Journal of Antimicrobial Chemotherapy (1975) 1 (Suppl.), 63-69
Antiviral activity of ribavirin on influenza infection in ferrets
Karen P. Schofield, C. W. Potter, Dorothy Edey, R. Jennings and J. S. Oxford*
The effect of l-P-D-ribofuranosyl-1-2, 4-triazoIe-3-carboxamide (Ribavirin) on influenza virus infection was studied in ferrets. Ferrets were given lOOmg/kg of ribavirin intraperitoneally 1 day before, 2 h before and daily for 5 days following intranasal infection with 103-0 ferret infective doses of A/Port Chalmers/73 virus. Ribavirin-treated animals showed no temperature response, no increase of nasal wash protein and did not produce nasal antibody following virus infection indicating an inhibitory effect on virus infection. In addition, drug-treated ferrets had markedly reduced titres of virus in nasal washings and did not produce serum antibody following virus infection. The absence of an antibody response in the presence of virus in the upper respiratory tract was attributed to an immunosuppressive action of the compound. Introduction
The compound l-P-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (ribavirin) has been reported to inhibit the replication of a number of DNA and RNA viruses (Sidwell et al., 1972), including influenza virus infection in vitro (Huffman, Sidwell & Khare, 1973; Oxford, 1975a) and in vivo (Khare, Sidwell, Witkowski, Simon & Robins, 1973). In the present study, the effects of ribavirin on influenza virus A/Port Chalmers/73 infection of ferrets was examined, since influenza in this species closely approximates the disease in man (Haff, Schriver & Stewart, 1966; Potter, Oxford, Shore, McLaren & StuartHarris, 1972) and this recommends the ferret for the investigation of influenza-inhibiting compounds. Thus, ferrets infected with influenza viruses show a sharp febrile response, produce high titres of virus, produce nasal wash and serum antibody and show a marked increase in nasal wash protein; using these parameters, the effects of antiviral compounds on influenza virus infection can be measured qualitatively (Cochran, Maassab, Tsunoda & Berlin, 1965; Squires, 1970; Potter et al, 1972; Haff & Pinto, 1973; Potter & Schofield, 1975). Methods and materials Virus and virus vaccines Influenza virus A/Port Chalmers/73 (H3N2) was obtained from Dr G. C. Schild, National Institute for Medical Research, Mill Hill, London. A virus pool was prepared •Present address: Division of Virology, National Institute for Biological Standards and Control, Holly Hill, London NW3, England. 63
Downloaded from http://jac.oxfordjournals.org/ at OCLC on June 21, 2015
Academic Division of Pathology {Virology), University of Sheffield Medical School, Sheffield S10 2RX, England
64
K. P. Scbofield a al.
in 10-day embryonated eggs and the infectivity of the virus for ferrets was established by titration in normal animals. Using the temperature response, virus isolation and the serum antibody response as indices of infection, the 50% ferret infective dose (FID50) was calculated as 10*0EID80. Ferrets were lightly anaesthetized with ether and inoculated with lC^FIDgoof the A/Port Chalmers/73 virus; the virus was given dropwise intranasally in a 10 ml volume of PBS. Ribavirin Ribavirin was kindly supplied by Dr R. W. Sidwell, ICN Nucleic Acid Research Institute, Irvine, California and by Lederle Labs., Gosport, Hants., and was stored at room temperature. The drug was weighed out fresh each day and dissolved in phosphate buffered saline, pH 7-2, at a concentration of 100 mg/ml. Ferrets were inoculated daily by the intraperitoneal route with 100 mg/kg during the period of treatment.
Virus isolation Bovine serum albumin, at a final concentration of 2-0% (v/v) and antibiotics (250 units/ ml of penicillin and 200 ug/ml of streptomycin) were added to nasal washings taken for 6 days after virus infection; the washings were collected in 10 ml of PBS as described previously (Potter et al., 1972) and stored at — 80°C. The titre of virus in these specimens was determined by titration in whole, 10-day embryonated eggs inoculated by the allantoic route (Potter et al, 1972). Serological tests (1) Haemagglutination inhibition (HI) antibody tests. Ferret serum specimens were tested for HI antibody by the microtitre technique (Sever, 1962). Before testing, the sera were treated with 5 vol. of cholera filtrate (Burroughs Wellcome Ltd) for 18 h at 37°C, and then heated for 1 h at 56°C. (2) Single radial diffusion (SRD) tests. Post-infection ferret sera were analysed for antibodies to influenza virus haemagglutinin by SRD, as described previously (Schild, Oxford & Virilizier, 1975). The haemagglutinin for mouse immunization experiments was obtained from purified X31 recombinant influenza virus by treatment with bromelain; the freed haemagglutinin was purified in sucrose gradients (Schild et al, 1975).
