title: Detection of hemagglutinin variants of the pandemic influenza A (H1N1) 2009 virus by pyrosequencing.
abstract: For influenza viruses, pyrosequencing has been successfully applied to the high-throughput detection of resistance markers in genes encoding the drug-targeted M2 protein and neuraminidase. In this study, we expanded the utility of this assay to the detection of multiple receptor binding variants of the hemagglutinin protein of influenza viruses directly in clinical specimens. Specifically, a customized pyrosequencing protocol that permits detection of virus variants with the D, G, N, or E amino acid at position 222 in the hemagglutinin of the 2009 pandemic influenza A (H1N1) virus was developed. This customized pyrosequencing protocol was applied to the analysis of 241 clinical specimens. The use of the optimized nucleotide dispensation order allowed detection of mixtures of variants in 10 samples (4.1%) which the standard cyclic nucleotide dispensation protocol failed to detect. The optimized pyrosequencing protocol is expected to provide a more accurate tool in the analysis of virus variant composition.
authors.affiliation: Virus Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Mail Stop G-16, 1600 Clifton Road, Atlanta, GA 30333, USA.
authors.forename: Marnie
authors.initials: M
authors.lastname: Levine
authors.forename: Tiffany G
authors.initials: TG
authors.lastname: Sheu
authors.forename: Larisa V
authors.initials: LV
authors.lastname: Gubareva
authors.forename: Vasiliy P
authors.initials: VP
authors.lastname: Mishin
mesh_terms: D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000821:virology*; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral