title: High-Efficiency Capture of Drug Resistant-Influenza Virus by Live Imaging of Sialidase Activity.
abstract: Influenza A and B viruses possess a neuraminidase protein that shows sialidase activity. Influenza virus-specific neuraminidase inhibitors (NAIs) are commonly used for clinical treatment of influenza. However, some influenza A and B viruses that are resistant to NAIs have emerged in nature. NAI-resistant viruses have been monitored in public hygiene surveys and the mechanism underlying the resistance has been studied. Here, we describe a new assay for selective detection and isolation of an NAI-resistant virus in a speedy and easy manner by live fluorescence imaging of viral sialidase activity, which we previously developed, in order to achieve high-efficiency capture of an NAI-resistant virus. An NAI-resistant virus maintains sialidase activity even at a concentration of NAI that leads to complete deactivation of the virus. Infected cells and focuses (infected cell populations) of an oseltamivir-resistant virus were selectively visualized by live fluorescence sialidase imaging in the presence of oseltamivir, resulting in high-efficiency isolation of the resistant viruses. The use of a combination of other NAIs (zanamivir, peramivir, and laninamivir) in the imaging showed that the oseltamivir-resistant virus isolated in 2008 was sensitive to zanamivir and laninamivir but resistant to peramivir. Fluorescence imaging in the presence of zanamivir also succeeded in selective live-cell visualization of cells that expressed zanamivir-resistant NA. Fluorescence imaging of NAI-resistant sialidase activity will be a powerful method for study of the NAI resistance mechanism, for public monitoring of NAI-resistant viruses, and for development of a new NAI that shows an effect on various NAI-resistant mutations.
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Yuuki
authors.initials: Y
authors.lastname: Kurebayashi
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Tadanobu
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Tamoto
authors.affiliation: Shizuoka Institute of Environment and Hygiene, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Sahara
authors.affiliation: Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, Kure-shi, Hiroshima, Japan.
authors.forename: Tadamune
authors.initials: T
authors.lastname: Otsubo
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yokozawa
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.|Shizuoka City Institute of Environmental Sciences and Public Health, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Nona
authors.initials: N
authors.lastname: Shibahara
authors.affiliation: Shizuoka City Institute of Environmental Sciences and Public Health, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Hirohisa
authors.initials: H
authors.lastname: Wada
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Minami
authors.affiliation: Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, Kure-shi, Hiroshima, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ikeda
authors.affiliation: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka-shi, Shizuoka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004285:Dogs; D024882:Drug Resistance, Viral; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000201:enzymology*; D020128:Inhibitory Concentration 50; D061985:Madin Darby Canine Kidney Cells; D009439:Neuraminidase / Q000378:metabolism*; D061848:Optical Imaging; D053139:Oseltamivir / Q000494:pharmacology
chemical_list: D053139:Oseltamivir; D009439:Neuraminidase