Exacerbation of Influenza Virus Infections in Mice by Intranasal ...

Exacerbation of Influenza Virus Infections in Mice by Intranasal Treatments and Implications for Evaluation of Antiviral Drugs Donald F. Smee,a Mark von Itzstein,b Beenu Bhatt,b and E. Bart Tarbeta Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, USA,a and Institute for Glycomics, Griffith University, Southport, Queensland, Australiab

Compounds lacking oral activity may be delivered intranasally to treat influenza virus infections in mice. However, intranasal treatments greatly enhance the virulence of such virus infections. This can be partially compensated for by giving reduced virus challenge doses. These can be 100- to 1,000-fold lower than infections without such treatment and still cause equivalent mortality. We found that intranasal liquid treatments facilitate virus production (probably through enhanced virus spread) and that lung pneumonia was delayed by only 2 days relative to a 1,000-fold higher virus challenge dose not accompanied by intranasal treatments. In one study, zanamivir was 90 to 100% effective at 10 mg/kg/day by oral, intraperitoneal, and intramuscular routes against influenza A/California/04/2009 (H1N1) virus in mice. However, the same compound administered intranasally at 20 mg/ kg/day for 5 days gave no protection from death although the time to death was significantly delayed. A related compound, Neu5Ac2en (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), was ineffective at 100 mg/kg/day. Intranasal zanamivir and Neu5Ac2en were 70 to 100% protective against influenza A/NWS/33 (H1N1) virus infections at 0.1 to 10 and 30 to 100 mg/kg/ day, respectively. Somewhat more difficult to treat was A/Victoria/3/75 virus that required 10 mg/kg/day of zanamivir to achieve full protection. These results illustrate that treatment of influenza virus infections by the intranasal route requires consideration of both virus challenge dose and virus strain in order to avoid compromising the effectiveness of a potentially useful antiviral agent. In addition, the intranasal treatments were shown to facilitate virus replication and promote lung pathology.

E

valuation of new compounds for antiviral activity begins first by identifying promising inhibitors in cell culture. Compounds with sufficient activity are later studied in infected animals. Although several animal species are available that can be infected with influenza viruses (1, 10), mice are the species of first choice for initial evaluations due to cost and size (requiring less compound for testing) considerations. Initial testing for toxicity is performed in uninfected mice prior to selecting appropriately safe doses for antiviral studies. If pharmacokinetic information regarding oral bioavailability is not available in the beginning, compounds are generally tested by intraperitoneal route and given two or more times per day. In the process of preclinical development of an active antiviral agent, it may be discovered that the compound has little oral activity. At that point, testing may shift to studying the effectiveness of intranasal treatments or, less commonly, aerosol administration. Intranasal treatments of influenza virus infections in mice exacerbate the infections. This phenomenon was described by Taylor in 1941 (16), who showed that intranasal treatments postinfection resulted in increased lung virus production and greater mortality. Takano et al. (14) referred to the exacerbating effect of intranasal liquid treatment as “drowning” the mice. Taylor (16) was more accurate in suggesting that intranasal liquid helps to spread the virus inside the lungs. However, Taylor did not show progressive pathogenesis in intranasally treated mice versus untreated animals. His work mainly focused on virus titer and mortality, with a description of lung histopathology near the time of death. Takano et al. (14) found that interferon administered intranasally postinfection was ineffective against influenza virus infections, but aerosol administration resulted in improved efficacy. Studies of antivirals published in the 1990s briefly mention difficulties encountered with intranasal treatments as investigators realized they had to reduce their virus challenge dose to lessen

