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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Virology. Author manuscript; available in PMC May 12, 2009.
Published in final edited form as:
PMCID: PMC2680697
NIHMSID: NIHMS47611

Injectable Peramivir Mitigates Disease and Promotes Survival in Ferrets and Mice Infected with the Highly Virulent Influenza Virus, A/Vietnam/1203/04 (H5N1)

Abstract

The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16-18 day period resulted from peramivir treatment, with improved survival of 40-64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets.

Keywords: Influenza A virus, avian influenza, pathogenicity, antiviral agents, animal model, virulence, peramivir

Introduction

Influenza A viruses are negative-sense, single-stranded segmented RNA viruses that belong to the virus family Orthomyxoviridae. This segmented genome structure allows influenza viruses to evolve by reassortment of gene segments exchanged between different influenza A strains, allowing highly virulent variants to emerge in birds and swine and to cross species barriers to infect humans (Ellis et al., 2004; Neumann and Kawaoka, 2006; Normile, 2006; Shinya et al., 2005; Sturm-Ramirez et al., 2004; Subbarao and Katz, 2000; Webster, Shortridge, and Kawaoka, 1997). Over 330 human cases of highly pathogenic avian influenza of the H5N1 subtype in Southeast Asia in the periods 1997 and 2003-2005 and in Turkey and the Middle East in 2005-2006, have been confirmed by the World Health Organization (Beigel et al., 2005; WHO, 2006b; 2006c). Of these cases, over 60% were fatal (WHO, 2006b). The avian H5N1 influenza viruses that are currently circulating in Asia represent the most immediate threat of a possible influenza pandemic (Cinatl, Michaelis, and Doerr, 2007a; Lee et al., 2006; Poland, Jacobson, and Targonski, 2007).

Encephalitis, as a direct complication of human influenza A viral infections is not very common (Boyd et al., 2006). However, in contrast to the limited virulence of epidemic human influenza A viruses, some avian H5N1 viruses have proven to be highly pathogenic in humans (de Jong et al., 2005a) as well as in various naturally and experimentally infected birds and mammals, e.g., ferrets, mice, cats and other exotic animals (Kuiken et al., 2006a; Kuiken et al., 2006b; Kuiken et al., 2004; Kuiken et al., 2003; Rimmelzwaan et al., 2006; van Riel et al., 2006; Zitzow et al., 2002) and have caused systemic infections with involvement of the central nervous system (Beigel et al., 2005; Lee et al., 2006; Lye, Ang, and Leo, 2007). This highlights the prospect for continued emergence of neuroinvasive and/or neurovirulent avian influenza A variants that are capable of crossing species barriers to infect humans.

Further H5N1 pathogenesis studies are warranted, due to unique observations in cases of human infection with avian influenza: 1) a high incidence of diarrhea (in 80% of cases), which occurs prior to the onset of respiratory symptoms (Beigel et al., 2005), and 2) case presentation with diarrhea in the absence of respiratory symptoms, followed by the rapid onset of coma. Several vaccine candidates against highly pathogenic avian influenza A H5N1 viruses have been developed and are currently undergoing clinical trials (WHO, 2006a). Manufacturing of influenza vaccines, however, represents a substantial limitation to the timely production of vaccine stocks for use during the outbreak of avian influenza (Cox, 2005; Gerdil, 2003; Hampson, 2006). Currently, the only treatment options for potential use in epidemic avian influenza are antiviral drugs developed for seasonal human influenza viruses. Only two classes of antiviral drugs are approved for influenza A treatment, M2 inhibitors (amantadine, rimantadine) and neuraminidase inhibitors (oseltamivir, zanamivir) (Gubareva, Kaiser, and Hayden, 2000). The influenza virus neuraminidase inhibitor, oseltamivir carboxylate (Tamiflu, Roche Laboratories, Inc.; Nutley, NJ) is currently approved for use in humans > 1 year of age for treatment of uncomplicated acute (within 2 days of the occurrence of symptoms) seasonal influenza (FDA, 2006) and is administered orally as an inactive prodrug (Mendel et al., 1998). Oseltamivir has been used for treatment of human cases of avian influenza, with variable results (Beigel et al., 2005). In addition, inhaled zanamivir is approved for prophylaxis or treatment of seasonal influenza (Jefferson et al., 2006).

