We further cultured this variant in the presence of a high laninamivir concentration (100 M) and obtained good cytopathic effects. of laninamivir resistance should be considered. The objectives of the present study were, first, to evaluate the activity of laninamivir against a collection of NAI-resistant seasonal A(H1N1), A(H3N2), and 2009 pandemic A(H1N1)pdm09 viruses. Second, we aimed to generate and characterize laninamivir-resistant influenza A(H1N1) and A(H3N2) computer virus variants following passaging under laninamivir pressure. MATERIALS AND METHODS Cells culture. ST6Gal1 Madin-Darby canine kidney cells, overexpressing the 2 2,6 sialic acid receptors (MDCK 2,6; kindly provided by (S)-3-Hydroxyisobutyric acid Y. Kawaoka from the University of Wisconsin, Madison, WI), and human embryonic kidney 293T cells (ATCC) were maintained in Dulbecco’s altered Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA), supplemented with 10% fetal IL20RB antibody bovine serum (Invitrogen, Carlsbad, CA). Madin-Darby canine kidney (MDCK) cells were sourced from the European Collection of Cell Cultures (ECACC; Wiltshire, United Kingdom). These cells were maintained to generate cell bank stocks in minimal essential medium without l-glutamine (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Drug susceptibility testing. A selection of seasonal A(H1N1), A(H3N2), and A(H1N1)pdm09 viruses harboring NAI-resistant NA mutations (Table 1) was used for assessing susceptibility to laninamivir (R-125489) (Biota Scientific Management, Notting Hill, Australia), oseltamivir carboxylate (Hoffmann-La Roche, Basel, Switzerland), zanamivir (GlaxoSmithKline, Stevenage, United Kingdom), and peramivir (BioCryst, Birmingham, United States) by NA inhibition assays, as previously described (17) with minor modifications. Briefly, viruses were standardized to an NA activity level 10-fold higher than that of the background, as measured by the production of a fluorescent product from the 2-(4-methylumbelliferyl)–d-N-acetylneuraminic acid (MUNANA; Sigma, St-Louis, MO) substrate. Drug susceptibility profiles were determined by the extent of NA inhibition after incubation with 3-fold serial dilutions of NAIs at final concentrations ranging from 0 to 10,800 nM. The 50% inhibitory concentrations (IC50s) were determined from the dose-response curve. TABLE 1 Laninamivir susceptibility profiles of influenza A(H1N1)pdm09, A(H1N1), and A(H3N2) viruses harboring NA substitutions mediating resistance to other neuraminidase inhibitors assessments. RESULTS Laninamivir susceptibility profiles of influenza viruses harboring mutations of resistance to other NAIs. The IC50s of laninamivir against various NAI-resistant influenza A computer virus variants as determined by NA inhibition assays are summarized in Table 1. All viruses that were susceptible to zanamivir also had a susceptible phenotype to laninamivir, including oseltamivir-resistant A(H1N1) variants made up of H275Y and N295S substitutions as well as the A(H3N2) variant with the E119V change. Influenza A(H1N1)pdm09 variants made up of the E119V/G and Q136K substitutions, which conferred (S)-3-Hydroxyisobutyric acid resistance to zanamivir, exhibited reduced or highly reduced inhibition to laninamivir. Of note, a multidrug resistance phenotype to laninamivir, zanamivir, peramivir, and oseltamivir was observed for the (S)-3-Hydroxyisobutyric acid E119V A(H1N1)pdm09 recombinant variant. Selection of laninamivir-resistant variants < 0.001), 1.0% (< 0.001), and 1.1% (< 0.001), respectively, compared to the WT protein. Of note, we cannot distinguish between decreased activity or expression based on the current assay. The G147E substitution alone did not significantly affect the relative total NA activity (104%) or susceptibility to laninamivir (7-fold decrease in IC50 compared to WT). TABLE 4 Susceptibility profiles to neuraminidase inhibitors of a recombinant A(H1N1)pdm09 computer virus harboring the E119A neuraminidase substitution < 0.001, compared to the WT NA activity. DISCUSSION NAIs are expected to play a major role in the control of seasonal and eventual pandemic influenza computer virus infections. However, the emergence and spread of NAI-resistant variants is usually a serious concern. The identification of amino acid substitutions conferring resistance to NAIs from studies may help us to understand mechanisms of resistance and to predict clinical cases of resistance to this class of antivirals. In fact, the well-known NA changes conferring resistance to oseltamivir in humans, including the H1N1 H275Y variant and the H3N2 E119V and R292K variants, were previously predicted by studies (21,C23). In the present study, we used an approach to investigate mechanisms of resistance to laninamivir, a novel NAI. By testing several A(H1N1)pdm09 as well as seasonal A(H1N1) and A(H3N2) variants, previously found to be resistant to at least one NAI, we exhibited a similar pattern of susceptibility between laninamivir and zanamivir. More specifically, laninamivir was shown to be active against oseltamivir-resistant H1N1-H275Y computer virus, as reported previously (24), and H3N2-E119V variants. Therefore, laninamivir could constitute an antiviral option for the treatment of severe oseltamivir-resistant cases. In contrast, the recombinant A(H1N1)pdm09 viruses containing E119A/G/V framework NA substitutions exhibited reduced or highly reduced susceptibility to zanamivir.