MultiDrug-Resistant 2009 Pandemic Influenza A(H1N1) Viruses Maintain Fitness and Transmissibility in Ferrets

Influenza A viruses are significant human pathogens, both in their sporadic pandemic and endemic (seasonal) forms. Antiviral therapies are commonly used to treat both severe illness in high-risk patients and self-limited influenza infection. Widespread emergence of antiviral resistance among seasonal influenza A viruses began during the 2004–2005 influenza season, when a significant increase in the prevalence of adamantane resistance among H3N2 influenza viruses was observed [1, 2]. In most cases, a single amino acid substitution in the M2 protein (S31N) was responsible for this resistance, having no effect on viral fitness [3, 4]. During the subsequent season in 2005–2006, H3N2 adamantane resistance surpassed 90%, rendering this class of drugs ineffective as a viable treatment for seasonal H3N2 influenza infections [3]. By the time H3N2 adamantane resistance became prominent, a shift in practice was already occurring for a number of reasons, and despite seasonal H1N1 strains remaining adamantane susceptible, use of these drugs decreased and the neuraminidase inhibitors (NAIs), particularly oseltamivir, became the primary influenza antiviral of choice. Widespread resistance to oseltamivir was deemed unlikely because NAIs closely resemble the sialic acids that are the natural substrates for the neuraminidase's active site [5], and that any mutation leading to resistance was expected to lead to reduced fitness of the virus [6, 7]. When NAI resistance mutations were first identified through both in vitro selection under drug pressure and during oseltamivir treatment of experimentally infected individuals [8–10], the neuraminidase (NA) changes at position 275, 292, and 119 identified in H3N2 and H1N1 viruses led to a significant reduction in the ability of the viruses to replicate and transmit in animal models [7, 9, 11, 12]. This correlated well with the low incidence of NAI resistance of 0.32% in the adult population observed during 2005–2006, when most circulating seasonal viruses retained susceptibility to oseltamivir [6]. During 2007–2009, the unexpected emergence of a dominant clade of oseltamivir-resistant seasonal H1N1 virus demonstrated that NAI resistance could occur in the context of an otherwise fit and transmissible virus. By the end of the 2008–2009 influenza season, ∼98% of seasonal H1N1 isolates worldwide exhibited resistance to oseltamivir [13]. Recent studies have identified permissive changes in the NA of seasonal H1N1 that might account for the widespread circulation of oseltamivir-resistant strains [14]. Over the past 4 years, a smaller number of NAI-resistant H3N2 viruses have been identified, some containing the H274Y change (equivalent mutation in N2 subtype NA) or other newly identified NA amino acid changes. In animal models, these resistant H3N2 viruses demonstrated equivalent fitness and transmissibility to wild-type viruses, suggesting that widespread seasonal H3N2 NAI resistance is also a threat [15–17]. Recently, the 2009 pandemic H1N1 influenza virus emerged in Mexico and the southwestern United States [18, 19] and rapidly spread worldwide, causing increased morbidity and mortality among children and young adults throughout most of 2009 and early 2010. Similar to seasonal H3N2 viruses, the 2009 pandemic viruses are resistant to the adamantanes [20], making NAIs the drug class of choice. The concern for increased levels of severe disease and need for a parenteral therapy for patients unable to achieve adequate NAI levels orally or via inhalation prompted the US Food and Drug Administration (FDA) to release a new parenteral NAI, peramivir, on an emergency use authorization for treatment of severe pandemic H1N1 infections. After only a few months into the pandemic, reports of sporadic NAI resistance emerged, leading to the possibility of a virus with limited susceptibility to available antivirals transmitting in the community [21–23]. Recently, we isolated 2 adamantine-resistant pandemic H1N1 viruses from 2 different immunocompromised patients and found that, after a period of 9–14 days of NAI treatment, the viruses from these patients independently acquired the NA H275Y mutation [23]. We further demonstrated that the H275Y change in these pandemic H1N1 viruses conferred resistance not only to oseltamivir, but also to peramivir, suggesting that for those at risk of severe disease with NAI resistant infections, intravenous peramivir may not be an adequate treatment option. In this report, we characterized the multidrug-resistant pandemic influenza A(H1N1) viruses isolated from the 2 individuals described previously [23], to determine how great a potential exists for widespread multidrug antiviral resistance and transmissibility. We examined their viral genomes for other significant amino acid substitutions and then evaluated the virulence, fitness, and transmissibility in a mammalian host with use of a ferret transmission model.