Development and testing of alternatives to the use of oral and/or inhaled treatments are necessary due to the limitations of using inhaled treatments to halt an epidemic as well as due to potential for an elevated rate of drug resistance to occur following widespread usage of the available antivirals (Cinatl, Michaelis, and Doerr, 2007b; Hurt, Ho, and Barr, 2006). In addition, studies of the efficacy of these approved anti-viral drugs against highly pathogenic avian influenza A H5N1 variants are limited (Govorkova et al., 2007; Govorkova et al., 2001; Gubareva, Kaiser, and Hayden, 2000; Leneva et al., 2000; Yen et al., 2005) and further dosing schedules to optimize these treatment regimens in animal models of H5N1 are necessary to provide treatment guidelines for clinicians and to present appropriate drug supply recommendations to public health and government officials. Inbred mice (female BALB/c strain) and outbred ferrets (Mustela putorius furo) represent widely accepted animal models for the study of influenza A. Studies of oseltamivir treatment of six-week-old female mice infected intranasally with 5 times the mouse fifty percent lethal dose of influenza A H5N1 (A/Vietnam/1203/04), indicate that an eight day course of treatment, but not a five day course, protected mice from lethal disease (Yen et al., 2005). Several studies suggest that the influenza A H5N1 isolate from a fatal human case in 2004 (A/Vietnam/1203/04) may be more virulent than certain 1997 Hong Kong isolates and that rapid infection of and replication in the central nervous system may account for this difference (Bright et al., 2003; Dybing et al., 2000; Gao et al., 1999; Katz et al., 2000; Leneva et al., 2000; Maines et al., 2005; Rowe et al., 2003; Zitzow et al., 2002). Due to the highly virulent, rapid systemic dissemination of highly pathogenic H5N1 avian influenza, particularly CNS involvement, an injectable neuraminidase inhibitor may provide a more efficient means of inhibiting the primary replication and multi-organ spread of the highly lethal influenza virus, thereby alleviating illness and ultimately promoting survival.

Peramivir (BCX-1812) is a cyclopentane analogue that is structurally different from zanamivir and oseltamivir, and is a potent and selective inhibitor of influenza NA (Barnard, 2000). Previously, it was demonstrated that orally-administered peramivir is active against various influenza A and B viruses, including H5N1 viruses, in vitro, in vivo (Babu et al., 2000; Bantia et al., 2001; Drusano et al., 2001; Govorkova et al., 2001; Sidwell et al., 2001; Smee et al., 2001) and in human challenge experiments. (Barroso et al., 2005). In order to overcome limited bioavailability, parenteral formulations of peramivir are currently under preclinical and clinical investigation. Recent studies demonstrate that a single i.m. injection of peramivir (10 mg/kg) fully protects against lethal outcome, and is equivalent to five days oral oseltamivir therapy in the influenza A/H1N1 mouse model (Bantia et al., 2006).

At present, no published information about the efficacy of injectable peramivir against highly pathogenic avian influenza A H5N1 is available. Thus, we investigated the therapeutic efficacy of a modified dosing schedule of peramivir delivered by i.m. injection in influenza A/Vietnam/1203/04 (H5N1)-infected mice and ferrets. This isolate was shown to be highly virulent in experimental infection of BALB/c mice and in 3-5 month-old ferrets (Govorkova et al., 2005; Maines et al., 2005) and can cause lethal infection in both animal species without prior adaptation. Here, we report on the post-exposure efficacy of peramivir in the ferret model. To characterize the disease in ferrets caused by this virus, we have performed multiple experiments with both cell- and egg-grown virus prior to evaluating peramivir. Herein, we describe the pathogenesis of this H5N1 strain in mice and ferrets, and the effects of peramivir treatment on survival and mitigation of disease.

Results

Post-exposure therapeutic efficacy of intramuscular peramivir against influenza A H5N1 (A/Vietnam/1203/04) in the mouse model

Pharmacokinetic analysis indicated that the parenteral formulation of peramivir was rapidly introduced into the circulation of these mice following intramuscular inoculation (Fig. 1A). In addition, peramivir was effective in inhibiting the NA activity of influenza A/Vietnam/1203/04 in vitro (Hurt et al., 2006). In six-week-old mice, we evaluated survival and disease development following i.n. infection with H5N1 (1 × 10-1 - 1 × 103 TCID50 per animal). The post-exposure therapeutic efficacy of antiviral drug treatment was subsequently evaluated by measurement of survival (Fig. 1B), disease severity (Fig. 1C & 1D), body temperature (Suppl. Fig. 1A), and body weight (Suppl. Fig. 1B) of 10-week-old mice infected with influenza A H5N1 (A/Vietnam/1203/04) virus via intranasal (i.n.) route. For this peramivir efficacy study in mice, we selected a dose that killed 75% of the mice in our H5N1 dose-response studies (data not shown), which was equivalent to 6 × 102 TCID50 based on back-titration of the inoculum. At the end of the 15 day study period, animal survival was 70-80% in the groups receiving either a single or multiple doses of peramivir, and 70% in the oseltamivir treatment group, as compared to 36% in control (vehicle) group (Fig. 1B). The difference in median survival between peramivir or oseltamivir treatment and control groups was not statistically significant. Peramivir treatment marginally reduced the frequency of paralysis (Fig. 1C). The encephalitis rate was 20% (2 of 11) for the single dose peramivir group and 30% (3 of 11) for the multiple dose peramivir group in comparison to 27% (3 of 11) in the control, but was even higher for the oral oseltamivir treated mice (50%, 5 of 10). However, the disease onset occurred later for the peramivir-treated mice, at 11 dpi vs. 8 dpi (Suppl. Fig. 1C). An interesting finding was that H5N1-infection resulted in death of mice in the absence of apparent disease symptoms and this occurred with a lower frequency among peramivir- treated mice in either single or multi-dose groups (10%, 1/10) compared to the untreated control group (36%, 4/11) and did not occur in the oseltamivir group. Body weight loss 1-2 days prior to death of these mice was similar for the control (21-23% of baseline) and the single dose peramivir treatment groups, but was not as pronounced (loss of 15%) for the multiple dose peramivir group (Suppl. Fig. 1C).