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the severity of the infection. In a 1994 study, Ryan et al. (7) reduced their normal dose of influenza A/Singapore/57 virus by 10-fold in order to treat mice intranasally with zanamivir. Judd et al. (4) reported an initial experiment with 106 cell culture 50% infectious doses (CCID50) of influenza A/PR/8/34 (H1N1) virus and found that the infection was quite severe. They performed a second experiment at 104 CCID50 that was more successful in demonstrating the antiviral effect of an intranasally administered zinc finger peptide (4). Having learned the necessity of reducing the virus challenge dose, Sidwell et al. (9) later used influenza A/NWS/33 (H1N1) virus in mice at a 100-fold lower dose than usual for intranasal treatment studies with zanamivir. None of the aforementioned investigations alluded to any concerns with regard to virulence of one virus strain versus another when intranasal treatments were involved. With the advent of the 2009 influenza A H1N1 pandemic virus, we began evaluating a number of antiviral compounds, including known active drugs, as controls. It was during such studies that we observed that the influenza A/California/04/2009 (H1N1) virus was essentially untreatable with intranasal zanamivir. This led to investigations of other virus strains that might be more suitable, one of which was influenza A/NWS/33 (H1N1) virus based upon the report of Sidwell et al. (9). We also wanted to better understand the effects of the intranasal liquid treatment on the infection; thus, patho-

Received 13 August 2012 Returned for modification 12 September 2012 Accepted 25 September 2012 Published ahead of print 1 October 2012 Address correspondence to Donald F. Smee, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.01664-12

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Intranasal Treatment Enhances Inﬂuenza Virus Virulence

genesis studies were performed that went beyond the work performed by Taylor (16). The results of these studies further substantiated the work of prior investigators to markedly reduce the virus challenge dose for evaluation of antiviral agents by intranasal treatment (4, 7, 9, 14). It also identified influenza A/California/04/ 2009 (H1N1) virus (and perhaps other strains of the 2009 pandemic virus) as not appropriate for use in testing intranasally administered compounds because the infections are too severe. MATERIALS AND METHODS Animals. Female 17- to 19-g BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA) for this investigation. The animals were maintained on standard rodent chow and tap water ad libitum. The animals were quarantined for at least 48 h prior to use. Viruses. Influenza A/NWS/33 (H1N1) virus was originally obtained over 30 years ago from the University of Michigan (Ann Arbor, MI). This virus, as received, was already adapted to cause lethal infections in mice. The influenza A/California/04/2009 (H1N1) virus strain was kindly provided by Elena Govorkova (St. Jude Children’s Research Hospital, Memphis, TN). The virus was adapted to mice by Natalia Ilyushina and colleagues (2) at the same institution. Influenza A/Victoria/3/75 (H3N2) virus was purchased from the American Type Culture Collection (Manassas, VA) and adapted by seven serial passages in mice. Influenza A/ Mississippi/03/2001 (H1N1) virus containing an oseltamivir resistance mutation (H274Y) in the viral neuraminidase gene was obtained from the Neuraminidase Inhibitor Surveillance Network (Melbourne, Australia). The virus was passaged seven times in mice to adapt it for virulence, and its genotype was confirmed by sequence analysis (12). A low-pathogenic avian influenza A/Duck/MN/1525/81 (H5N1) virus was obtained from Robert Webster (St. Jude Children’s Research Hospital, Memphis, TN) and required only three passages in mice to adapt it. The viruses were later propagated in Madin-Darby canine kidney (MDCK) cells, followed by titration for lethality in mice in conjunction with intranasal liquid treatments that were given twice a day (at 12-h intervals) for 5 days. These titrations were necessary to determine proper virus challenge doses under the conditions where intranasal liquid treatments exacerbate the infection. Compounds. Zanamivir was purchased from Haorui Pharma-Chem (Edison, NJ). Neu5Ac2en (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) was prepared at Griffith University (Gold Coast, Australia). Both compounds were prepared to the appropriate dosages by dilution in sterile saline. Sterile saline served as the placebo control, administered intranasally. In one study involving three different routes of drug administration (see Table 2), zanamivir-treated mice were compared to an untreated group. Experiment design for animal studies. The experiments were conducted in accordance with the approval of the Institutional Animal Care and Use Committee of Utah State University dated 20 September 2010. The work was performed in the AAALAC-accredited Laboratory Animal Research Center of Utah State University. The U.S. Government (National Institutes of Health) approval was renewed 1 April 2010 (PHS assurance number A3801-01) in accordance with the Guide for the Care and Use of Laboratory Animals endorsed by the National Institutes of Health (5). Mice were anesthetized by intraperitoneal injection of ketamine-xylazine (50/5 mg/kg) and then intranasally exposed to a 90-␮l suspension of influenza virus. Other investigators use a 50-␮l infection volume. The higher volume delivers more virus to the lungs (13) since the nasal cavity can contain only a certain amount of administered liquid. A high-dose challenge of influenza A/Victoria virus (where no intranasal treatments were given) equated to approximately 2 ⫻ 105 CCID50 per mouse. A low-dose challenge of the same virus was about 200 CCID50 per mouse. The low-dose virus challenge accompanied by intranasal liquid treatments and the high-dose virus challenge gave complete mortality and equated to approximately 6 mouse 50% lethal challenge doses (mLD50)


