Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine

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1 Antiviral Chemistry & Chemotherapy 15: Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine Donald F Smee, Miles K Wandersee, Min-Hui Wong, Kevin W Bailey and Robert W Sidwell Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT, USA Corresponding author: Tel: ; Fax: ; dsmee@cc.usu.edu Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins. The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models. When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived. Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses. Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus. Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin). When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day. Ribavirin was active at 20, 40 and 80 mg/kg/day. Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day. Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted. Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7 11 of the infection. Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered. Ribavirin alone was very effective (90 100% survival of mice) when treatments started as late as 3 days after infection. Forty percent survival was evident even when treatments started 4 days post-infection. Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time. The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model. The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies. Keywords: antiviral, influenza A, influenza B, collectins, combination chemotherapy Introduction Identifying improved treatments for influenza virus infections is important in light of their continuing annual emergence and the subsequent morbidity and mortality, particularly in elderly populations (Hayden, 1997). The threat of worldwide pandemics by new influenza viral strains is increasing due to the emergence of highly pathogenic avian influenza H5N1 and H7N7 strains in Asia and Europe, respectively (Navani, 2004; Watts, 2004). Some of these new viruses are naturally resistant to two of the four approved antiviral drugs, amantadine and rimantadine (Trampuz et al., 2004). Other approved drugs used to treat influenza include zanamivir (administered by nasal spray) and oseltamivir (given orally) (Dunn & Goa, 1999; Lew et al., 2000; Dreitlein et al., 2001). Of these, oseltamivir appears to be the preferred drug based upon ease of administration. Experimentally, ribavirin has been found to be effective in treating influenza A and B infections in mouse models (Khare et al., 1973; Durr et al., 1975; Sidwell et al., 2001). Viramidine, the 3-carboxamidine derivative of ribavirin, also shows anti-influenza virus activity in mice 2004 International Medical Press /02/$

2 DF Smee et al. (Witkowski et al., 1973), although its potency is somewhat less than that of ribavirin. One of the purposes of the present set of experiments was to develop an influenza B virus infection model using a more recent strain of virus than was used in the past for studying treatment with antiviral agents. Previously in our laboratory, we have used either influenza B/Lee/40 or B/Hong Kong/5/72 isolated in 1940 and 1972, respectively. Both of these require nearly full-strength virus preparations to cause lethality in mice. The strains are also removed immunologically from the more recent clinical isolates that cause human disease. The newest B virus strain in our possession at the onset of these investigations was B/Sichuan/379/99, isolated in This virus was studied for its ability to kill mice, and later evolved into a model for evaluation of antiviral agents. We found that infection of mice with B/Sichuan/379/99 could be greatly enhanced by co-exposure to mannan, administering the infectious virus inoculum intranasally in medium containing mannan. Mannan is a polysaccharide preparation from Saccharomyces cerevisiae that other investigators have shown will enhance infections with certain influenza A viruses (Reading et al., 1997). Virus titres were increased in mannan-treated animals relative to those exposed only to virus. The mode of action of mannan is to bind host collectins that otherwise would bind to the virus and neutralize infectivity (Reading et al., 1997; Hartshorn et al., 2000). Once we established the mouse model with B/Sichuan/379/99, the infection was then used to evaluate antiviral substances, either used alone or in combination. One way to potentially improve antiviral efficacy