Fig. 1
Effects of post-exposure peramivir treatment on survival and disease development in mice infected with influenza A H5N1. A) Pharmacokinetic (PK) analysis of peramivir in mice

Body weight trends, including detailed body weights for individual animals, as well as summary data by treatment groups, are presented in Suppl. Fig. 1. To facilitate comparison of the disease development in H5N1-infected mice with published studies, we have included analysis of body weight loss as severe disease, which we define as the loss of ≥20% of their initial body mass (Fig. 1D). In prior reports of influenza drug efficacy, animals were euthanized when loss of >25% of their original body weight was recorded (Bright et al., 2003; Dybing et al., 2000; Gao et al., 1999; Katz et al., 2000; Leneva et al., 2000; Maines et al., 2005; Rowe et al., 2003; Zitzow et al., 2002). We evaluated the effect of peramivir treatment on disease outcome, with severe disease similarly defined as ≥20% loss of body weight. Severe disease over the 15 day monitoring period was significantly reduced in the treatment groups, as compared to the control group (logrank test; p=0.0006). Although further studies in this mouse model are required to demonstrate a statistically significant effect on mortality, either a single or multiple doses of parenteral peramivir improved the outcome of infection with highly pathogenic avian influenza.

Pathogenesis of avian influenza A H5N1 virus (A/Vietnam/1203/04) in ferrets

Initial fifty-percent lethal dose (LD50) studies were performed using virus grown in cell culture. Intranasal inoculation resulted in dose-dependent lethality and severity of the disease (LD50 between 1 × 102 and 1 × 103 TCID50), with signs of encephalitic disease occurring between day 4 and 10 (data not shown). This provided the basis for dose selection for the first peramivir trial in ferrets (trial 1, detailed below). To achieve a higher titer stock for use in the antiviral trials, the virus was grown in eggs, sequence confirmed (Suppl. Virus Sequencing) and the lethality confirmed by a repeat LD50 study (Fig. 2). In this LD50 study, however, ferrets were euthanized upon development of incapacitating disease, e.g., paralysis. Fifty-seven percent of ferrets infected with a dose of 1.89 × 102 or 1.89 × 104 TCID50 developed paralysis by day 10; of ferrets that received the lowest dose (1.89 × 102 TCID50), 100% developed paralysis by day 10. The latter stock was utilized for subsequent peramivir trials (trial 2 and 3, detailed below), and a higher dosage was used for these ferret infections.

Fig. 2
Dose-dependent survival and disease development in ferrets infected with influenza A H5N1. A) Cell-grown stock

Post-exposure therapeutic efficacy of intramuscular peramivir against influenza A H5N1 (A/Vietnam/1203/04) in the ferret model

The post-exposure therapeutic efficacy of peramivir was also evaluated in outbred animals (ferrets), as pharmacokinetic analysis of peramivir in ferrets showed rapid uptake into the circulation following i.m. inoculation (Fig. 3). For the peramivir efficacy studies in ferrets, we selected a dose at which >75% of the ferrets died or developed paralysis, equivalent to >1 × 103 TCID50 based on back-titration of the inoculum. Pre-inoculation hemagglutination inhibition test (data not shown) and hematological analysis (white blood cell count, WBC, data not shown) indicated that these ferrets were not naturally infected with a circulating strain of influenza and that the animals did not have any abnormal hematological changes prior to infection.

