title: Evolution of oseltamivir resistance mutations in Influenza A(H1N1) and A(H3N2) viruses during selection in experimentally infected mice.
abstract: The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC50s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC50 level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses.
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Andrés
authors.initials: A
authors.lastname: Pizzorno
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Yacine
authors.initials: Y
authors.lastname: Abed
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Pier-Luc
authors.initials: PL
authors.lastname: Plante
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Julie
authors.initials: J
authors.lastname: Carbonneau
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Mariana
authors.initials: M
authors.lastname: Baz
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Marie-Ève
authors.initials: MÈ
authors.lastname: Hamelin
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada.
authors.forename: Jacques
authors.initials: J
authors.lastname: Corbeil
authors.affiliation: Research Center in Infectious Diseases of the CHU of Québec and Laval University, Québec City, Quebec, Canada guy.boivin@crchul.ulaval.ca.
authors.forename: Guy
authors.initials: G
authors.lastname: Boivin
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D004285:Dogs; D024882:Drug Resistance, Viral / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019143:Evolution, Molecular; D006389:Hemagglutinins, Viral / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000235:genetics; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects* / Q000235:genetics; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D009439:Neuraminidase / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000188:drug therapy / Q000821:virology; D053139:Oseltamivir / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic; D017423:Sequence Analysis, RNA; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D006389:Hemagglutinins, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D053139:Oseltamivir; D009439:Neuraminidase
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