against influenza disease is to combine drugs with differing modes of antiviral action (Hayden 1986; Hayden 1996; Zhirnov 1987). Such a study was reported with ribavirin and the neuraminidase inhibitor peramivir (BCX-1812, RWJ ) (Smee et al., 2002). Improvements in survival were noted against an influenza A/NWS/33 (H1N1) infection in mice when the two substances were given at varying combined doses. Amantadine or rimantadine could potentially combine with oseltamivir against influenza A virus infections, although no studies have yet been reported. Since amantadine and rimantadine do not inhibit influenza B viruses, drug combination studies using inhibitors with different modes of action are restricted to using a compound such as ribavirin and a neuraminidase inhibitor. Thus, combinations of ribavirin and oseltamivir were part of the present investigations. As an extension of the studies with influenza B/Sichuan/379/99, the spectrum of influenza A and B viruses that could be enhanced in virulence when combined with mannan was also determined. Results of these experiments indicate that certain other types of influenza viruses can be studied in mouse models of infection by using mannan, viruses that otherwise caused minimal or no disease in the animals. Materials and methods Antiviral compounds Oseltamivir was purchased from a local pharmacy in capsule form. Ribavirin and viramidine (also referred to as ribamidine in early literature) were provided by ICN Pharmaceuticals (Costa Mesa, Calif., USA). The compounds were dissolved in water for oral treatment of infected mice. Mannan This material is a polysaccharide preparation from S. cerevisiae purchased from Sigma Chemical Co (St Louis, Miss., USA). It was dissolved in cell culture medium at twice its final concentration, then combined in equal volume with twice the infecting virus challenge dose for intranasal administration to anaesthetized mice. A final concentration of 10 mg/ml (0.5 mg/mouse) was used for most experiments based upon results of these studies and those reported by other investigators (Reading et al., 1997). Viruses and cells The sources of the majority of the influenza virus strains used in these experiments have been reported previously (Smee et al., 2001b). Influenza B/Sichuan/379/99 and B/Hong Kong/330/01 were obtained from the Centers for Disease Control and Prevention (Atlanta, Ga., USA). The viruses were propagated in Madin Darby canine kidney (MDCK) cells obtained from the American Type Culture Collection (Manassas, Va., USA). The cells were grown in Eagle s medium (MEM) containing 5% fetal bovine serum (FBS), 0.1% sodium bicarbonate and no antibiotics in a 5% CO 2 environment. Viral propagation and assays were done using MDCK cells in MEM, no FBS, 0.18% sodium bicarbonate, 10 units of trypsin/ml, 1 µg EDTA/ml, and 50 µg gentamicin/ml. Further passage of the B/Sichuan/379/99 virus in mice is described later in the text. Animal studies Female g specific pathogen-free BALB/c mice were obtained from Charles River Labs (Wilmington, Mass., USA). The animals were quarantined 48 h before use and maintained on standard mouse chow and tap water ad libitum. Intranasal infections were done by anaesthetizing the mice with ketamine (100 mg/kg, ip) followed by intranasal instillation of 50 µl of virus-containing solution per mouse (with or without mannan present). The viral challenge doses given for determining lethality (as indicated in Table 3) were the highest allowable, based upon International Medical Press

3 Treatment of mannan-enhanced influenza B virus in mice virus titre of the pools. For antiviral studies, the B/Sichuan/379/99 virus infecting dose was % cell culture infectious doses (CCID 50 ) combined with mannan at 0.5 mg/mouse. For antiviral studies, 10 mice per drug treatment group and animals in the placebo group were held to determine the death rate from the infection. Oral treatments for the initial experiments were twice daily for 5 days beginning 4 h pre-virus exposure. Subsequent evaluations varied the time of initiation of first treatment relative to virus challenge. The animals were observed 21 days for death. Arterial oxygen saturation (SaO 2 ) levels in infected mice were determined on days 3 11 using the Ohmeda Biox 3800 pulse oximeter (Ohmeda, Louisville, OH, USA). The ear probe attachment was used and placed on the thigh of the animal, with the slow instrument mode selected. Readings were made after a 30 s stabilization time on each animal. Mice dead on