Fig. 3
Pharmacokinetic (PK) analysis in ferrets

Trial 1

The therapeutic efficacy of i.m. peramivir was evaluated by measurement of survival (Fig. 4A) and disease development (Table 1) of seven-week-old female ferrets infected with 1.5 × 103 TCID50 of influenza A H5N1 (A/Vietnam/1203/04) virus via intranasal (i.n.) route prior to peramivir treatment. The percent survival of peramivir-treated ferrets was higher than in the control group; 86% in peramivir treatment group, as compared to 43% in control (vehicle) group (Fig. 4A). By day 5, disease symptoms were present and/or death occurred, with or without paralysis/encephalitis in the control group. The difference in survival between the peramivir and control groups was not statistically significant. Summary disease data is shown in Table 1. In the peramivir treatment group, one of seven ferrets developed encephalitis and/or paralysis (Table 1) and died the following day (data not shown). In vehicle treatment group, two of seven ferrets developed paralysis and/or encephalitis (Table 1) and died on the same or a subsequent day (data not shown); two of seven ferrets died without development of encephalitis/paralysis.

Fig. 4
Effects of post-exposure peramivir treatment on survival in six- to eight-week-old ferrets infected with influenza A H5N1. Survival curves are shown for animals tested in three independent post-exposure efficacy trials: A) trial 1, B) trial 2, and C) ...
Table 1
Effects of post-exposure peramivir treatment on disease development in six-to eight-week-old ferrets infected with influenza A H5N1

Trial 2

A second trial of post-exposure peramivir efficacy in seven-week-old female ferrets was performed similarly to trial 1, but using a higher H5N1 challenge dose (1.5 × 104 TCID50) and a smaller number of ferrets. In this trial (Fig. 4B), there was a statistically significant difference in the median survival time between groups (logrank, p=0.0041). Survival was 75% for H5N1-infected peramivir-treated animals, as compared to 11% for untreated animals infected at the same dose (Fig. 4B). Peramivir extended the survival time with the majority of treated animals surviving the entire 16-day study period, whereas the median survival time for untreated H5N1-infected animals was 5 days. Disease development is summarized in Table 1. In the untreated group, encephalitis onset occurred between day 3 and 8, and in one case, persisted up to day 11 post-infection; paralysis developed either concurrently (majority of ferrets) or within 3 days of encephalitis (one ferret). By the end of the observation period of 16 days, 44% of untreated ferrets developed paralysis (Table 1), whereas none of the peramivir treated animals developed paralysis. Fewer symptoms were observed among peramivir-treated ferrets, and only 2 of 8 ferrets (25%) developed encephalitis; encephalitis also occurred relatively later than for the untreated group: one on day 5 and the other on day 7. For untreated ferrets, a variety of symptoms, e.g., ataxia, seizure, febrile and/or moribund animals, occurred between day 2 and 10 post-infection (data not shown). In the control group, 100% of animals inoculated with uninfected egg stock survived and no disease development was observed (data not shown).

Trial 3

A third trial of post-exposure peramivir efficacy was performed with seven-week-old ferrets and a similar H5N1 challenge dose to trial 2 (1.7 × 104 TCID50). In this trial, there was a statistically significant difference in the median survival time between groups (logrank, p=0.0373). Survival was 70% for H5N1-infected peramivir-treated animals, as compared to 30% for vehicle-treated animals infected at the same dose (Fig. 4C). Peramivir extended the survival time with the majority of treated animals surviving the entire 18-day study period, whereas the median survival time for vehicle-treated H5N1-infected animals was 8 days. Disease development is summarized in Table 1. Disease development occurred at a lower rate for the peramivir-treated group, with 5/10 (50%) and 1/10 (10%) developing encephalitis and paralysis, respectively, in contrast to the encephalitis and paralysis rate for vehicle-treated controls of 7/10 (70%) and 6/10 (60%), respectively (Table 1). Among the vehicle-treated controls, encephalitis onset occurred between day 3 and 9; paralysis developed either concurrently or within 2-5 days of encephalitis (data not shown). For the peramivir treatment group, the peak percentage of encephalitic animals was 30% on day 7 whereas for the vehicle-treated group, the peak percentage was 40% on day 4-5; for the peramivir-treated group, the peak percentage of paralyzed animals was 10% on day 10, whereas the peak for the vehicle-treated group was 30% on day 5 (data not shown).

We evaluated the level of infectious virus at 4 and 6 days following H5N1-infection in the lungs, brain and nasal washes of peramivir- and vehicle-treated ferrets, which were randomly pre-selected (N=5/group, Fig 5). In nasal washes, the rate of virus detection among peramivir-treated ferrets was identical to that of the vehicle-treated control group: 100% negative (5/5) on day 4 and 80% negative (4/5) on day 6; however, for the vehicle-treated ferret, the titer was 75-fold higher (1 × 105 TCID50/g) in comparison to the peramivir-treated ferret (1.3 × 103 TCID50/g). For peramivir-treated ferrets, virus was undetectable in lung or brain on either day 4 or day 6 post-infection (5/5, 100% negative). In contrast, the average titer for the vehicle-treated ferret brains on day 4 and 6 was 2.6 × 106 and 3.4 × 105 TCID50/g, respectively. In the brains of vehicle-treated ferrets, on day 4, 40% (2/5) of the mock-treated ferrets tested positive and on day 6, 20% (1/5) tested positive. For vehicle-treated ferrets, 60% (3/5) of the lungs were positive on day 6 and the average lung titer was 2.5 × 104 TCID50/g. These results indicate that the virus spread systemically from the initial site of inoculation (nasal cavity) and virus was not as frequently detected in the lungs or brain of peramivir-treated ferrets. Of the six ferrets that developed paralysis during the study (Table 1), four developed paralysis by day 5-6; the two others were paralyzed on day 8-9. The former were euthanized at the time of death and the organ titers evaluated. For these vehicle-treated ferrets, the average titers for brains, lungs and nasal washes were 4.2 × 109 TCID50/g, 2.9 × 107 TCID50/g and 3.2 × 103 TCID50/ml, respectively. In contrast, virus was undetectable in the lungs, brains or nasal washes of any of the ferrets that survived to day 18 post-infection.

