Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1) pdm09 Influenza Viruses

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide. Author Summary Antimicrobial resistance is an increasing problem for the treatment of infectious diseases. In 2007-2008 human seasonal A(H1N1) influenza viruses rapidly acquired resistance to the most commonly used anti-influenza drug oseltamivir, via a H275Y amino acid mutation within the neuraminidase (NA) protein. In 2009 the oseltamivir sensitive A(H1N1)pdm09 virus (encoding NA 275H) emerged in the human population, rapidly replacing the oseltamivir resistant seasonal A(H1N1) virus. However, there is increasing concern that currently circulating A(H1N1)pdm09 viruses may similarly acquire oseltamivir resistance (via the NA H275Y mutation) and become widespread. Here we demonstrate that two novel amino acid changes present in virtually all recent A(H1N1)pdm09 viruses (NA V241I and N369K) enable the acquisition of the NA H275Y oseltamivir resistance mutation without compromising viral fitness. As such recent A(H1N1)pdm09 viruses are now one step closer to acquiring widespread oseltamivir resistance.