically inhibitory doses of ribavirin and mouse IFN were used in chequerboard assays to detect either a synergistic, additive or antagonistic inhibitory effect of ribavirin and IFN against SFV. 15000 L-cells were seeded into each well of 96well plates. After formation of the monolayers, the medium was discarded and then dilutions of IFN (11640, 11960, 111280, 111920, 112560 and 1/3840) were added and incubated for 24 h at 37°C. Control mono layers were incubated with medium alone. Therafter dilutions (4-fold) of ribavirin (0, 5, 7.5, 10, 15, 20 en 30 !tg/ml) and SFV (3000,6000 and 12000 PFU/well) were added and incubated for 19 h at 37°C. Subsequently, the supernatant fluids of identical wells (n = 4) were pooled and the infectivity of each pool was determined by plaque titration. The monolayers were fixed with glutaraldehyde and subsequently SFV was detected by direct EIA. The results of plaque titration and EIA are presented in Tables 1 and 2. In Table 1, the inhibition of SFV multiplication by IFN alone and by ribavirin alone are shown for each MOL As indicated in Table 1, the results for the three MOIs did not differ greatly and therefore the combined results have been presented in Table 2. As shown in Tables 1 and 2, inhibition by either IFN alone or ribavirin alone was dependent on the concentration of inhibitor in both the plaque titration assay and EIA. EIA which measures only cellassociated virus at one timepoint seemed to be as sensitive as plaque titration. Combinations of critical dilutions of IFN (113840, 112560) and ribavirin (5 and 7.5 !tg per ml) had a clear additive effect on inhibition in the EIA when addition was defined as a greater inhibition by combinations of both agent than inhibition by either agent alone. A combined inhibitory effect was also measured by plaque titration. Discussion In this study, direct EIA of SFV in cell culture and plaque titration were utilized simultaneously to determine the combined inhibitory activity of ribavirin and IFN on the multiplication of SFV in L cells. Use of critically inhibitory concentrations of IFN and ribavirin revealed an additive inhibition measurable with either assay. The results suggest that inhibition of SFV by ribavirin is superimposed on inhibition by IFN. Obviously, the inhibitory effect of ribavirin is not affected positively or negatively by preincubation with IFN. An objection which could be put forward against use of direct EIA of SFV in glutaraldehyde-fixed L cells is that it only measures cell-associated virus at the end of the infection period. In contrast, plaque titration measures the production of infectious virus particles during the whole infection period although thermo-inactivation could diminish the amount of PFU per ml culture fluid. The range of dilutions in which inhibition could be measured is considerably greater for plaque titration than for the direct EIA as indicated in Tables 1 and 2. On the other hand, direct EIA proved to be as sensitive as plaque titration, however only towards the end of infection. Furthermore, in contrast to plaque titration, direct EIA allows the performance of large-scale experiments and is therefore excellently suited for research on antiviral agents. EIA in cell culture is feasible for viruses other than SFV e.g. with encephalomyocarditis virus (10), mumps virus (9) and influenza viruses. By means of the direct EIA, the combined inhibitory effect of ribavirin and amantadine on replication of influenza A viruses could be measured in a similar way as described in the present paper (unpublished results).