Fig. 5
Effects of post-exposure peramivir treatment on levels of infectious virus in the lungs, brain and nasal washes in six- to eight-week-old ferrets infected with influenza A H5N1

Discussion

Recent outbreaks of highly pathogenic avian influenza A viruses and their high lethality in infected humans have re-emphasized the importance of effective antiviral drugs for the protection of human population in the case of a pandemic (Beigel et al., 2005;2006c). Multiple approaches are ongoing to develop antiviral drugs and vaccines against H5N1 viruses (Stephenson et al., 2004). At the time of our studies, no preclinical or clinical trial had yet been published demonstrating efficacy against A/Vietnam/1203/04 isolate, one of the most virulent human isolates. Recently, oral oseltamivir has been shown to be efficacious against this isolate (Govorkova et al., 2007). In this study presented here, we have evaluated the post-exposure therapeutic efficacy of injectable peramivir, which is an effective neuraminidase inhibitor in vitro, against the human H5N1 isolate (A/Vietnam/1203/04) in mice and ferrets. We present encouraging data in two animal models; in ferrets, two of three independent trials of i.m. peramivir treatment resulted in a significant improvement of 40-63% in survival following H5N1-infection. Surprisingly, shedding of virus in the nasal washes of the ferrets was low overall in this study, in comparison to published data (Govorkova et al., 2007). This may be explained by several factors that differ in our study from this prior publication: 1) the lower dose of virus inoculum used, 2) the younger age of ferrets, 3) our use of the TCID50 assay, which is not directly comparable to their EID50 titration assay, and which is likely to represent a more sensitive means of titrating virus.

Mice are naturally resistant to most seasonal human influenza A viruses unless the virus is adapted, which can be accomplished by serial passaging of the virus in the target animal species (Sidwell and Smee, 2000). In contrast to human influenza A viruses, highly pathogenic avian influenza A viruses (H5N1) cause severe disease in adult mice (four- to eighteen-weeks-old) without prior adaptation, characterized by loss of body weight, hypothermia, hyperventilation and encephalitis that results in paralysis in some animals (Maines et al., 2005). Some of the recent isolates from Vietnam, such as the virus that was used in this study, are highly neuroinvasive and neurotropic in mice and grow to high titers in brains of infected mice, frequently causing lethal encephalitis. Previous mouse studies of H5N1 infection demonstrated that both the loss of body weight and the development of hypothermia correlated with the encephalitic phase in the end stage of the disease (Bright et al., 2003; Dybing et al., 2000; Gao et al., 1999; Lipatov et al., 2003; Lu et al., 1999; Rowe et al., 2003). We have also used these symptoms in our study to determine the severity of the disease in mice. Our results have shown that the majority of mock-treated animals lost more than 20% body weight while peramivir-treated mice did not. In addition, a single injection treatment with peramivir was comparable to five oral treatments with oseltamivir, although reduced doses of oseltamivir were not tested.

The ferret model is considered to be a more appropriate model for comparison of the pathogenesis of H5N1 in humans since the receptors for attachment of H5N1 virus in the lower respiratory tract are similar in humans and ferrets (Maher and DeStefano, 2004; van Riel et al., 2006). Also, in contrast to mice, ferrets develop symptoms analogous to humans, e.g., fever and nasal discharge as well as diarrhea and encephalitis, upon infection with influenza A viruses (Coates et al., 1985; Coates, Sweet, and Smith, 1986; Maher and DeStefano, 2004). Unfortunately, due to space and biosafety limitations for work performed in the BSL-4 laboratory, which is mandated for studies using the A/Vietnam/1203/04 isolate at University of Texas Medical Branch, only limited information was captured about the disease caused by this virus in ferrets. However, our results compare favorably with these other published studies, which had smaller study size of 2-4 ferrets per dose group (Govorkova et al., 2005; Maines et al., 2005); lethality in our study varied from 75%-100% and we have observed a spectrum of disease symptoms.

