title: A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus.
abstract: A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Huan
authors.initials: H
authors.lastname: Fan
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Xian
authors.initials: X
authors.lastname: Qi
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Xiling
authors.initials: X
authors.lastname: Guo
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Yiyue
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Kangchen
authors.initials: K
authors.lastname: Zhao
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wu
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Shan
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Minghao
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Zhiyang
authors.initials: Z
authors.lastname: Shi
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China.
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Lab of Enteric Pathogenic Microbiology (Ministry of Health), Institute of Pathogenic Microbiology, Jiangsu Center for Disease Prevention and Control, 172 Jiangsu Road, Nanjing 210009, PR China. Electronic address: lbcui@jscdc.cn.
authors.forename: Lunbiao
authors.initials: L
authors.lastname: Cui
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D002681:China; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D064766:Influenza A Virus, H7N9 Subtype / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D053139:Oseltamivir / Q000494:pharmacology; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D014764:Viral Proteins; D053139:Oseltamivir; C487630:NA protein, influenza A virus; D009439:Neuraminidase
keywords: Avian influenza A (H7N9) virus; E120V mutation; NAIs resistance; Pyrosequencing