TABLE 1 Lethal dose comparisons between mice treated intranasally with saline and untreated animals following virus exposure Lethal challenge dose by treatment group (log10 CCID50)a Virus strain

Treated miceb

Untreated mice

A/California/04/2009 (H1N1) A/Mississippi/3/2001 (H1N1) H274Yc A/NWS/33 (H1N1) A/Victoria/3/75 (H3N2) A/Duck/MN/1525/81 (H5N1)d

1.7 1.0 2.0 1.5 1.7

3.5 3.9 4.0 4.5 3.5

a Virus challenge dose per mouse equal to 1 mLD50 (50% mouse lethal dose). Results are from single determinations conducted for the purpose of determining proper virus challenge doses to administer in future antiviral experiments. Eight animals per group and four to five virus dosage groups were used per lethality determination. b Intranasal treatments (50 ␮l) were given under anesthesia twice a day for 5 days starting 2 h prior to intranasal infection. c Oseltamivir-resistant virus containing the indicated mutation in the viral neuraminidase gene. d A low-pathogenic avian virus strain whose replication is confined to the lungs.

under the conditions of infection and treatment. Influenza A/NWS/33 (H1N1) and A/California/04/2009 (H1N1) viruses were infected at about 500 CCID50 per mouse, or 5 to 10 mLD50. For each experiment there were initially 10 mice per compound-treated group and 20 placebos. For studies involving intranasal treatments, mice were anesthetized as described above and treated with a 50-␮l volume of saline alone (placebo) or saline containing test compound. One mouse out of a group of 10 died during the early treatment phase and was excluded from the results (leaving 9 animals for the results reported in Table 2). Antiviral compounds and placebo were administered twice a day for 5 days (at 12-h intervals) starting either 2 h prior to or 4 h after virus exposure, as indicated for each table or figure. Determination of lung infection parameters. On various days after virus infection, groups of 4 to 5 mice were sacrificed and necropsied for analysis of lung parameters (lung hemorrhage scores, weights, and virus titers). The lungs were weighed on a precision balance and then scored for lung hemorrhage on a scale of 0 (normal) to 4 (maximum plum coloration over entire lung) (8) in 0.5-unit increments, followed by freezing at ⫺80°C. At a later date the lungs were homogenized and titrated for the presence of virus by the endpoint dilution method (6) in 96-well microplates (11). Virus titers are reported as log10 CCID50/g of tissue. Statistical analysis. Survival curves were initially compared by the Mantel-Cox log rank test. When statistical significance was found, pairwise comparisons of survival curves were made using the Gehan-BreslowWilcoxon test with Bonferroni’s corrected threshold of significance for multiple groups. Lung weight and lung virus titer analyses were performed by one-way analysis of variance (ANOVA) followed by TukeyKramer multiple comparisons tests. Lung hemorrhage score comparisons (nonparametric) were performed by a Kruskal-Wallis test followed by Dunn’s test for pairwise comparisons. Calculations were made using Prism, version 5.0, software program (GraphPad Software, San Diego, CA). Statistical comparisons were made between treated and placebo groups.

