Oseltamivir-Resistant Variants of the 2009 Pandemic H1N1 Influenza A Virus Are Not Attenuated in the Guinea Pig and Ferret Transmission Models

Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.Oseltamivir resistance was rare until 2008, when resistant seasonal H1N1 viruses were found circulating in the general Scandinavian population (15). Soon after, studies from other countries in Europe also reported the isolation of oseltamivir-resistant viruses, and eventually, oseltamivir resistance was recognized as a global phenomenon (9, 27). Prior to 2008, resistant viruses were primarily isolated from patients with nonresponsive influenza virus infections or from infected patients who received a low-dose prophylaxis regiment prior to symptom onset. At the time, these resistant isolates accounted for 1% of the circulating H1N1 virus. Drug resistance mutations were identified during oseltamivir development, including a histidine-to-tyrosine mutation at position 275 (H275Y) in N1 neuraminidase (NA). This mutation in particular was shown to attenuate virus growth and pathology in ferrets (17). Additionally, oseltamivir-resistant viruses with a nearby mutation in N2 neuraminidase transmitted less efficiently than oseltamivir-sensitive viruses in the guinea pig transmission model (4). Surprisingly, the seasonal 2008 H1N1 viral isolates that spread around the world had the same tyrosine mutation, which was previously associated with iatrogenic infections and attenuation. Furthermore, epidemiological studies concluded that this resistant virus developed independently of drug selection, suggesting that compensatory adaptations allowed an attenuating mutation to become permissible (3, 18). The ability of resistant 2008 isolates to perform on par with nonresistant 2008 isolates in growth curves, in mean plaque size, and in a transmission model was recently confirmed (2). Currently, 99% of seasonal H1N1 viruses are oseltamivir resistant; however, the prevalence of these viruses is very low due to replacement by a novel reassortant H1N1 virus (6, 8). This novel reassortant was originally identified in Mexico by doctors concerned about a jump in the number of influenza cases during the month of March in 2009 (7). Later referred to as swine-origin influenza virus, novel H1N1 virus, or 2009 pandemic H1N1 virus, this virus would continue to efficiently transmit around the world, even during the summer months of the northern hemisphere. Its robust transmission was later confirmed in aerosol transmission models, in which 86% of ferrets and 100% of guinea pigs exposed to infected animals contracted pandemic influenza (22, 28, 31). Oseltamivir was used broadly during the outbreak, treating those with complications and prophylactically treating close contacts of confirmed cases. The use of oseltamivir in this manner provided ample opportunity for oseltamivir-resistant viruses to develop. More than 225 cases of oseltamivir-resistant infections have been confirmed from the beginning of the pandemic, including four incidents of suspected aerosol transmission (21, 32, 33). Fortunately, these clinical isolates never progressed into stable transmission in the general public. This study seeks to evaluate if introducing the H275Y mutation into the pandemic 2009 H1N1 virus attenuates virus replication in vitro or in vivo using the guinea pig model and the ferret model to test aerosol transmission efficiency. Furthermore, this study evaluates if a reassortant between the circulating novel H1N1 virus and seasonal neuraminidase (NA) forms a well-adapted, resistant virus capable of efficient transmission.Currently, oseltamivir is the drug of choice for treating novel H1N1 complications and outpatient prophylaxis. Therefore, it is of great importance to study the in vitro replication and transmission phenotypes of oseltamivir-resistant novel H1N1 viruses to understand why broad oseltamivir resistance has not occurred or whether we should expect it to occur in the future.