1 : 640 1 : 960 1 : 1280 1: 1920 1: 2560 1 : 3840
30 ""g/ml 20 ""g/ml 15 ""g/ml 10 ""g/ml 7.5 ""g/ml 5 ""g/ml
IFN
Ribavirin
0.115 0.154 0.180 0.314 0.458 0.580
0.132 0.130 0.112 0.247 0.484 0.615
0.121 0.729
A450
MOl
0.2
101 94 90 68 44 23
98 99 102 79 39 17
100 0
(%)
Inhibition
3.6 4.4 4.7 5.5 6.2 6.0
3.0 3.4 3.7 4.9 6.1 5.8
6.6
Log lO PFU/ml
0.109 0.206 0.237 0.470 0.666 0.849
0.126 0.138 0.228 0.431 0.734 0.823
0.112 0.882
~50
MOl 0.4
100 88 83 53 27 2
98 97 85 58 18 6
100 0
(%)
Inhibition
3.3 4.9 4.9 5.3 6.3 6.4
0.8 3.1 4.6 5.1 5.3 6.1
6.9
Log lO PFU/ml
0.111 0.261 0.385 0.705 0.767 0.910
0.114 0.119 0.215 0.728 0.878 0.930
0.109 0.997
A4s0
MOl
0.8
100 83 69 33 26 10
99 99 88 30 13 8
100 0
(%)
Inhibition
3.7 5.1 5.7 6.1 6.0 6.0
2.9 3.5 4.1 5.2 5.5 6.5
6.9
Log lO PFU/ml
a
Experiment performed as described in the text. Control absorbance values are the mean of 12 determinations; all other values are the mean of 4 determinations.
cell control virus control
None
Inhibitor
Table 1. Inhibition by interferon and ribavirin of SFV multiplication in L-cells at different multiplicities of infection measured by direct EIA and plaque titration a
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Combined results obtained with three MOIs are presented. See text and Table 1.
3.5±0.2 101±1
3A±0.2 102±1
3.2±0.1 101±1
3.5±0.1 100±1
30.0 30.0
Plaque test EIA
3.9±0.2 97±1
3.8±0.2 96±3
4.1±0.1 91±2
4.8±0.2 88±3
20.0 20.0
Plaque test EIA
4.5±0.3 92±2
4A±0.1 88±5
4.3±0.1 86±5
5.1±0.3 81±6
15.0 15.0
4.8±0.2 87±5
4.9±OA 74±9
Plaque test EIA
4.7±0.1 64±9
5.6±0.2 50±10
10.0 10.0
4.9±0.3 82±7
5.3±0.1 60±13
Plaque test EIA
5.6±0.2 45±8
6.2±0.1 32±6
7.5 7.5
Plaque test EIA
5.2±0.1 69±5
5A±0.1 54±5
5.6±0.3 27±7
6.1±0.1 12±6
5.0 5.0
Plaque test EIA
0
5.1±0.1 56±14
5.6±0.2 23±7
6.1±0.2 10±3
6.8±0.2
1920
2560
3840
-
control
---------"-_.,-
3.0±0.1 103±1
3.7±0.2 100±1
3.8±0.1 98±1
2.7±0.1 102±1
3.3±0.3 100±1
3A±OA 101±1
3A±OA 100±2
3.6±0.3 100±2
4.2±0.1 93±3 3.8±0.2 94±4
3A±OA 101±1
3.3±0.1 98±1
960
4.2±0.1 89±4
4.1±0.3 92±5
1280
= 3) of
2.0±OA 100±2
1.9±0.5 102±1
2.1±0.3 102±1
2.6±0.2 102±1
2.8±0.1 101±1
2.7±0.1 101±1
2.2±0.7 98±1
640
Log lO PFU titre (Mean ± SD; n = 3) in culture fluid and percentage inhibition (Mean ± SD; n absorbance at serial dilutions of IFN and ribavirin
0.0 0.0
Concentration of ribavirin (!-tg/ml )
Plaque test EIA
Semliki Forest virus measured by:
Table 2. Chequerboard titration to measure inhibition of SFV multiplication by combinations of IFN and ribavirin a
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Inhibition of Semliki Forest Virus Multiplication
391
References 1. Garoff, H., A. M. Frischauf, K. Simons, H. Lehrach, and H. Delius: Nucleotide sequ-
ence of cDNA coding for Semliki Forest virus membrane glycoproteins. Nature (Lond.) 288 (1980) 236-241 2. Kraaijeveld, C.A., M. Harmsen, and B.]. Boutahar-Trouw: Delayed-type hypersensitivity against Semliki Forest virus in mice. Infect. Immun. 23 (1979) 219-223 3. Kraaijeveld, C. A., W. Kamphuis, B.]. Benaissa-Trouw, M. Harmsen, and H. Snippe: Modulation of adjuvant-enhanced delayed-type hypersensitivity by the interferon inducers poly I: C and Newcastle disease virus. Int. Arch. Allergy app!. Immuno!. 79 (1986) 86-89 4. Nakane, P. K. and A. Kawaoi: Peroxidase-labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22 (1974) 1084--1091 5. Oosterlaken, T. A. M., M. Harmsen, C. Tangerman, P. Schielen, C. A. Kraaijeveld, and H. Snippe: A neutralization-inhibition enzyme immunoassay for anti-idiotypic antibodies that block monoclonal antibodies neutralizing Semliki Forest virus. J. Immuno!. Meth. 115 (1988) 255-261 6. Van Tiel, F. H., W. A. M. Boere, ]. Vinje, T. Harmsen, B.]