Downloaded from http://jac.oxfordjournals.org/ at OCLC on June 21, 2015
Experimental design Adult ferrets, aged 4-8 months and weighing 500-800 g, were obtained from accredited dealers and housed in individual cages for 7 days prior to experimentation. During this period, nasal washings were collected for protein estimation and temperatures taken to establish normal values. Ferrets were test bled and inoculated intraperitoneally with 100 mg/kg of ribavirin. The following day, the ferrets were given a second dose of ribavirin, and 2 h later the animals and a group of control ferrets were infected with influenza virus A/Port Chalmers/73. The drug-treated ferrets were inoculated with ribavirin daily for 5 days after virus infection. Following virus infection, ferret temperatures were taken twice daily for 4 days, and once daily for a further 3 days. Nasal washings were collected daily for 6 days after virus infection for virus isolation and on alternate days from day 5-15 for protein and neutralizing antibody studies. Blood samples were taken 10 and 21 days after virus infection to determine the serum antibody response to infection.
Activity of ribavirin on influenza
65
(3) Neutralization tests. Nasal washings collected for neutralizing antibody and protein determinations were shaken with glass beads and centrifuged at 3000 r.p.m. for 10 min. The supernatant fluid was then concentrated 10-fold by dialysis against 30% Carbowax. The concentrated nasal wash specimens were tested for neutralizing antibody by the allantois-on-shell (AOS) method (Fazekas de St Groth, Witchell & Lafferty, 1958) using standard methods.
Ribavirin - treatment Control 41
E o
39 I
-
i
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0
i
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I
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I
3
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7
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I I 4 5 6
7
Time after infection (days)
Figure 1. Temperature response of ferrets treated with Ribavirin and infected with influenza virus A/Port Chalroers/73.
Results Temperature response to infection with A/Port Chalmersj73 virus All 4 control ferrets inoculated with lO^FIDso of A/Port Chalmers/73 virus showed a sharp febrile response to virus infection, which was maximal at 24-48 h after infection. The temperature profile for 2 of the ferrets is shown in Figure 1. Thus, for each animal the temperature rose ^ 1 ° C above the mean, pre-infection level to a peak of ^ 4 0 0 ° C on 2 occasions; this was taken as indicating both a significant temperature and a significant rise in temperature (Potter et al., 1972). A second peak of temperature was observed in 2 of the control ferrets; this had been seen in previous experiments as an infrequent and random observation. In contrast to the control ferrets, ferrets given 7 daily doses of 100 mg/kg of ribavirin intraperitoneally exhibited no febrile response to infection with A/Port Chalmers/73 virus. Thus, none of the ferrets in this group developed either a significant temperature or a significant rise in temperature following virus infection. Virus isolations following infection with influenza virus Nasal washings were collected daily for 6 days following virus infection and were titrated for infective virus in 10-day embryonated eggs. The results are shown in Figure 2. For control ferrets, viruses were detected 24 h after infection and peak titres were found 2-3 days after infection; after this time the titres of virus recovered progressively declined. Ferrets treated with ribavirin for 7 days showed the same result, but on each day the titre of virus recovered was 10-50-fold less than that found on control ferrets.
Downloaded from http://jac.oxfordjournals.org/ at OCLC on June 21, 2015
£40
Q.
66
K. P . SchofieJd et aL
Time after infection (days) Ribavirin treatment
Concentration of nasal wash protein
Nasal washings were collected from all control and ribavirin treated ferrets on alternate days from days 5-15 after virus infection. The specimens were concentrated 10-fold and the amount of protein measured by the method of Lowry et al. (1951). The results are shown in Figure 3. The concentration of protein in nasal washings from control ferrets increased to a maximum at 5-7 days after virus infection; at this time, the concentration was 2-4 times greater than that found in nasal washings collected prior to infection. After day 7 following virus infection the concentration of protein declined and by day Control
Ribovirin -treated (x7 )
16 *l-2 _ _ rf>8 «
rr.