the day of reading were assigned an SaO 2 value of 75, which was the lowest level generally seen in a seriously ill animal. Use of this device for measuring effects of the influenza virus infection on SaO 2 has been previously described (Sidwell et al., 1992). On day 6 of some of the infections, groups of five mice were sacrificed for the determination of lung scores, weights and virus titres, as in previous studies (Sidwell et al., 2001). Lungs were given a consolidation score ranging from 0 4 based upon the percentage of the lung exhibiting a plum discolouration. Tissues were prepared by homogenization in 1 ml of cell culture medium in stomacher bags. Virus titrations of lung homogenates were performed in MDCK cells as previously described (Sidwell et al., 1986; Smee et al., 2001b). Statistical analysis Increases in survivor number were evaluated by the Fisher exact test. Mean day of death and lung parameters (score, weight, virus titre and SaO 2 ) differences between drugtreated and placebo groups were analysed by the Mann Whitney U-test. All analyses were two-tailed. Standard deviations were provided for mean values where indicated in tables. In one study, animal weights were determined on days 0 and 8 of the infection, and weight changes between the 2 days were calculated. Because mice were weighed as a group instead of individually, no statistical comparisons could be performed. Results These studies were initially conducted in an attempt to develop a virulent influenza B virus infection using a recent clinical isolate, which at the time of study initiation was B/Sichuan/379/99. Two passages of the virus were done in mice, with low amounts of virus recovered from lungs and no deaths observed. Then published studies were noted indicating that enhancement of influenza virus infection could be achieved with mannan (Reading et al., 1997). Incorporation of mannan into experimental influenza B/Sichuan/379/99 virus infections was subsequently initiated. The mouse passage 2 virus was lethal to mice at CCID 50 per animal when mannan was co-administered intranasally, but completely non-lethal without mannan. Three more passages of the virus were done using mannan to enhance virus titre production as was shown previously with influenza A virus (Reading et al., 1997). The fifth mouse passage pool was then titrated for lethality in mice (Table 1). The highest dose of virus that could be given to mice from this pool ( CCID 50 /animal) was not lethal when mannan was absent during virus exposure. Nearly all animals receiving a viral challenge dose of or higher with mannan (0.5 mg/mouse) died from infection. With mannan, three out of eight mice died at a virus dose of /animal. This represented over a 100-fold enhancement of virulence when mannan was co-administered during the infection. It also indicates the probable importance of host collectins (absorbed by mannan in the lethal infection) in fighting the infection. Table 1. Lethality of influenza B/Sichuan/379/99 virus in mice exposed to mannan (0.5 mg/animal) Infecting virus challenge dose Mannan exposure Survivors/total Mean day of death 5.7 8/ / /8 9.1 ± /8 8.4 ± / ± / /8 The virus underwent two passages in mice without co-exposure to mannan followed by three more mouse passages using mannan. Log 10 CCID 50 /mouse. For mice that died prior to day 21 of the infection. Antiviral Chemistry & Chemotherapy 15:5 263

4 DF Smee et al. The next experiment was designed to determine the amount of mannan necessary to induce lethality in the animals. Mice exposed to CCID 50 of virus were treated with varying amounts of mannan (Table 2). The minimum effective dose inducing complete lethality was 0.16 mg/mouse, corresponding to a concentration of 3.2 mg/ml in the virus inoculum. Uninfected mice treated with mannan at the highest dose remained healthy. Based on the results of this study, it was decided that 0.5 mg of mannan (10 mg/ml in the inoculum) would be used for all subsequent experiments. Since mannan worked well in enhancing the virulence of influenza B/Sichuan/379/99 in mice, it was of interest to determine whether the virulence of other influenza A and B viruses could be similarly enhanced. A panel of nonmouse adapted viruses was selected using the highest available virus titre (Table 3). Weight loss was measured as an indicator of disease enhancement, along with death. Mannan by itself in uninfected mice did not induce weight loss, thus it was judged to be non-toxic. Mannan enhanced disease of three