. Benaissa- Trouw, C. A. Kraaijeveld, and H. Snippe: Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labelled monoclonal antibodies specific for glycoproteins E1 and E2 • J. Clin. Microbio!. 20 (1984) 387-390 7. Van Tiel, F.H., W.A.M. Boere, M. Harmsen, B.]. Benaissa-Trouw, C.A. Kraaijeveld, and H. Snippe: Enzyme immunoassay of interferon with peroxidase labelled virus specific monoclonal antibodies. J. Gen. Viro!. 66 (1986) 1353-1356 8. Van Tiel, P. H., M. Harmsen, C. A. Kraaijeveld, and H. Snippe: Inhibition of Semliki Forest virus multiplication by ribavirin detected with virus specific monoclonal antibody: a potential method for the monitoring of antiviral agents in serum. J. Viro!' Meth. 14 (1986) 119-126 9. Van Tiel, P. H., C. A. Kraaijeveld,]. Baller, T. Harmsen, T. A. M. Oosterlaken, and H. Snippe: Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies. J. Viro!. Meth. 22 (1988) 99-108 10. Vlaspolder, P., T. Harmsen, D. van Veenendaal, C. A. Kraaijeveld, and H. Snippe: Application of immunoassay of encephalomyocarditis virus in cell culture with enzymelabelled virus-specific monoclonal antibodies for rapid detection of virus, neutralizing antibodies and interferon. J. Clin. Microbio!. 26 (1988) 2593-2597 Dr. Cornelis A. Kraaijeveld, Eijkman-Winkler Laboratory for Medical Microbiology, Heidelberglaan 100, G 04.614, NL-3584 CX Utrecht, The Netherlands
Inhibition of Semliki Forest Virus Multiplication in L-Cells by Combinations of Interferon and Ribavirin as Measured by Plaque Titration and Direct Enzyme Immunoassay THEO HARMSEN, DIDY VAN VEENENDAAL, and CORNELIS A. KRAAIJEVELD" From the Eijkman-Winkler Laboratory for Medical Microbiology, Utrecht University, Utrecht, The Netherlands
Received July 13, 1992 . Revision received April 26, 1993 . Accepted June 4, 1993
Summary Inhibition of Semliki Forest virus (SFV) multiplication in L-cell monolayers by combinations of mouse interferon (IFN) and ribavirin was measured by plaque titration and by direct enzyme immunoassay of SFV in L-cells. When critically inhibitory quantities of IFN and ribavirin were combined, an additive inhibitory effect was observed in either assay. Zusammenfassung Die Hemmung der Vermehrung von Semliki-Forest-Virus (SFV) in L-Zellen-Monolayers durch Kombination von murinem Interferon (IFN) und Ribavirin wurde mittels PlaqueTitration und directem Enzym-Immunoassay von SFV in L-Zellen gemessen. Bei einer Kombination von hemmenden Mengen von IFN und Ribavirin wurde in beiden Priifungen eine additive Hemmwirkung beobachtet. Introduction Combinations of antiviral agents might have either an antagonistic, additive or a synergistic effect on virus multiplication. The inhibitory effects of antiviral agents are mostly measured by yield reduction assays, e.g. by plaque titration of culture fluids. Earlier, we have developed enzyme immunoassays (EIAs) for detection of Semliki Forest virus (SFV) in glutaraldehyde-fixed L-cell monolayers using horseradish peroxidase-labelled SFV-specific monoclonal antibodies (MAbs) (6). These EIAs proved to * Corresponding author
Inhibition of Semliki Forest Virus Multiplication
387
be suitable for the measurement of the antiviral effect of IFN and ribavirin (7, 8). In the present study, we investigated whether direct EIA of SFV could be used to measure the inhibitory effect of combinations of IFN and ribavirin by comparing it with the aid of plaque titration. EIA measures cell-associated virus at the end of the infection period while plaque titration of supernatant fluids measures the production of infectious virus (minus thermo-inactivation) over the entire period.