RESULTS

Comparison of lethal virus challenge doses in mice. Over the years we have conducted lethality titrations of viruses in mice for purposes of studying intranasal treatments and other routes of compound administration (commonly oral [by gavage] or intraperitoneal). The dose of virus required to kill 50% of mice is nearly 100 to 1,000 times lower in animals receiving intranasal liquid treatments than in nontreated mice (Table 1). A lower virus challenge dose is necessary for intranasal treatment experiments in

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order to demonstrate compound efficacy, as exemplified by the studies of Judd et al. (4). Pathogenesis of virus infections with and without intranasal treatments. Although the exacerbating effect of intranasal treatments during influenza virus infection was described decades ago by Taylor (16), experiments showing what actually occurs during such infections are lacking in detail. We chose influenza A/Victoria/ 3/75 (H3N2) virus to investigate this effect further. There is a 1,000-fold difference between the lethal virus challenge dose used with intranasal liquid treatment and the dose of virus for intraperitoneal or oral treatments. A comparison was made of viral pathogenesis in mice infected with a high virus challenge dose compared to animals receiving a low virus challenge dose but treated with intranasal liquid. A control group consisted of mice infected with the low virus challenge dose that were not subsequently treated intranasally with saline (Fig. 1). All animals died that were infected with the high dose of virus or were infected with the low dose of virus and treated intranasally with saline (Fig. 1A). Untreated mice infected with the low virus challenge dose survived through day 21 when the experiment was terminated. Body weights in groups that died had similar weight reductions (Fig. 1B), whereas weight loss was not evident in mice infected with the low-dose virus challenge that were not intranasally treated. Groups of animals were sacrificed during these infections, and their lungs were analyzed. Lung hemorrhage and lung weight increases appeared sooner in the high-dose virus challenge group (Fig. 1C and D). These parameters were delayed but eventually rose to high levels in the low-dose virus challenge group treated with saline, and they remained low in the untreated group infected with a low virus titer. Lung virus titers peaked at day 1 in mice receiving the high virus challenge dose compared to day 3 in the low-dose virus challenge group treated with saline (Fig. 1E). Virus titers in the untreated group infected with low-titer virus never reached the high levels achieved in the other groups. Zanamivir treatment of a low-dose influenza A/Victoria/ 3/75 (H3N2) virus infection in mice. Zanamivir was used intranasally to treat an H3N2 virus infection in mice challenged with a low virus inoculum. It was observed to be completely protective at 10 mg/kg/day but only partially protective at 1 mg/kg/day (Fig. 2A). Minimal body weight loss was seen in the group treated with a high dose of zanamivir (Fig. 2B). In a separate experiment, a 30-mg/kg/day dose of zanamivir was 100% protective whereas all mice that received placebos died by day 9 (data not shown). Thus, this infection was treatable with intranasally administered zanamivir. Treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections. Initially, we investigated the treatment of influenza A/California/04/2009 H1N1 virus infection by intraperitoneal, intramuscular, and oral (by gavage) routes (Table 2). A 10-mg/kg/day dose of compound was 90 to 100% protective by any of these regimens, but lower doses were less effective or ineffective. These results demonstrate that zanamivir administered by different routes can be used effectively to treat a pandemic H1N1 virus infection in mice. Subsequently, a study was performed in which zanamivir (also known as 4-deoxy-4-guanidinoNeu5Ac2en) was compared to Neu5Ac2en by the intranasal treatment route. Surprisingly, neither compound was able to prevent death of the animals (Fig. 3A) although there was a significant delay in the time to death in the zanamivir (20 mg/kg/day) group. In a separate study, intranasal zanamivir at 10 mg/kg/day resulted

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FIG 1 Effects of intranasal saline treatment on survival (A), body weight (B), lung hemorrhage score (C), lung weight (D), and lung virus titer (E) during an influenza A/Victoria/3/75 (H3N2) virus infection in mice. Saline was administered twice a day for 5 days starting 2 h prior to infection. The low-dose virus challenge equated to approximately 200 CCID50/mouse, and the high-dose virus challenge was 1,000 times higher at 2 ⫻ 105 CCID50/mouse. Mice infected with the low virus challenge dose and not treated intranasally (i.n.) with saline survived until the end of the study period (day 21). For panels A and B, the low-virus inoculum group had 10 mice. The other two groups were averaged from two studies of 20 mice per study. In panels C, D, and E there were 4 to 5 mice per time point sacrificed for evaluation of lung parameters, except for day 9 where only 1 or 2 animals were available in groups experiencing mortality from the infection. Standard deviation error bars are shown in panels B to E.