influenza A (H1N1) viruses in mice based upon weight loss, with the A/New Caledonia/20/99 virus causing 80% mortality when co-administered with mannan. Four influenza A (H3N2) viruses were resistant to enhancement by mannan as indicated by minimal or no weight loss. One of three influenza B viruses was enhanced by mannan, namely B/Hong Kong/330/01. This virus, isolated 2 years after the B/Sichuan/379/99 virus in 2001, is related in virulence to the earlier virus. The lack of enhancement of virulence of certain viruses by mannan suggests that they may not be responsive to neutralization by host collectins in vivo. The antiviral studies with oseltamivir, ribavirin and viramidine were conducted with the B/Sichuan/379/99 virus infection enhanced by mannan. In the first experiment, oseltamivir and ribavirin were given twice daily for 5 days Table 2. Effects of mannan exposure at varying concentrations given at the time of infection on the severity of an influenza B/Sichuan/379/99 virus infection in mice Mannan dose, Survivors/ Mean day mg/mouse total of death 0.5 (10) 0/ ± (3.2) 0/6 9.3 ± (1.0) 2/ ± (0.32) 6/ (0.1) 6/6 ( ), mg/ml present in virus inoculum of CCID 50 /mouse. For mice that died prior to day 21 of the infection. Uninfected animals exposed to mannan (0.5 mg/mouse) were unaffected by such treatment. starting 4 h before virus challenge (Table 4). All three doses of each compound were highly protective, with % survival. Ribavirin reduced mean lung scores significantly at the two highest doses, and lung weights were reduced at three doses. Virus titres were only moderately reduced on day 6, however. Oseltamivir was less effective than ribavirin in reducing lung scores and lung weights, with minimal virus titre reduction seen on the day of assay. Arterial oxygen saturation levels were statistically significantly better than placebo for oseltamivir- and ribavirin-treated groups on days 7 11 of the infection (Figure 1). In the second experiment, viramidine was evaluated at three doses twice daily for 5 days starting 4 h before infection (Table 4). The 160 and 80 mg/kg/day doses were % protective, but the 40 mg/kg/day dose failed to have an impact on survival. Significant reductions in lung scores were noted with treatment at the two highest doses. Lung weights and lung virus titres were also reduced by these treatments but not significantly on the day of assay. Arterial oxygen saturation levels for viramidine-treated mice were greater than placebo on days 7 11, with the lowest dose causing the least beneficial effect (Figure 2). A drug combination study was conducted with oseltamivir and ribavirin, the intent being to determine how long after infection treatments could be given and still achieve a beneficial effect. Treatments were initiated as late as 5 days after infection (Table 5). In this experiment, ribavirin remained effective when treatments started as late as day 3 after virus exposure. Forty percent survival was noted when ribavirin treatments started as late as 4 days after infection. Oseltamivir had only a moderate effect with treatments starting on days 1, 2 and 3. The combination of ribavirin and oseltamivir appeared to be no better than what was seen using ribavirin alone. Discussion In previously published work, mannan was shown to enhance influenza virus infections in mice as determined by significant increases in lung virus titre production (Reading et al., 1997). The effect was attributed to the action of mannan in absorbing host collectins that would otherwise neutralize virus infectivity. We showed that mannan was able to enhance infections of mice with three strains of influenza A (H1N1) and two strains of influenza B viruses. With influenza A/New Caledonia/20/99 (H1N1), influenza B/Sichuan/379/99 and influenza B/Hong Kong/330/01, enhancement of disease resulted in death of the majority of the infected animals. Mannan-induced enhancement of infections by A/New Jersey/8/76 (H1N1) and A/Texas/36/91 (H1N1) led to severe weight loss without death. With all of the influenza A (H3N2) virus infections studied here, mannan caused no appreciable International Medical Press