-
T i t r « of leutrali ring ont body
5 7 Flgur* 3
9
II
15 5 7 13 Timi o f t e r infection (day«)
16 _ Control
A ft 1*1 ft 9
II
13
15
Ribavirin - treated ( x 7 )
8
4
Antiviral activity of ribavirin on influenza infection in ferrets
Karen P. Schofield, C. W. Potter, Dorothy Edey, R. Jennings and J. S. Oxford*
The effect of l-P-D-ribofuranosyl-1-2, 4-triazoIe-3-carboxamide (Ribavirin) on influenza virus infection was studied in ferrets. Ferrets were given lOOmg/kg of ribavirin intraperitoneally 1 day before, 2 h before and daily for 5 days following intranasal infection with 103-0 ferret infective doses of A/Port Chalmers/73 virus. Ribavirin-treated animals showed no temperature response, no increase of nasal wash protein and did not produce nasal antibody following virus infection indicating an inhibitory effect on virus infection. In addition, drug-treated ferrets had markedly reduced titres of virus in nasal washings and did not produce serum antibody following virus infection. The absence of an antibody response in the presence of virus in the upper respiratory tract was attributed to an immunosuppressive action of the compound. Introduction
The compound l-P-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (ribavirin) has been reported to inhibit the replication of a number of DNA and RNA viruses (Sidwell et al., 1972), including influenza virus infection in vitro (Huffman, Sidwell & Khare, 1973; Oxford, 1975a) and in vivo (Khare, Sidwell, Witkowski, Simon & Robins, 1973). In the present study, the effects of ribavirin on influenza virus A/Port Chalmers/73 infection of ferrets was examined, since influenza in this species closely approximates the disease in man (Haff, Schriver & Stewart, 1966; Potter, Oxford, Shore, McLaren & StuartHarris, 1972) and this recommends the ferret for the investigation of influenza-inhibiting compounds. Thus, ferrets infected with influenza viruses show a sharp febrile response, produce high titres of virus, produce nasal wash and serum antibody and show a marked increase in nasal wash protein; using these parameters, the effects of antiviral compounds on influenza virus infection can be measured qualitatively (Cochran, Maassab, Tsunoda & Berlin, 1965; Squires, 1970; Potter et al, 1972; Haff & Pinto, 1973; Potter & Schofield, 1975). Methods and materials Virus and virus vaccines Influenza virus A/Port Chalmers/73 (H3N2) was obtained from Dr G. C. Schild, National Institute for Medical Research, Mill Hill, London. A virus pool was prepared •Present address: Division of Virology, National Institute for Biological Standards and Control, Holly Hill, London NW3, England. 63
Downloaded from http://jac.oxfordjournals.org/ at OCLC on June 21, 2015
Academic Division of Pathology {Virology), University of Sheffield Medical School, Sheffield S10 2RX, England
64
K. P. Scbofield a al.
in 10-day embryonated eggs and the infectivity of the virus for ferrets was established by titration in normal animals. Using the temperature response, virus isolation and the serum antibody response as indices of infection, the 50% ferret infective dose (FID50) was calculated as 10*0EID80. Ferrets were lightly anaesthetized with ether and inoculated with lC^FIDgoof the A/Port Chalmers/73 virus; the virus was given dropwise intranasally in a 10 ml volume of PBS. Ribavirin Ribavirin was kindly supplied by Dr R. W. Sidwell, ICN Nucleic Acid Research Institute, Irvine, California and by Lederle Labs., Gosport, Hants., and was stored at room temperature. The drug was weighed out fresh each day and dissolved in phosphate buffered saline, pH 7-2, at a concentration of 100 mg/ml. Ferrets were inoculated daily by the intraperitoneal route with 100 mg/kg during the period of treatment.
Virus isolation Bovine serum albumin, at a final concentration of 2-0% (v/v) and antibiotics (250 units/ ml of penicillin and 200 ug/ml of streptomycin) were added to nasal washings taken for 6 days after virus infection; the washings were collected in 10 ml of PBS as described previously (Potter et al., 1972) and stored at — 80°C. The titre of virus in these specimens was determined by titration in whole, 10-day embryonated eggs inoculated by the allantoic route (Potter et al, 1972). Serological tests (1) Haemagglutination inhibition (HI) antibody tests. Ferret serum specimens were tested for HI antibody by the microtitre technique (Sever, 1962). Before testing, the sera were treated with 5 vol. of cholera filtrate (Burroughs Wellcome Ltd) for 18 h at 37°C, and then heated for 1 h at 56°C. (2) Single radial diffusion (SRD) tests. Post-infection ferret sera were analysed for antibodies to influenza virus haemagglutinin by SRD, as described previously (Schild, Oxford & Virilizier, 1975). The haemagglutinin for mouse immunization experiments was obtained from purified X31 recombinant influenza virus by treatment with bromelain; the freed haemagglutinin was purified in sucrose gradients (Schild et al, 1975).
