title: Profiling and characterization of influenza virus N1 strains potentially resistant to multiple neuraminidase inhibitors.
abstract: Neuraminidase inhibitors (NAIs) have been widely used to control influenza virus infection, but their increased use could promote the global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened a known mutation(s) that could confer multidrug resistance to the currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA-encoding genes (catalytic residues R152K and R292K, framework residues E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single and double mutant viruses recovered in the backbone of pH1N1, four variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition by all of the NAIs and two variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the nine recombinant H5N1 viruses, four variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition by all of the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance on pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection.
  
  
  OBJECTIVE
  There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition by all of the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo. Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza virus variants, as well as the development of alternative therapeutics.
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Yun Hee
authors.initials: YH
authors.lastname: Baek
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Min-Suk
authors.initials: MS
authors.lastname: Song
authors.affiliation: Infection and Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Lee
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Young-il
authors.initials: YI
authors.lastname: Kim
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Eun-Ha
authors.initials: EH
authors.lastname: Kim
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Su-Jin
authors.initials: SJ
authors.lastname: Park
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Kuk Jin
authors.initials: KJ
authors.lastname: Park
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Hyeok-il
authors.initials: HI
authors.lastname: Kwon
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Philippe Noriel Q
authors.initials: PN
authors.lastname: Pascua
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Gyo-Jin
authors.initials: GJ
authors.lastname: Lim
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea.
authors.forename: Semi
authors.initials: S
authors.lastname: Kim
authors.affiliation: Viral Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Youseong-gu, Daejeon, Republic of Korea.
authors.forename: Sun-Woo
authors.initials: SW
authors.lastname: Yoon
authors.affiliation: Infection and Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea Biosystems and Bioengineering Program, University of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Myung Hee
authors.initials: MH
authors.lastname: Kim
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Webby
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Seowon-gu, Cheongju, Republic of Korea choiki55@chungbuk.ac.kr.
authors.forename: Young-Ki
authors.initials: YK
authors.lastname: Choi
mesh_terms: D000146:Acids, Carbocyclic; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D042822:Genomic Instability; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000201:enzymology; D053124:Influenza A Virus, H5N1 Subtype / Q000187:drug effects* / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D053139:Oseltamivir / Q000494:pharmacology; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D014774:Virulence; D014779:Virus Replication; D053243:Zanamivir / Q000494:pharmacology
chemical_list: D000146:Acids, Carbocyclic; D000998:Antiviral Agents; D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D006146:Guanidines; D050505:Mutant Proteins; D014764:Viral Proteins; D053139:Oseltamivir; C487630:NA protein, influenza A virus; D009439:Neuraminidase; D053243:Zanamivir; C414210:peramivir