Materials and Methods Semliki Forest virus (SFV). The prototype strain of SFV was obtained from H. GaTOff (1). After one passage over L-cells, the culture fluid contained 109 plaque forming units (PFU) per ml and was stored in small portions at -70 0e. Plaque titration of SFV and other general virological methods have been described earlier (2). Cells and media. L-cells, a continuous line of mouse fibroblasts, were maintained in Dulbecco's minimal essential medium (DMEM), buffered with 0.01 M N-2-hydroxyethylpiperazine-N'-ethane sulphonic acid, and supplemented with 10% calf serum, 0.2% tryptose (Difco Laboratories, Detroit, USA) and antibiotics. Cells, SFV, interferon and ribavirin were diluted to the required concentrations in the same medium with 5% calf serum. Interferon (IFN). IFN was induced in L-cell monolayers with ultraviolet light-inactivated Newcastle disease virus as described previously (3). Culture medium containing L-cell (mouse) IFN was divided in to small portions and stored at -20°e. Ribavirin. Ribavirin was obtained from Servo Feinbiochemica, Heidelberg, Germany. Monoclonal antibody (MAb). SFV in cell culture was detected with horseradish peroxidase (HRPO)-Iabelled MAb UM 1.4. This SFV-neutralizing MAb (IgG2a) is specific for the E2 membrane protein of SFV (5). Horseradish peroxidase was conjugated to MAb UM 1.4 (ascitic fluid) by the periodate method (4). The conjugate was diluted 1: 10 in phosphate buffered saline (PBS) of pH 7.2 with 1 % bovine serum albumin and stored in small portions in sterile siliconized plastic tubes at 4°C. Immediately before use, the conjugate was diluted to a final dilution of 1: 40000 in PBS with 0.5% Tween 20. Determination of the combined antiviral activity of IFN and ribavirin by direct enzyme immunoassay of SFV in cell culture. To each well of 96-well plates (catalogue No. 3596, Costar Plastics, Cambridge, MA, USA), 15000 L-cells (0.1 ml) were seeded to form monolayers. After 3 h incubation at 37"C, the medium was discarded. Dilutions (0.05 ml per well) of IFN were added to the monolayers followed by incubation for 24 h at 37°e. Then dilutions (0.05 ml) of ribavirin and SFV (0.05 ml) were added to the wells. Suitable infectious doses of SFV proved to be 3000 to 12000 PFU per well equalling to multiplicities of infection (MOIs) of 0.2 to 0.8. After an infection period of 19 h at 37°C, the supernatant fluids (0.15 ml) were transfered individually to wells of other plates, stored at -20°C and used later for plaque titration (pools of 4 identical wells). The monolayers were fixed with 0.05% glutaraldehyde (E. Merck AG, Darmstadt, Germany) for 10 min at room temperature. Thereafter, these mono layers were washed with tap water, rinsed with PBS and shaken until dry. Subsequently 0.1 ml aliquots of SFV-specific HRPO-Iabelled MAb UM 1.4 (1: 40000) were pipetted into the wells and incubated for 1 h at 37°e. The plates were washed again with PBS, shaken until dry, and then 0.1 ml volumes of a solution of 3'3'5'5' tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO, USA) and ureaperoxide (Organon, Teknika, Boxtel, The Netherlands) were added. After 30 min incubation at room temperature, the enzyme reaction was stopped with 0.1 ml of 0.19 M H 2 S0 4 per well. The colour reaction was measured as optical density at 450 nm with a multichannel photometer (Flow Laboratories, Irvine, Scotland, U.K.). Inhibition of virus multiplication was calculated as percentage of control: % of inhibition = 100% - [(~50 of inhibitor - ~50 of cell control) /(~50 of virus control - A450 of cell control)] X 100%. Titres were arbitrarily defined as dilutions causing 50% inhibition.
