The resistance of influenza viruses to neuraminidase (NA) inhibitors (NAIs; i.e. oseltamivir, zanamivir, peramivir and laninamivir) can be associated with several NA substitutions, with differing effects on viral fitness. To identify novel molecular markers conferring multi-NAI resistance, the NA gene of oseltamivir-resistant (H275Y, N1 numbering) 2009 pandemic influenza [A(H1N1)pdm09] virus was enriched with random mutations. This randomly mutated viral library was propagated in Madin–Darby canine kidney (MDCK) cells under zanamivir pressure and gave rise to additional changes within NA, including an I436N substitution located outside the NA enzyme active site. We generated four recombinant A(H1N1)pdm09 viruses containing either wild-type NA or NA with single (I436N or H275Y) or double (H275Y-I436N) substitutions. The double H275Y-I436N mutation significantly reduced inhibition by oseltamivir and peramivir and reduced inhibition by zanamivir and laninamivir. I436N alone reduced inhibition by all NAIs, suggesting that it is a multi-NAI resistance marker. I436N did not affect viral fitness in vitro or in a murine model; however, H275Y and I436N together had a negative impact on viral fitness. Further, I436N alone did not have an appreciable impact on viral replication in the upper respiratory tract or transmissibility in ferrets. However, the rg-H275Y-I436N double mutant transmitted less efficiently than either single mutant via the direct contact and respiratory droplet routes in ferrets. Overall, these results highlight the usefulness of a random mutagenesis approach for identifying potential molecular markers of resistance and the importance of I436N NA substitution in A(H1N1)pdm09 virus as a marker for multi-NAI resistance.
Centers for Disease Control and Prevention (CDC) Update: influenza activity - United States, September 28, 2008-January 31, 2009. MMWR Morb Mortal Wkly Rep2009; 58:115–119[PubMed]
HurtAC, HardieK, WilsonNJ, DengYM, OsbournM et al. Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia. J Infect Dis2012; 206:148–157 [View Article][PubMed]
Centers for Disease Control and Prevention (CDC) Oseltamivir-resistant 2009 pandemic influenza A (H1N1) virus infection in two summer campers receiving prophylaxis-North Carolina, 2009. MMWR Morb Mortal Wkly Rep2009; 58:969–972[PubMed]
LeQM, WertheimHF, TranND, van DoornHR, NguyenTH et al. A community cluster of oseltamivir-resistant cases of 2009 H1N1 influenza. N Engl J Med2010; 362:86–87 [View Article][PubMed]
HurtAC, BesselaarTG, DanielsRS, ErmetalB, FryA et al. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014-2015. Antiviral Res2016; 132:178–185 [View Article][PubMed]
MeijerA, Rebelo-de-AndradeH, CorreiaV, BesselaarT, Drager-DayalR et al. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2012-2013. Antiviral Res2014; 110:31–41 [View Article][PubMed]
TakashitaE, MeijerA, LackenbyA, GubarevaL, Rebelo-de-AndradeH et al. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014. Antiviral Res2015; 117:27–38 [View Article][PubMed]
IvesJA, CarrJA, MendelDB, TaiCY, LambkinR et al. The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. Antiviral Res2002; 55:307–317 [View Article][PubMed]
de JongMD, TranTT, TruongHK, VoMH, SmithGJ et al. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med2005; 353:2667–2672 [View Article][PubMed]
Okomo-AdhiamboM, Demmler-HarrisonGJ, DeydeVM, SheuTG, XuX et al. Detection of E119V and E119I mutations in influenza A (H3N2) viruses isolated from an immunocompromised patient: challenges in diagnosis of oseltamivir resistance. Antimicrob Agents Chemother2010; 54:1834–1841 [View Article][PubMed]
PirallaA, Gozalo-MargüelloM, FiorinaL, RovidaF, MuzziA et al. Different drug-resistant influenza A(H3N2) variants in two immunocompromised patients treated with oseltamivir during the 2011-2012 influenza season in Italy. J Clin Virol2013; 58:132–137 [View Article][PubMed]
BabadyNE, LaplanteJM, TangYW, St GeorgeK. Detection of a transient R292K mutation in influenza A/H3N2 viruses shed for several weeks by an immunocompromised patient. J Clin Microbiol2015; 53:1415–1418 [View Article][PubMed]
NguyenHT, TrujilloAA, SheuTG, LevineM, MishinVP et al. Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States. Antiviral Res2012; 93:381–386 [View Article][PubMed]
LittleK, LeangSK, ButlerJ, BaasC, HarrowerB et al. Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring. Euro Surveill2015; 20: [View Article][PubMed]
TuV, AbedY, BarbeauX, CarbonneauJ, FageC et al. The I427T neuraminidase (NA) substitution, located outside the NA active site of an influenza A(H1N1)pdm09 variant with reduced susceptibility to NA inhibitors, alters NA properties and impairs viral fitness. Antiviral Res2017; 137:6–13 [View Article][PubMed]
KimCU, LewW, WilliamsMA, LiuH, ZhangL et al. Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J Am Chem Soc1997; 119:681–690 [View Article][PubMed]
TamuraD, DebiasiRL, Okomo-AdhiamboM, MishinVP, CampbellAP et al. Emergence of multidrug-resistant influenza A(H1N1)pdm09 virus variants in an immunocompromised child treated with oseltamivir and zanamivir. J Infect Dis2015; 212:1209–1213 [View Article][PubMed]
van der VriesE, StelmaFF, BoucherCA. Emergence of a multidrug-resistant pandemic influenza A (H1N1) virus. N Engl J Med2010; 363:1381–1382 [View Article][PubMed]
L'HuillierAG, AbedY, PettyTJ, CordeyS, ThomasY et al. E119D neuraminidase mutation conferring pan-resistance to neuraminidase inhibitors in an A(H1N1)pdm09 isolate from a stem-cell transplant recipient. J Infect Dis2015; 212:1726–1734 [View Article][PubMed]
HurtAC, ChotpitayasunondhT, CoxNJ, DanielsR, FryAM et al. Antiviral resistance during the 2009 influenza A H1N1 pandemic: public health, laboratory, and clinical perspectives. Lancet Infect Dis2012; 12:240–248 [View Article][PubMed]
ColmanPM, VargheseJN, LaverWG. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature1983; 303:41–44 [View Article][PubMed]
HarionoM, AbdullahN, DamodaranKV, KamarulzamanEE, MohamedN et al. Potential new H1N1 neuraminidase inhibitors from ferulic acid and vanillin: molecular modelling, synthesis and in vitro assay. Sci Rep2016; 6:38692 [View Article][PubMed]
RussellRJ, HaireLF, StevensDJ, CollinsPJ, LinYP et al. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature2006; 443:45–49 [View Article][PubMed]
MaratheBM, LévêqueV, KlumppK, WebsterRG, GovorkovaEA. Determination of neuraminidase kinetic constants using whole influenza virus preparations and correction for spectroscopic interference by a fluorogenic substrate. PLoS One2013; 8:e71401 [View Article][PubMed]