A limited number of publications indicate that there may be variability in the virulence of these avian influenza isolates in ferrets (Govorkova et al., 2005; Maines et al., 2005) and mice (Bright et al., 2003; Dybing et al., 2000; Gao et al., 1999; Maines et al., 2005; Rowe et al., 2003) as well as in children (Peiris, de Jong, and Guan, 2007), with increased virulence associated with isolates that efficiently infect the brain. In ferrets and mice, this may partially be observed because the inoculum is delivered intranasally under experimental conditions, which may enable the virus to penetrate the CNS rapidly via the olfactory route. However, if aerosol is a main source of infection for humans while having contact with contagious birds, the lower respiratory tract could potentially become the target for the initial infection and not the nasal cavity. In that case, the virus would potentially have to spread systemically to the brain, which may explain the lower rate of encephalitis associated with H5N1 infections in humans.

Experiments are ongoing in our laboratory to better characterize the ability of this virus to penetrate the CNS in the ferret model and the immune response. In particular, our aim is to bypass the nasal cavity in the infection process in order to more accurately represent the initiation of infection in humans. These studies we describe here indicate that once this virus infects the ferret brain, a high titer of virus coincides with paralysis. The encephalitic phase does not always lead to paralysis or death and in our experiments, some animals that presented with ataxia or local seizures (hind-limb) recovered and survived the infection; no infectious virus could be detected in the lungs and brains of diseased animals that survived and were euthanized at the end of the study.

Parenteral peramivir is neuraminidase inhibitor that is worthy of further investigation, as it would offer an alternative therapy in instances where oral treatments may be limited or unacceptable. As observed in the pharmacokinetic studies of i.m. injections performed in ferrets and modeled in mice (Figs. 3 and and1A,1A, respectively), this formulation can achieve maximum plasma concentrations (Cmax) of up to 20,000 –fold above the IC50 for influenza A and B isolates, including currently circulating strains of H5N1 in Southeast Asia (0.37 nM +/- 0.26 nM) including the highly pathogenic A/Vietnam/1203/04 isolate used in these studies (0.171 nM) (Hurt et al., 2006). This strategy quickly introduces significant concentrations of peramivir into the circulation, which may be beneficial for treating highly pathogenic avian influenza infections that spread systemically.

Since our results indicate that some peramivir-treated ferrets and mice developed encephalitis, the ability of peramivir to effectively treat influenza infections within the CNS requires further investigation. Additional studies are needed to better understand the circulation of peramivir in ferrets as well as its potential to act against the virus once in the brain. In all, our study indicates that injectable peramivir effectively reduces the severity of the disease and/or promotes survival in ferrets and mice infected with the highly pathogenic influenza isolate A/Vietnam/1203/04 (H5N1). As potential exists for selection of H5N1-variants that are resistant to the available neuraminidase-inhibitor based drugs (de Jong et al., 2005b; Le et al., 2005), including peramivir (Abed et al., 2002; Baz, Abed, and Boivin, 2007), we are currently conducting studies to evaluate this question. Nonetheless, collectively, these data suggest that injectable peramivir might be useful for the treatment of highly pathogenic avian influenza infections. Both intravenous and i.m. formulations of peramivir have been evaluated recently in volunteer subjects, and multinational Phase 2 clinical studies for both formulations are under investigation for the treatment of acutely-ill and hospitalized patients infected with seasonal and life-threatening strains of influenza. Additional preclinical as well as human trials are ongoing to further evaluate the potential of injectable peramivir as an effective treatment for both seasonal and avian strains of influenza.

Materials & Methods

Tissue and egg culture

Madin-Darby canine kidney (MDCK) cells were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in minimal essential medium (MEM) with 10% fetal bovine serum with antibiotics. SPAFAS Specific Pathogen Free premium eggs were supplied by Charles River Laboratories (Wilmington, MA).

Viruses

Influenza A/Vietnam/1203/04 was provided by the Influenza Laboratory at the U.S. Centers for Disease Control and Prevention in Atlanta, Georgia. All work with this virus isolate was approved by institutional and federal agencies (CDC/USDA) and was performed in the Robert E. Shope Laboratory at BSL-4 at the University of Texas Medical Branch (Galveston, TX). Virus stock for inoculation of animals was obtained by cultivation for 48 hours in MDCK cells or by cultivation in embryonated chicken eggs (Charles River Laboratories, Wilmington, MA) at 37°C for 20-36 hours. Aliquots of harvested virus were stored at -80°C until animal infection. Viral dose was determined, as indicated below. All infection studies were performed using cell grown virus stock unless indicated.