388
T. Harmsen, D. van Veenendaal, and C. A. Kraaijeveld
Results Predetermined critically inhibitory doses of ribavirin and mouse IFN were used in chequerboard assays to detect either a synergistic, additive or antagonistic inhibitory effect of ribavirin and IFN against SFV. 15000 L-cells were seeded into each well of 96well plates. After formation of the monolayers, the medium was discarded and then dilutions of IFN (11640, 11960, 111280, 111920, 112560 and 1/3840) were added and incubated for 24 h at 37°C. Control mono layers were incubated with medium alone. Therafter dilutions (4-fold) of ribavirin (0, 5, 7.5, 10, 15, 20 en 30 !tg/ml) and SFV (3000,6000 and 12000 PFU/well) were added and incubated for 19 h at 37°C. Subsequently, the supernatant fluids of identical wells (n = 4) were pooled and the infectivity of each pool was determined by plaque titration. The monolayers were fixed with glutaraldehyde and subsequently SFV was detected by direct EIA. The results of plaque titration and EIA are presented in Tables 1 and 2. In Table 1, the inhibition of SFV multiplication by IFN alone and by ribavirin alone are shown for each MOL As indicated in Table 1, the results for the three MOIs did not differ greatly and therefore the combined results have been presented in Table 2. As shown in Tables 1 and 2, inhibition by either IFN alone or ribavirin alone was dependent on the concentration of inhibitor in both the plaque titration assay and EIA. EIA which measures only cellassociated virus at one timepoint seemed to be as sensitive as plaque titration. Combinations of critical dilutions of IFN (113840, 112560) and ribavirin (5 and 7.5 !tg per ml) had a clear additive effect on inhibition in the EIA when addition was defined as a greater inhibition by combinations of both agent than inhibition by either agent alone. A combined inhibitory effect was also measured by plaque titration. Discussion In this study, direct EIA of SFV in cell culture and plaque titration were utilized simultaneously to determine the combined inhibitory activity of ribavirin and IFN on the multiplication of SFV in L cells. Use of critically inhibitory concentrations of IFN and ribavirin revealed an additive inhibition measurable with either assay. The results suggest that inhibition of SFV by ribavirin is superimposed on inhibition by IFN. Obviously, the inhibitory effect of ribavirin is not affected positively or negatively by preincubation with IFN. An objection which could be put forward against use of direct EIA of SFV in glutaraldehyde-fixed L cells is that it only measures cell-associated virus at the end of the infection period. In contrast, plaque titration measures the production of infectious virus particles during the whole infection period although thermo-inactivation could diminish the amount of PFU per ml culture fluid. The range of dilutions in which inhibition could be measured is considerably greater for plaque titration than for the direct EIA as indicated in Tables 1 and 2. On the other hand, direct EIA proved to be as sensitive as plaque titration, however only towards the end of infection. Furthermore, in contrast to plaque titration, direct EIA allows the performance of large-scale experiments and is therefore excellently suited for research on antiviral agents. EIA in cell culture is feasible for viruses other than SFV e.g. with encephalomyocarditis virus (10), mumps virus (9) and influenza viruses. By means of the direct EIA, the combined inhibitory effect of ribavirin and amantadine on replication of influenza A viruses could be measured in a similar way as described in the present paper (unpublished results).