5 Treatment of mannan-enhanced influenza B virus in mice Table 3. Lethality determination of influenza A and B viruses in mice exposed to mannan Infectious Mean day Host weight Virus dose Mannan Survivors/total of death change (g) Influenza A (H1N1) New Jersey/8/ / /5 3.7 New Caledonia/20/ / /5 6.3 ± Texas/36/ / /5 4.1 Influenza A (H3N2) Los Angeles/2/ / /5 0.9 Johannesburg/33/ / / Panama/2007/ / / Sydney/05/ / / Influenza B Beijing/184/ / / Harbin/07/ / /5 0.7 Hong Kong/330/ / /5 9.2 ± Uninfected 5/ / Log 10 CCID 50 /mouse. 0.5 mg/mouse present in virus inoculum. For mice that died prior to day 21 of the infection. Determined between days 0 and 8 of the infection. enhancement of disease severity. These data suggest that for these unaffected viruses, the activities of host collectins may not lead to anti-influenza virus immunity in mice. In contrast, Reading et al. (1997) showed data on other types of H3N2 viruses where neutralization by mouse or rat serum was inhibited by mannose in vitro and where higher lung virus titres were developed using mannan in vivo. Those investigations did not determine whether the same viruses combined with mannan induced signs of disease in the mice. In one of the present experiments, we were able to define more precisely doses of mannan that were effective in causing disease enhancement. Using mannan as a disease-enhancing agent, the antiviral activities of oseltamivir, ribavirin and viramidine were studied against influenza B/Sichuan/379/99 infections. Viramidine was the least potent of the compounds, its potency being about one fourth that of ribavirin. The relative potencies of ribavirin and viramidine are similar to what has been reported for the two compounds against Punta Toro virus (Sidwell et al., 1988a,b). Viramidine may be less potent, but it also exhibits decreased toxicity relative to ribavirin. Thus, the relative therapeutic indices between ribavirin and viramidine are about the same. Viramidine has been shown to distribute in the body differently to ribavirin, particularly to the liver (Lin et al., 2003, 2004). For this reason it may be more suited than ribavirin as an anti-hepatitis C virus drug, but possibly not against influenza infections. Ribavirin has been given by small particle aerosol for treatment of human respiratory syncytial virus infections (Ottolini & Hemming, 1997), and has US FDA approval for that indication. Trials with aerosolized ribavirin against influenza infections have also been conducted (Gilbert et al. 1985; Bernstein et al., 1988), but real benefits from such treatment were inconclusive. Delivery of drugs such as ribavirin by small particle aerosol has not received widespread acceptance, making orally active drugs highly sought after. Both oseltamivir and ribavirin were efficacious when given early to combat the influenza B/Sichuan 379/99 virus infection. However, the activity of oseltamivir waned considerably when treatments began after infection. In contrast, ribavirin was highly effective when starting Antiviral Chemistry & Chemotherapy 15:5 265

6 DF Smee et al. Table 4. Effects of twice-daily oral treatment with ribavirin, oseltamivir and viramidine on an influenza B/Sichuan/379/99 virus infection in mice made more severe by mannan exposure Mean lung parameters (Day 6) Compound Survivors/ Mean day (mg/kg/day) total of death Score Weight (mg) Virus titre Experiment 1: Oseltamivir (10) 10/ ± ± ±0.1 Oseltamivir (5) 8/ ± ± ± ±0.6 Oseltamivir (2.5) 9/ ± ± ± ±0.2 Ribavirin (80) 10/ ± ± ±0.4 Ribavirin (40) 10/ ± ± ±0.3 Ribavirin (20) 9/ ± ± ± ±0.7 Placebo 0/ ± ± ± ±0.4 Experiment 2: Viramidine (160) 9/ ± ± ± ±0.2 Viramidine (80) 10/ ± ± ±0.1 Viramidine (40) 1/ ± ± ± ±0.1 Placebo 0/ ± ± ± ±0.6 Mannan (0.5 mg/mouse) was administered intranasally directly with the infecting virus. For mice that died prior to day 21. Log 10 CCID 50 /g. P<0.05, P<0.01, P< Table 5. Effects of twice-daily oral treatment with ribavirin, oseltamivir or the two drugs combined, on an influenza B/Sichuan/379/99 virus infection in mice made more severe by mannan exposure Compound (mg/kg/day) Treatment starting day Survivors/total Mean day of death Oseltamivir (5) 1 6/ ±1.0 Ribavirin (40) 1 10/10 Oseltamivir (5) + ribavirin (40) 1 10/10 Oseltamivir (5) 2 3/ ±1.6 Ribavirin (40) 2 8/ ±0.0 Oseltamivir (5) + ribavirin (40) 2 10/10 Oseltamivir (5) 3 4/ ±1.7 Ribavirin (40) 3 9/ ±0.0 Oseltamivir (5) + ribavirin (40) 3 9/ ±0.0 Oseltamivir (5) 4 1/ ±1.4 Ribavirin (40) 4 4/ ±2.3 Oseltamivir (5) + ribavirin (40) 4 4/ ±1.0 Oseltamivir (5) 5 2/ ±0.9 Ribavirin (40) 5 2/ ±3.1 Oseltamivir (5) + ribavirin (40) 5 1/ ±2.1 Placebo 1 0/ ±1.6 Mannan (0.5 mg/mouse) was administered intranasally directly with the infecting virus. For mice that died prior to day 21. P<0.05, P<0.01. treatments as late as 3 days after infection, with some activity seen with treatments starting