title: Rapid discrimination of oseltamivir-resistant 275Y and -susceptible 275H substitutions in the neuraminidase gene of pandemic influenza A/H1N1 2009 virus by duplex one-step RT-PCR assay.
abstract: Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir-resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir-resistant A/H1N1pdm viruses possessing H275Y, a duplex one-step RT-PCR assay (H275Y RT-PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT-PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty-four clinical specimens were tested after RNA purification using the H275Y RT-PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy-three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT-PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir-resistant A/H1N1pdm virus activity.
authors.affiliation: Influenza Virus Research Center, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan.
authors.forename: Mina
authors.initials: M
authors.lastname: Nakauchi
authors.forename: Makoto
authors.initials: M
authors.lastname: Ujike
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Obuchi
authors.forename: Emi
authors.initials: E
authors.lastname: Takashita
authors.forename: Ikuyo
authors.initials: I
authors.lastname: Takayama
authors.forename: Miho
authors.initials: M
authors.lastname: Ejima
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Oba
authors.forename: Nami
authors.initials: N
authors.lastname: Konomi
authors.forename: Takato
authors.initials: T
authors.lastname: Odagiri
authors.forename: Masato
authors.initials: M
authors.lastname: Tashiro
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kageyama
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004285:Dogs; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics*; D007251:Influenza, Human / Q000188:drug therapy / Q000453:epidemiology / Q000235:genetics* / Q000821:virology; D007564:Japan; D058889:Molecular Typing / Q000379:methods*; D020125:Mutation, Missense; D009439:Neuraminidase / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology*; D058873:Pandemics; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014764:Viral Proteins / Q000235:genetics*; D053243:Zanamivir / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D014764:Viral Proteins; D053139:Oseltamivir; D009439:Neuraminidase; D053243:Zanamivir