1 : 640 1 : 960 1 : 1280 1: 1920 1: 2560 1 : 3840
30 ""g/ml 20 ""g/ml 15 ""g/ml 10 ""g/ml 7.5 ""g/ml 5 ""g/ml
IFN
Ribavirin
0.115 0.154 0.180 0.314 0.458 0.580
0.132 0.130 0.112 0.247 0.484 0.615
0.121 0.729
A450
MOl
0.2
101 94 90 68 44 23
98 99 102 79 39 17
100 0
(%)
Inhibition
3.6 4.4 4.7 5.5 6.2 6.0
3.0 3.4 3.7 4.9 6.1 5.8
6.6
Log lO PFU/ml
0.109 0.206 0.237 0.470 0.666 0.849
0.126 0.138 0.228 0.431 0.734 0.823
0.112 0.882
~50
MOl 0.4
100 88 83 53 27 2
98 97 85 58 18 6
100 0
(%)
Inhibition
3.3 4.9 4.9 5.3 6.3 6.4
0.8 3.1 4.6 5.1 5.3 6.1
6.9
Log lO PFU/ml
0.111 0.261 0.385 0.705 0.767 0.910
0.114 0.119 0.215 0.728 0.878 0.930
0.109 0.997
A4s0
MOl
0.8
100 83 69 33 26 10
99 99 88 30 13 8
100 0
(%)
Inhibition
3.7 5.1 5.7 6.1 6.0 6.0
2.9 3.5 4.1 5.2 5.5 6.5
6.9
Log lO PFU/ml
a
Experiment performed as described in the text. Control absorbance values are the mean of 12 determinations; all other values are the mean of 4 determinations.
cell control virus control
None
Inhibitor
Table 1. Inhibition by interferon and ribavirin of SFV multiplication in L-cells at different multiplicities of infection measured by direct EIA and plaque titration a
\0
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S' :::r §'
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Combined results obtained with three MOIs are presented. See text and Table 1.
3.5±0.2 101±1
3A±0.2 102±1
3.2±0.1 101±1
3.5±0.1 100±1
30.0 30.0
Plaque test EIA
3.9±0.2 97±1
3.8±0.2 96±3
4.1±0.1 91±2
4.8±0.2 88±3
20.0 20.0
Plaque test EIA
4.5±0.3 92±2
4A±0.1 88±5
4.3±0.1 86±5
5.1±0.3 81±6
15.0 15.0
4.8±0.2 87±5
4.9±OA 74±9
Plaque test EIA
4.7±0.1 64±9
5.6±0.2 50±10
10.0 10.0
4.9±0.3 82±7
5.3±0.1 60±13
Plaque test EIA
5.6±0.2 45±8
6.2±0.1 32±6
7.5 7.5
Plaque test EIA
5.2±0.1 69±5
5A±0.1 54±5
5.6±0.3 27±7
6.1±0.1 12±6
5.0 5.0
Plaque test EIA
0
5.1±0.1 56±14
5.6±0.2 23±7
6.1±0.2 10±3
6.8±0.2
1920
2560
3840
-
control
---------"-_.,-
3.0±0.1 103±1
3.7±0.2 100±1
3.8±0.1 98±1
2.7±0.1 102±1
3.3±0.3 100±1
3A±OA 101±1
3A±OA 100±2
3.6±0.3 100±2
4.2±0.1 93±3 3.8±0.2 94±4
3A±OA 101±1
3.3±0.1 98±1
960
4.2±0.1 89±4
4.1±0.3 92±5
1280
= 3) of
2.0±OA 100±2
1.9±0.5 102±1
2.1±0.3 102±1
2.6±0.2 102±1
2.8±0.1 101±1
2.7±0.1 101±1
2.2±0.7 98±1
640
Log lO PFU titre (Mean ± SD; n = 3) in culture fluid and percentage inhibition (Mean ± SD; n absorbance at serial dilutions of IFN and ribavirin
0.0 0.0
Concentration of ribavirin (!-tg/ml )
Plaque test EIA
Semliki Forest virus measured by:
Table 2. Chequerboard titration to measure inhibition of SFV multiplication by combinations of IFN and ribavirin a
w
0:
< r1l
~. '(;.