Downloaded from http://jac.oxfordjournals.org/ at OCLC on June 21, 2015
Experimental design Adult ferrets, aged 4-8 months and weighing 500-800 g, were obtained from accredited dealers and housed in individual cages for 7 days prior to experimentation. During this period, nasal washings were collected for protein estimation and temperatures taken to establish normal values. Ferrets were test bled and inoculated intraperitoneally with 100 mg/kg of ribavirin. The following day, the ferrets were given a second dose of ribavirin, and 2 h later the animals and a group of control ferrets were infected with influenza virus A/Port Chalmers/73. The drug-treated ferrets were inoculated with ribavirin daily for 5 days after virus infection. Following virus infection, ferret temperatures were taken twice daily for 4 days, and once daily for a further 3 days. Nasal washings were collected daily for 6 days after virus infection for virus isolation and on alternate days from day 5-15 for protein and neutralizing antibody studies. Blood samples were taken 10 and 21 days after virus infection to determine the serum antibody response to infection.
Activity of ribavirin on influenza
65
(3) Neutralization tests. Nasal washings collected for neutralizing antibody and protein determinations were shaken with glass beads and centrifuged at 3000 r.p.m. for 10 min. The supernatant fluid was then concentrated 10-fold by dialysis against 30% Carbowax. The concentrated nasal wash specimens were tested for neutralizing antibody by the allantois-on-shell (AOS) method (Fazekas de St Groth, Witchell & Lafferty, 1958) using standard methods.
Ribavirin - treatment Control 41
E o
39 I
-
i
I
1
0
i
1
I
2
II
I
3
4
5
6
7
I -
1
I 0
1
I 2
I 3
I I 4 5 6
7
Time after infection (days)
Figure 1. Temperature response of ferrets treated with Ribavirin and infected with influenza virus A/Port Chalroers/73.
Results Temperature response to infection with A/Port Chalmersj73 virus All 4 control ferrets inoculated with lO^FIDso of A/Port Chalmers/73 virus showed a sharp febrile response to virus infection, which was maximal at 24-48 h after infection. The temperature profile for 2 of the ferrets is shown in Figure 1. Thus, for each animal the temperature rose ^ 1 ° C above the mean, pre-infection level to a peak of ^ 4 0 0 ° C on 2 occasions; this was taken as indicating both a significant temperature and a significant rise in temperature (Potter et al., 1972). A second peak of temperature was observed in 2 of the control ferrets; this had been seen in previous experiments as an infrequent and random observation. In contrast to the control ferrets, ferrets given 7 daily doses of 100 mg/kg of ribavirin intraperitoneally exhibited no febrile response to infection with A/Port Chalmers/73 virus. Thus, none of the ferrets in this group developed either a significant temperature or a significant rise in temperature following virus infection. Virus isolations following infection with influenza virus Nasal washings were collected daily for 6 days following virus infection and were titrated for infective virus in 10-day embryonated eggs. The results are shown in Figure 2. For control ferrets, viruses were detected 24 h after infection and peak titres were found 2-3 days after infection; after this time the titres of virus recovered progressively declined. Ferrets treated with ribavirin for 7 days showed the same result, but on each day the titre of virus recovered was 10-50-fold less than that found on control ferrets.
Downloaded from http://jac.oxfordjournals.org/ at OCLC on June 21, 2015
£40
Q.
66
K. P . SchofieJd et aL
Time after infection (days) Ribavirin treatment
Concentration of nasal wash protein
Nasal washings were collected from all control and ribavirin treated ferrets on alternate days from days 5-15 after virus infection. The specimens were concentrated 10-fold and the amount of protein measured by the method of Lowry et al. (1951). The results are shown in Figure 3. The concentration of protein in nasal washings from control ferrets increased to a maximum at 5-7 days after virus infection; at this time, the concentration was 2-4 times greater than that found in nasal washings collected prior to infection. After day 7 following virus infection the concentration of protein declined and by day Control
Ribovirin -treated (x7 )
16 *l-2 _ _ rf>8 «
rr.
-
T i t r « of leutrali ring ont body
5 7 Flgur* 3
9
II
15 5 7 13 Timi o f t e r infection (day«)
16 _ Control
A ft 1*1 ft 9
II
13
15
Ribavirin - treated ( x 7 )
8
4