at 4 days. The results with this virus infection differ from a previous study which reported that ribavirin was not active against an influenza A/NWS/33 (H1N1) infection when treatments began 24 h after infection or later (Sidwell et al., 1998). On the other hand, oseltamivir (GS4104) was found to be effective when treatments started as late as 60 h after onset of infection with influenza A/NWS/33 (H1N1) (Sidwell et al., 1998). These data point out that different strains of influenza virus react differently to treatment by these inhibitors. In general, much less work has been done with compounds against influenza B virus infections than influenza A, particularly timing experiments like those presented here. The combination studies with ribavirin and oseltamivir were designed to determine how late in an infection the International Medical Press

7 Treatment of mannan-enhanced influenza B virus in mice Figure 1. Arterial oxygen saturation (SaO 2 ) levels during an acute influenza B/Sichuan/379/99 virus infection plus mannan (0.5 mg/mouse) in animals treated with oseltamivir (A) and ribavirin (B) Figure 2. Arterial oxygen saturation (SaO 2 ) levels during an acute influenza B/Sichuan/379/99 virus infection plus mannan (0.5 mg/mouse) in animals treated with viramidine A 90 Mean % SaO2 level B Days after virus exposure Mean % SaO2 level Days after virus exposure Oral gavage treatments were given twice-daily for 5 days starting 4 h pre-virus exposure. ( ) Normal uninfected animals; viramidine at ( ) 160, ( ) 80 and ( ) 40 mg/kg/day; ( ) placebo. Standard deviation values around each data point ranged from 3 6%. P<0.05, P<0.01, P< Mean % SaO2 level Days after virus exposure Oral gavage treatments were given twice daily for 5 days starting 4 h pre-virus exposure. (A) ( ) Normal uninfected animals; oseltamivir at ( ) 10, ( ) 5 and ( ) 2.5 mg/kg/day; ( ) placebo. (B) ( ) Normal uninfected animals; ribavirin at ( ) 80, ( ) 40 and ( ) 20 mg/kg/day; ( ) placebo. Standard deviation values at each data point ranged from 3 6%. P<0.05, P<0.01, P< combination could be given and still exhibit a beneficial effect. The concentration of each drug was in the highly effective range when given early in the course of the infection. In these experiments, ribavirin exhibited the majority of the survival benefit, and the combination of the two inhibitors appeared to be no better than ribavirin alone. Previous combination studies have focused on using suboptimal doses of inhibitors at early time points to determine whether the low dose combinations could achieve a superior response. For example, ribavirin was shown to combine well with the neuraminidase inhibitor peramivir (BCX-1812, RWJ ) in preventing mortality in mice infected with influenza A/NWS/33 (H1N1) (Smee et al., 2002). Ribavirin has not proven to be clinically effective in the treatment of influenza virus infections in humans, although it is very effective in mice, both in the mannan model presented here and in infections with mouse-adapted viruses (Khare et al., 1973; Durr et al., 1975; Sidwell et al., 2001). This should in no way negate the value of animal models in identifying candidate compounds for human testing. Ribavirin undergoes considerably greater phosphorylation to its active antiviral state (ribavirin 5 -triphosphate) in mouse cells compared with primate cells (Smee et al., 2001a), and consequently is much more potent as an antiviral agent in mouse cells than in cells of higher species. This may partly explain why the anti-influenza virus effect of the drug seen in mice was not realized in humans. The studies with mannan showed that certain viruses that were not adapted or only minimally adapted to mice could be enhanced in virulence by mannan treatment. This allowed antiviral studies to be done with one of them, the Antiviral Chemistry & Chemotherapy 15:5 267

8 DF Smee et al. B/Sichuan/379/99 virus. The infections so induced were treatable with clinically approved and/or known active antiinfluenza compounds. Thus, the use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in infection models in rodents. Acknowledgements Supported by contracts NO1-AI and NO1-AI from the Virology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The investigators adhered to the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (National Institutes of Health Publication No , revised 1985), and used facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. 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