1'0
~
.
>-
0
::l p..
1'0
J?..
1'0
::l p..
r1l
::l
~ r1l
::l
1'0
t:I <
?
r1l
CF>
1'0
.::r:8
:-l
0
\J;)
Inhibition of Semliki Forest Virus Multiplication
391
References 1. Garoff, H., A. M. Frischauf, K. Simons, H. Lehrach, and H. Delius: Nucleotide sequ-
ence of cDNA coding for Semliki Forest virus membrane glycoproteins. Nature (Lond.) 288 (1980) 236-241 2. Kraaijeveld, C.A., M. Harmsen, and B.]. Boutahar-Trouw: Delayed-type hypersensitivity against Semliki Forest virus in mice. Infect. Immun. 23 (1979) 219-223 3. Kraaijeveld, C. A., W. Kamphuis, B.]. Benaissa-Trouw, M. Harmsen, and H. Snippe: Modulation of adjuvant-enhanced delayed-type hypersensitivity by the interferon inducers poly I: C and Newcastle disease virus. Int. Arch. Allergy app!. Immuno!. 79 (1986) 86-89 4. Nakane, P. K. and A. Kawaoi: Peroxidase-labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22 (1974) 1084--1091 5. Oosterlaken, T. A. M., M. Harmsen, C. Tangerman, P. Schielen, C. A. Kraaijeveld, and H. Snippe: A neutralization-inhibition enzyme immunoassay for anti-idiotypic antibodies that block monoclonal antibodies neutralizing Semliki Forest virus. J. Immuno!. Meth. 115 (1988) 255-261 6. Van Tiel, F. H., W. A. M. Boere, ]. Vinje, T. Harmsen, B.]. Benaissa- Trouw, C. A. Kraaijeveld, and H. Snippe: Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labelled monoclonal antibodies specific for glycoproteins E1 and E2 • J. Clin. Microbio!. 20 (1984) 387-390 7. Van Tiel, F.H., W.A.M. Boere, M. Harmsen, B.]. Benaissa-Trouw, C.A. Kraaijeveld, and H. Snippe: Enzyme immunoassay of interferon with peroxidase labelled virus specific monoclonal antibodies. J. Gen. Viro!. 66 (1986) 1353-1356 8. Van Tiel, P. H., M. Harmsen, C. A. Kraaijeveld, and H. Snippe: Inhibition of Semliki Forest virus multiplication by ribavirin detected with virus specific monoclonal antibody: a potential method for the monitoring of antiviral agents in serum. J. Viro!' Meth. 14 (1986) 119-126 9. Van Tiel, P. H., C. A. Kraaijeveld,]. Baller, T. Harmsen, T. A. M. Oosterlaken, and H. Snippe: Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies. J. Viro!. Meth. 22 (1988) 99-108 10. Vlaspolder, P., T. Harmsen, D. van Veenendaal, C. A. Kraaijeveld, and H. Snippe: Application of immunoassay of encephalomyocarditis virus in cell culture with enzymelabelled virus-specific monoclonal antibodies for rapid detection of virus, neutralizing antibodies and interferon. J. Clin. Microbio!. 26 (1988) 2593-2597 Dr. Cornelis A. Kraaijeveld, Eijkman-Winkler Laboratory for Medical Microbiology, Heidelberglaan 100, G 04.614, NL-3584 CX Utrecht, The Netherlands