Assessing the development of oseltamivir and zanamivir resistance in A(H5N1) influenza viruses using a ferret model
Introduction
Highly pathogenic A(H5N1) avian influenza viruses have infected poultry throughout parts of Asia, Middle East, Europe and Africa, resulting in the death or culling of many animals and significant economic loss in affected countries (Webster and Govorkova, 2006). On occasions, humans have also become infected with the A(H5N1) virus with the vast majority of infections having been caused by close contact with infected poultry (Abdel-Ghafar et al., 2008). Viruses circulating in different regions are antigenically and genetically distinct and have been classified into at least 10 separate phylogenetic clades and subclades (Abdel-Ghafar et al., 2008). A(H5N1) infections in humans typically cause severe pneumonia, which often progresses to acute respiratory distress syndrome, and on occasions can cause gastrointestinal symptoms, leukopenia and lymphopenia (de Jong et al., 2006). A(H5N1) infection has also been associated with a disseminated spread of the virus throughout the body, with infectious virus being detected in the blood and cerebrospinal fluid of some severely ill patients (de Jong et al., 2005b). As a result of the highly pathogenic nature of the virus, case fatality rates have been high. Over 490 confirmed human cases of A(H5N1) infection have occurred worldwide since 2003, resulting in 292 deaths. This gives an overall case fatality rate (CFR) of approximately 60% although this varies considerably between countries. Indonesia, where most cases of A(H5N1) infection have been reported, has a significantly higher CFR of 82% compared to the CFR for cases seen in Egypt of 31% (WHO, 2009).
Although human-to-human transmission of the virus has been rare to date, the potential for mutation of the A(H5N1) virus to facilitate transmission and rapid spread throughout the human population, leading to a pandemic, is still a possibility. Although A(H5N1) vaccine clinical trials have yielded promising results, they are not publically available for use, and therefore antiviral drugs remain the only immediately available measure for the control of A(H5N1) infections. The neuraminidase inhibitors (NAIs), oseltamivir (Tamiflu) and zanamivir (Relenza) are currently the most appropriate options for the treatment of A(H5N1) infection. Of these, oseltamivir has been the most widely used and has been demonstrated to improve patient survival, although early administration of the drug significantly improves its effectiveness (Abdel-Ghafar et al., 2008, de Jong and Hien, 2006). Previous animal studies have suggested that the oseltamivir dose and duration of treatment may need to be increased for A(H5N1) viruses compared to recommendations for seasonal influenza (Boltz et al., 2008, Govorkova et al., 2007, Yen et al., 2005). There is currently little data on the effectiveness of zanamivir treatment for prophylaxis of A(H5N1) infection in humans or in animal models.
The effectiveness of any antiviral drug can be significantly impaired if a virus develops resistance. Oseltamivir resistant A(H5N1) viruses containing a H274Y mutation (N2 numbering) in the neuraminidase (NA) gene have been detected in two patients undergoing oseltamivir treatment (de Jong et al., 2005a, Le et al., 2005), and may have been associated with the clinical deterioration and fatal outcomes in these individuals. We have previously conducted in vitro studies that selected for drug resistant variants during cell culture passage of A(H5N1) virus in increasing NAI concentrations (Hurt et al., 2009a). In that study we identified a range of mutations that can occur in A(H5N1) viruses under oseltamivir or zanamivir pressure, although in vitro experiments may not accurately predict the likelihood of these resistant strains occurring in humans during drug treatment. Here we use an in vivo ferret model to investigate the development of resistance to oseltamivir and zanamivir in A(H5N1) viruses by treating the animals with doses either equivalent to the normal human treatment dose (Ward et al., 2005) or a range of sub-optimal drug concentrations. Analysis of viruses shed by the NAI treated ferrets detected one zanamivir-resistant isolate that contained a novel glutamine (Q) to leucine (L) mutation at residue 136, an amino acid that has recently been demonstrated to play a role in zanamivir susceptibility following the detection of a Q to lysine (K) mutation at the same residue in cultured seasonal A(H1N1) viruses (Hurt et al., 2009c, Okomo-Adhiambo et al., 2010).
Section snippets
Viruses
Two A(H5N1) influenza viruses classified as Highly Pathogenic Notifiable Avian Influenza according to World Organisation for Animal Health (OIE) criteria, namely A/Vietnam/1203/2004 (Vn/1203) (haemagglutinin (HA) phylogenetic clade 1) and A/Chicken/Laos/26/2006 (Laos/26) (HA phylogenetic clade 2.3) (Abdel-Ghafar et al., 2008) (kindly supplied by Paul Selleck, Australian Animal Health Laboratory, Australia), were cultured in embryonated eggs to titres of 1 × 108.33 EID50/0.1 ml and 1 × 108.70 EID50/0.1
Effect of NA inhibitor treatment on survival and duration of viral shedding
Two different A(H5N1) influenza viruses, Vn/1203 and Laos/26, were used in this study to infect ferrets and to determine how readily resistance was generated under NAI selective pressure. Although both viruses fulfilled the OIE criteria for Highly Pathogenic Notifiable Avian Influenza in chickens they were found to cause significantly different morbidity in ferrets. Control ferrets that were not treated with either NAI and were infected with Vn/1203 showed severe disease and were euthanized on
Discussion
The NA inhibitors are essential drugs for the treatment of A(H5N1) infected patients, although concerns exist that resistance may reduce the effectiveness of antiviral therapy (White et al., 2009). In the absence of human clinical trial data, animal models provide a more realistic alternative to predicting the likelihood of resistant variants being selected under NAI pressure than in vitro cell culture experiments. In a previous study we generated NA inhibitor resistance in the same two A(H5N1)
Acknowledgements
Supported by Australian National Health and Medical Research Council award 400595. We also acknowledge the expert technical assistance of Tim Hancock and Jessica Klippel. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health and Ageing.
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