Median tissue culture infectious dose (TCID50) assay

Serial ten-fold dilutions of the virus stock or of a 10% tissue homogenate was prepared in MEM without serum. MDCK cells were grown to confluency in 96-well tissue culture plates, washed twice with 100 μl of DPBS, followed by inoculation of 100 μl of each virus dilution of virus into four replicate wells, or, as negative control, DPBS. Plates were incubated for 90 minutes at 37°C, 5% CO2, after which an additional 100 μl of MEM was added to each well. Plates were incubated for 4 days at 37°C, 5% CO2. HA assay (WHO, 2002) was performed by removing 50 μL of supernatant from each well and transferring it to a 96-well plate, followed by addition of 50 μL per well of a 0.5% solution of horse erythrocytes suspended in DPBS w/ Ca+ and Mg2+. Erythrocytes were allowed to settle and hemagglutination was documented for each replicate. Virus concentration of stocks for infection was determined as TCID50 per ml. For organ titrations, infectious virus titers were expressed as median tissue culture infectious (TCID50) dose per gram (g) of tissue (WHO, 2002).

Animals

BALB/c mice were purchased from Harlan (Indianapolis, Indiana). Ferrets (Mustela putorius furo), were purchased from Marshall Farms (North Rose, New York). Animals were housed in a specific pathogen-free environment for a minimum of 2-7 days until treated with antiviral drugs or infected with influenza A H5N1. Implantation of transponders for telemetric temperature recording was carried out in ABSL-2 and infection/drug treatment performed in ABSL-4. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and carried out according to NIH guidelines. Prior to infection, blood was drawn for routine hematological and serological testing. Screening of ferrets for potential infection with the circulating strains of influenza was performed using hemagglutination inhibition (HI) assay on serum samples obtained on day -2 and day -8 pre-infection. Prior to virus infection via intranasal (i.n.) route, anesthesia was performed using 5% isoflurane.

Hemagglutination inhibition (HI) test

HI antibodies to the circulating strains of influenza A were measured by a standard method (WHO, 2002), as follows. Antigen was prepared from allantoic fluid harvested from influenza A/Wyoming/03/03 (H3N2)-infected 10-day old embryonated chicken eggs (Charles River Laboratories, Wilmington, MA) and the total HA units of the stock was determined as described for TCID50 assay (WHO, 2002); antigen stock was diluted to a concentration of 8 HA units of antigen in 50 μl of DPBS. HI test was performed using antigen, or as negative controls, antigen or DPBS alone. As a positive control, pooled serum from ferrets infected with influenza A/New York/55/04 (H3N2) was used. Chicken erythrocytes stored in Alsever's solution (CBT Farms, Chestertown, MD) were washed and re-suspended at a 0.5% dilution in DPBS w/ Ca+ and Mg2+. Ferret test or control serum was mixed with Receptor Destroying Enzyme II (RDE II, serum:enzyme ratio of 1:3, Accurate Chemical and Scientific Corp., Westburg, NY) and was incubated at 37°C overnight. Subsequently, the samples were heat inactivated at 56°C for 45-60 minutes and diluted 1:10 in DPBS w/ Ca+ and Mg2+. Serum samples were then diluted 1:2 in DPBS with Ca+ and Mg2+ in a 96-well U-bottomed plate. An equal volume of antigen was added to all wells except for the DPBS control; the plate was then incubated at 37°C for 60 minutes. Fifty microliters of 0.5% chicken erythrocytes was added to each well, and the mixture was incubated for 15-30 min at room temperature. The HI titers of ferret serum samples are reported as the reciprocal of the highest dilution at which hemagglutination was inhibited.

Hematology

Ferret blood was collected in EDTA and standard hematological analysis was performed using the HEMAVET®1700, Drew Scientific, Inc. (Oxford, CT) on whole blood specimens to determine platelet and differential counts, in accordance with the manufacturer's recommendation.

Telemetry

For measurement of body temperature, animals were anaesthetized and implanted subcutaneously with BMDS IPTT-300 transponders (chips) purchased from Bio Medic Data Systems, Inc. (BMDS, Seaford, DE), using a trocar needle assembly. Animals were monitored for signs of infection or of migration of transponder for two days prior to transfer of animals into the ABSL-4 facility. Chips were scanned using a DAS-6007 transponder reader (BMDS). Downloading of digital temperature data was performed in accordance with the manufacturer's protocol.

Antiviral drugs

Peramivir (BCX-1812) and oseltamivir were synthesized by BioCryst Pharmaceuticals, Inc (Birmingham, AL), as previously described (Kim et al., 1997). Chemical structure and purity were verified by nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), CHN analysis, and infrared (IR) spectroscopy. For antiviral studies, these antiviral drugs were diluted in sterile saline (0.9% sodium chloride) and this diluent stock was used as the “vehicle” control.