in no protection from death although there was a statistically significant delay in the time to death (data not shown). An intranasal dose of 50 mg/kg/day of zanamivir had no effect in reducing lung virus titers and had only a minimal effect on lung hemorrhage scores and lung weights (Table 3). In addition, Neu5Ac2en treatments up to 200 mg/kg/day were completely ineffective. All groups had very high virus titers that we attribute to enhancement of the infection by intranasal treatment since lung virus titers did not achieve this high level in mice treated by other routes of administration (15). Treatment of seasonal influenza A/NWS/33 (H1N1) virus infection. Because of the unexpected results with the A/California/ 04/2009 (H1N1) virus infection, a separate study comparing zanamivir with Neu5Ac2en was performed using influenza A/NWS/33 (H1N1) virus. Previously Sidwell et al. (9) determined that zana-



FIG 2 Effects of zanamivir on survival (A) and body weights (B) during an influenza A/Victoria/3/75 (H3N2) virus infection in mice. Intranasal treatments were administered twice a day for 5 days starting 4 h after virus exposure. Mice were infected with a low intranasal virus challenge dose (approximately 200 CCID50/mouse). There were 10 mice per group treated with zanamivir and 20 placebos. Standard deviation error bars are shown in panel A.

FIG 3 Differential efficacy of zanamivir and Neu5Ac2en against influenza A/California/04/2009 (H1N1) (A) and influenza A/NWS/33 (H1N1) (B) virus infections in mice when administered by the intranasal route. Treatments were administered twice a day for 5 days starting 2 h before virus exposure. Mice were infected with low intranasal virus challenge doses (approximately 200 CCID50/mouse). There were 10 mice per group treated with antiviral compound and 20 placebos.

mivir was effective against this virus strain when a reduced virus challenge dose was given. Here, intranasal zanamivir provided complete protection from death at doses of 1 and 10 mg/kg/day, with 60% protection at 0.1 mg/kg/day (Fig. 3B). Neu5Ac2en was 70 and 100% protective at doses of 30 and 100 mg/kg/day, respectively. Lung infection parameters were determined on day 5 of the infection. Significant decreases in lung hemorrhage scores and lung weights were found in groups treated with zanamivir and

Neu5Ac2en compared to the placebo (Table 4). The highest two doses of zanamivir reduced lung virus titers significantly. The placebo group’s influenza A/NWS/33 (H1N1) virus titers in lungs were nearly 100-fold lower than those in lungs of mice infected with the A/California/04/2009 (H1N1) virus (Table 3). This may help explain why the former virus is more treatable with zanamivir and Neu5Ac2en than the latter virus. DISCUSSION

TABLE 2 Effects of zanamivir by three different systemic routes on survival during an influenza A/California/04/2009 (H1N1) virus infection in mice Treatment and routea Zanamivir (mg/kg/day) Intraperitoneal 10 3 1 Intramuscular 10 3 1 Oral gavage 10 3 1 Untreatedb

No. of survivors/ total no. of micec

Mean day of death ⫾ SDc

9/9** 2/10 1/10

14.0 ⫾ 2.2** 9.4 ⫾ 1.9

9/10 1/10 3/10*

13.0 12.0 ⫾ 5.1 10.0 ⫾ 3.2*

10/10** 3/10* 0/10

11.0 ⫾ 2.7* 9.7 ⫾ 1.0

1/20

8.6 ⫹ 3.7

a

Treatments were given twice a day for 5 days starting 4 h after virus exposure. These mice were not placebos because they were not treated since many different routes of zanamivir treatment were performed. c *, P ⬍ 0.05; **, P ⬍ 0.001 (compared to placebo). b


The studies presented here demonstrate that two subtypes of influenza virus exhibit the property of being more lethal to mice when intranasal saline treatments are administered daily after virus exposure. This was first investigated by Taylor (16), who observed enhanced virus replication and greater mortality in inTABLE 3 Effects of intranasal treatment with zanamivir and Neu5Ac2en on lung infection parameters during an influenza A/California/04/2009 (H1N1) virus infection in mice Mean value for the parameter ⫾ SD (day 6) Compound (dose [mg/kg])a

Hemorrhage scorec

Weight (mg)b

Virus titer (log10 CCID50/g)

