Supporting Information S1 Preparation of DNA and RNA synthetic ECs.  Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness BACKGROUND: Resistance to the neuraminidase inhibitor oseltamivir can be conferred by a well-characterized mutation in the neuraminidase gene, H275Y. In human H1N1 viruses that circulated in the first years of the 21st century, this mutation carried a fitness cost and resistant viruses were rare. During the 2007–08 influenza season, oseltamivir-resistant viruses of H1N1 phenotype emerged and predominated. March 2009 saw the emergence of a novel H1N1 influenza pandemic. We examined whether the H275Y mutation affected neuraminidase enzyme activity or replication of the pandemic influenza virus. METHODS: Using reverse genetics we engineered the H275Y mutation into the neuraminidase of a 2009 pandemic H1N1 virus and assessed the ability of this enzyme to desialylate mono- and multivalent substrates. The growth kinetics of wild-type and mutant viruses were assessed in Madin–Darby canine kidney (MDCK) and fully differentiated human airway epithelial (HAE) cells. RESULTS: The presence of H275Y was associated with a 1.3-fold decrease in the affinity of the neuraminidase for a monovalent substrate and a 4-fold compromise in desialylation of multivalent substrate. This was associated with a fitness cost to viral replication in vitro, which only became apparent during competitive replication in the mucus-rich HAE culture system. CONCLUSIONS: The neuraminidase protein of pandemic influenza isolates tolerates the H275Y mutation and this mutation confers resistance to oseltamivir. However, unlike seasonal H1N1 viruses isolated since 2007, the mutation is not associated with any fitness advantage and thus is unlikely to predominate without further antigenic drift, compensating mutations or intense selection pressure. March 2009 saw the emergence of a novel strain of influenza with the ability to transmit readily between humans. The rapid global spread of the new influenza A/H1N1 virus resulted in the first influenza pandemic in 40 years. 1 Two therapies are currently licensed to treat influenza infections: the M2 ion channel blocking adamantane drugs (amantadine and rimantadine) and the neuraminidase (NA) inhibitors (oseltamivir and zanamivir). Pandemic influenza A/H1N1 2009 virus (pH1N1) crossed into humans carrying a well-characterized amantadine-resistance mutation, S31N, within the M2 ion channel protein, rendering the adamantane class of antiviral drug ineffective against the virus. Initial isolates were susceptible to NA inhibitors, including oseltamivir and consequently this drug was used extensively in the treatment and prophylaxis of pandemic influenza. 2 Oseltamivir resistance emerged infrequently in pH1N1 2009 influenza viruses. 3, 4 In contrast, seasonal H1N1 influenza viruses from the 2007-08 season onwards were predominantly resistant to oseltamivir with resistance conferred by the H275Y mutation in the viral NA. 5 Traditionally, the H275Y mutation was associated with compromised viral fitness amongst H1N1 isolates. 6 However, isolates from the 2007 -08 season with this mutation suffered no attenuation. 7 It is likely that certain other sequence variations, such as the D344N (N1 numbering) change found within the NA gene of oseltamivir-resistant viruses from the 2007-08 season, counteract the decrease in enzyme function that H275Y confers. 8, 9 Residue 344 is tyrosine in most avian influenza NA genes but mutated to asparagine in two N1 viruses, which crossed from birds into mammals shortly thereafter (the 1918 pandemic virus and the Eurasian lineage swine H1N1 viruses). 10 During circulation in humans, an aspartic acid at residue 344 was eventually selected in seasonal H1N1 viruses. Mutation back to asparagine occurred in circulating human H1N1 viruses prior to the 2007 season and was associated with increased NA activity. 9 The NA enzyme of pH1N1 virus is derived from a Eurasian lineage H1N1 swine virus, and harbours asparagine at residue 344. This suggested that it may tolerate mutations such as H275Y that would concomitantly decrease NA activity and confer oseltamivir resistance. This study aimed to assess in vitro the effect of the H275Y mutation in the NA of a prototypic pH1N1 2009 isolate.