Pharmacokinetic (PK) analysis

PK data was obtained in ferrets and mice, as follows (Covance Laboratories, Inc, Madison, WI). Male ferrets (three per group) were injected via i.m. route with a dose of 1, 3 or 9 mg/kg peramivir (BioCryst, Birmingham, AL). Prior to dosing (time 0) and at time intervals between 0.083 and 72 hrs following dosing, blood samples of approximately 0.5 ml were collected and analyzed via liquid chromatography with tandem mass spectrophotometry (LS/MS/MS). Mice PK data were generated based upon modeling from a 10 mg/kg i.m. dose. PK parameters were calculated and modeling performed using WinNonlin® 5.0.1 (Pharsight Corp., Mountain View, CA). PK data were obtained following a single i.m. injection of ferrets (3 per group) with doses of 1, 3 or 9 mg/kg peramivir. Animals were monitored for peramivir concentration in plasma over a 72 hour period (Covance, Princeton, NJ).

Clinical disease

H5N1 infection and antiviral treatment studies in mice and in ferrets were performed as detailed in the sections below. Outcomes monitored were death, and the development of encephalitis or paralysis. Standardized data recording was performed using the following definitions: encephalitis, development of discoordination, ataxia or transient seizures with retention of the ability to drink and feed; paralysis, hind limb (hemiplegic) or quadriplegic paralysis with the inability to reach the feeder or water bottle.

Influenza A H5N1 infection in ferrets

For the H5N1 virulence study, seven-week-old ferrets were infected intranasally with influenza A H5N1 (A/Vietnam/1203/04) using a range of doses from 1 × 10-1 to 1 × 103 TCID50 of cell culture-grown virus (N=4 per group) or using a range of doses from 1.89 × 102 to 1.89 × 104 TCID50 of egg-grown virus per animal (N=7 per group). Following infection, animals were monitored daily for disease development and death for a period of 16-19 days following infection. In the latter study, animals that developed paralysis were euthanized with an overdose of pentobarbital delivered intraperitoneally. Daily telemetric monitoring of body temperature was performed for a period of 14 days.

Antiviral drug treatment

Mice

Ten-week-old mice (10-11 per group) were infected intranasally with a dose of 6 × 102 TCID50 of influenza A H5N1 (A/Vietnam/1203/04) grown in MDCK cells. Subsequently, mice were treated with peramivir (30 mg/kg) in a single dose at 1 h post-inoculation (+1 h) or multiple doses (+1 h, daily on day +1 through day +4) via i.m. injection. As control, mice were treated with drug diluent (“vehicle” at +1 h, daily on day +1 through day +4. For comparison, mice were treated orally (per os, p.o.) with oseltamivir (10 mg/kg/day) at +1 h and daily on day +1 through day +4. Animals were monitored daily for a period of 15 days for death, disease development, and body temperature. Body mass (weight) was recorded on days indicated in figure legend. Severe disease was defined as the loss of ≥20% of their initial body mass.

Ferrets

Six- to eight-week-old ferrets were infected intranasally with 1.5 × 103 TCID50 (trial 1), 1.5 × 104 TCID50 (trial 2), or 1.7 × 104 TCID50 of influenza A H5N1 (A/Vietnam/1203/04) using virus stock prepared in MDCK cells (trial 1) or in eggs (trial 2 and 3). Ferrets were then treated with multiple doses of peramivir (30 mg/kg) or, as control, were treated with vehicle at +1 h, and daily on day +1 through day +4 (trial 1 and 3) or were untreated (trial 2). Following infection and drug treatment, animals were monitored daily for a period of 16-18 days following infection for death and disease development. Daily telemetric monitoring of body temperature was performed. For trial 3, five animals per group were randomly pre-selected to be euthanized on day 4 and on day 6 post-infection for organ harvest and infectious virus titration.

Statistical analysis

Statistical analysis of survival for all groups over the indicated period as indicated in figure legends was performed using logrank test at a significant level of α<0.05 in GraphPad® Prism (San Diego, CA). For pairwise comparison of the survival of treated and untreated (or mock-treated) groups Fisher's Exact Test was performed at a significant level of α<0.05 in GraphPad® Prism.

Infectious virus titration in organs

Organs collected at the indicated time points were sagittally sectioned in half. One half of each brain was homogenized in MEM containing 10% fetal bovine serum, and a 10% suspension was made. The organ suspension was maintained at -80°C until further processing. The titer of infectious virus was calculated using TCID50 assay.

Supplementary Material

03

Acknowledgments

We are grateful to Dr. Phil Wyde for providing scientific input and technical training and to Anna H. Grund, Melinda Kelley, and Jennifer Smith for their technical assistance, to Jenna Linde and Nicolette Ward for aid in preparing the manuscript, and to Dr. Andrea Bertke for editorial suggestions. Funding was provided by NIAID contract #N01-AI-30065 T04. SP was supported by NIH K08 Award #AI059491-01.

Footnotes

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