Zanamivir (50) Neu5Ac2en (200) Neu5Ac2en (100) Neu5Ac2en (50) Placebo

2.5 ⫾ 1.5 3.0 ⫾ 1.0 3.8 ⫾ 0.3 3.6 ⫾ 0.5 3.7 ⫾ 0.4

186 ⫾ 43* 340 ⫾ 63 362 ⫾ 74 354 ⫾ 35 358 ⫾ 43

8.5 ⫾ 0.5 8.6 ⫾ 0.4 8.5 ⫾ 0.7 8.8 ⫾ 0.2 8.6 ⫾ 0.4

a Dose per treatment, given intranasally twice a day for 5 days starting 2 h prior to virus exposure. b Lung weights of uninfected mice weigh approximately 130 –140 mg. *, P ⬍ 0.001, compared to placebo. c Scores are based on a scale of 0 (normal) to 4 (maximum plum coloration over entire lung) in 0.5-unit increments.

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TABLE 4 Effects of intranasal treatments with Neu5Ac2en and zanamivir on lung infection parameters during an influenza A/NWS/33 (H1N1) virus infection in mice Mean value for the parameter ⫾ SD (day 5)c Compound (dose [mg/kg/day])a

Hemorrhage scored

Weight (mg)b

Virus titer (log10 CCID50/g)

Zanamivir (10) Zanamivir (1) Zanamivir (0.1) Neu5Ac2en (100) Neu5Ac2en (30) Placebo

0.8 ⫾ 0.2** 0.0 ⫾ 0.0** 0.6 ⫾ 0.2** 0.5 ⫾ 0.3** 0.6 ⫾ 0.2** 3.8 ⫾ 0.4

148 ⫾ 12*** 138 ⫾ 13*** 180 ⫾ 24*** 178 ⫾ 12*** 190 ⫾ 22*** 343 ⫾ 33

2.2 ⫾ 0.6*** 4.3 ⫾ 0.8*** 6.5 ⫾ 0.3 6.4 ⫾ 0.4 6.8 ⫾ 0.5 6.7 ⫾ 0.5

a

Intranasal treatments were given twice a day for 5 days starting 2 h prior to virus exposure. b Lung weights of uninfected mice are approximately 130 to 140 mg. c **, P ⬍ 0.01; ***, P ⬍ 0.001 (compared to placebo). d Scores are based on a scale of 0 (normal) to 4 (maximum plum coloration over entire lung) in 0.5-unit increments.

fected mice treated intranasally with saline. Apparently, influenza virus infections in mice primarily remain localized when virus challenge doses are low. As the virus challenge dose increases, the locations of infection also increase. At a certain threshold of virus challenge dose, the inflammatory response with accompanying fluid infiltration ensues and helps to spread the virus in the lungs. Low doses of virus below this threshold will not cause this effect, and the animals do not get severely ill. When mice are infected with a low challenge dose and then are given daily doses of intranasal liquid, this treatment acts to spread the virus infection rapidly, and the ensuing inflammatory response further exacerbates the infection, leading to lung consolidation and death. We used influenza A/Victoria/3/75 (H3N2) virus to study the pathogenesis of virus infection with high and low challenge doses to show the effects of intranasal saline treatments on the low-dose-challenged mice. The mortality and weight loss curves for the two infections were very similar (Fig. 1A and B). There were lags in the peak of virus production in the low-dose infection, but within a couple of days lung hemorrhage scores, lung weights, and lung virus titers were essentially the same as in the high-dose challenge group. Lung virus titers in the low-dosage group that was untreated never reached the threshold level to induce much lung hemorrhage or lung weight increase. Increase of virus titer in the low-dose challenge group treated with saline was about 100-fold, which was what Taylor (16) observed. These studies do not exclude the possibility that twice-daily anesthesia at 12-h intervals (given prior to intranasal treatments) may weaken the animals and cause some exacerbation of the infection and an earlier time to death. To prove this would require virus titrations involving infection followed only by twice-daily doses of anesthetic. Other scientists concur with our view that intranasal liquid causes exacerbation of the disease (4, 14, 16). In support of this hypothesis, it should be noted that fewer intranasal treatments than we gave (resulting in less stress from repeated anesthesia administration) induced a similar exacerbating effect on the disease process (4, 14, 16). Various investigators have reduced the virus challenge dose when intranasal treatments were given (4, 7, 9). Investigators who did not reduce the virus challenge dose reported reduced antiviral efficacy of their test materials (4, 14). In this report, zanamivir was effective by the intranasal route against a low-dose virus challenge

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of mice with influenza A/Victoria/3/75 (H3N2) virus. An equivalent dose of 10 mg/kg/day of zanamivir protected mice infected with influenza A/California/04/2009 (H1N1) virus by intraperitoneal, intramuscular, and oral routes. Originally, we included oral zanamivir, thinking that it would be a negative control, but antiviral activity was found. To our knowledge this is the first published work demonstrating oral activity of zanamivir in a mouse model. This is not relevant to humans since the drug is poorly absorbed orally. Zanamivir was ineffective (except for delaying the time to death) when administered intranasally to combat the A/California/04/2009 (H1N1) virus infection in mice at 20 mg/kg/ day. The related compound Neu5Ac2en was ineffective at 100 mg/kg/day. Yet the influenza A/NWS/33 (H1N1) virus infection was readily treatable with zanamivir (9) and Neu5Ac2en. Currently, the influenza A/California/04/2009 (H1N1) virus seems to be the pathogen of choice for evaluating new antiviral compounds. However, we strongly recommend against using it for intranasal treatment studies because an active compound may be doomed to fail in this model when multiple treatments are given. For reasons not understood, the virus replicates to high virus titers under these conditions whereas the A/NWS/33 (H1N1) virus does not. We note in a publication by Itoh et al. (3) that single daily doses of zanamivir (8 mg/kg/day for 5 days starting 1 h after virus exposure) reduced influenza A/California/04/2009 (H1N1) virus titers significantly on days 3 and 6 compared to a placebo given one time only. The authors used a virus that was not mouse adapted, and the indicated virus challenge dose was reported to be well below an mLD50. No survival or weight loss data were reported. Thus, those results cannot be directly compared with ours. The experiment with influenza A/NWS/33 (H1N1) virus demonstrates clearly what has been observed over the years with influenza virus infections in mice. That is, reduction in virus titer is not a direct correlation to survival, particularly when measured at a single time point. Table 4 shows that two doses of zanamivir reduced virus titers significantly on day 5 of the infection. All doses of zanamivir or Neu5Ac2en reduced lung hemorrhage scores and lung weights significantly, and all doses caused significant reductions in mortality (Fig. 3B). Had virus titers been evaluated earlier in the infection, there likely would have been some decrease in lung virus titers in animals treated with zanamivir and Neu5Ac2en. A few recommendations are provided to investigators studying antiviral compounds by intranasal treatment route. Although we have not studied the following to much extent, it seems logical that the number of intranasal treatments given postinfection will alter the infection. A single intranasal treatment per day may cause an infection to proceed at a lower rate than two treatments per day. An infection where treatments are given only for 2 days after infection may proceed more slowly than an infection where treatments are given for 4 days. Thus, prior to conducting the experiment, it is important to titrate the virus for lethality by giving intranasal saline under the future treatment regimen. Second, we have found that due to the weakened condition of the mice and the necessity to administer intranasal treatments to anesthetized mice, treatments are not recommended to be given after the fourth day of infection. Otherwise, some of the mice will not survive the treatment. Finally, in a study where one compound is given intranasally and another compound is administered by a



different route (e.g., orally), treat all mice by both routes, including placebos. These studies serve to highlight not only that it is important to select a proper low virus challenge dose for intranasal treatment experiments but also that the choice of virus strain may have a great influence on outcome. Virus replication kinetics favor one virus over another in these circumstances of intranasal treatment. The more aggressive the virus, the harder it will be to treat with an antiviral agent. Therefore, a compound with good virus-inhibitory potential may be discarded due to poorly designed or controlled intranasal experiments. ACKNOWLEDGMENTS This project was funded in part with federal funds from the Virology Branch and the Respiratory Diseases Branch, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract numbers N01-AI-30063 (awarded to Southern Research Institute) and HHSN272201000039I/HHSN27200005/A37. M.v.I. gratefully acknowledges the financial support of the Queensland State Government and the Griffith Enterprise Investment Fund.

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