title: The use of pyrosequencer-generated sequence-signatures to identify the influenza B-lineage and the subclade of the B/Yamataga-lineage viruses from currently circulating human influenza B viruses.
abstract: BACKGROUND
  Influenza B viruses belong to two antigenically and genetically distinct lineages which co-circulate in varying proportions in many countries.
  
  
  OBJECTIVE
  To develop simple, rapid, accurate and robust methods to detect and differentiate currently circulating B-lineage viruses in respiratory samples and virus isolates.
  
  
  METHODS
  Haemagglutinin (HA) gene sequences from more than 6300 influenza B strains were analysed to identify signature sequences that could be used to distinguish between B-lineages and sublineages.
  
  
  RESULTS
  Pyrosequencing and a real time PCR assays were developed to detect the major B-lineages (B/Victoria/2/87 or B/Yamagata/16/88) and pyrosequencing for a unique mutation was used to further differentiate the B/Yamagata viruses into two currently co-circulating subgroups. More than 300 influenza virus-containing samples, including original specimens, cell and egg grown viruses, were tested with a 100% accuracy. Furthermore, when the same PCR primers were used in an rRT-PCR assay, the two lineages could be differentiated by their distinct ranges of melting temperature with an overall accuracy of 99% for 158 samples tested.
  
  
  CONCLUSIONS
  These new pyrosequencing and rRT-PCR methods have the potential to aid the rapid identification of influenza B-lineages for surveillance purposes and to increase the available data for bi-annual selection of viruses for updating influenza vaccines.
authors.affiliation: WHO Collaborating Centre for Reference and Research on Influenza, 10 Wreckyn Street, North Melbourne, VIC 3051, Australia.
authors.forename: Yi-Mo
authors.initials: YM
authors.lastname: Deng
authors.forename: Pina
authors.initials: P
authors.lastname: Iannello
authors.forename: Natalie
authors.initials: N
authors.lastname: Caldwell
authors.forename: Lauren
authors.initials: L
authors.lastname: Jelley
authors.forename: Naomi
authors.initials: N
authors.lastname: Komadina
authors.forename: Chantal
authors.initials: C
authors.lastname: Baas
authors.forename: Anne
authors.initials: A
authors.lastname: Kelso
authors.forename: Ian G
authors.initials: IG
authors.lastname: Barr
mesh_terms: D005838:Genotype; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D009981:Influenza B virus / Q000145:classification* / Q000235:genetics*; D007251:Influenza, Human / Q000821:virology*; D017720:Molecular Epidemiology / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus
keywords: B-lineages; HA; Influenza B; MDCK; Madin-Darby canine kidney; NA; Pyrosequencing; Real-time RT-PCR; T(m); haemagglutinin; melting temperature; neuraminidase; rRT-PCR; real-time reverse transcriptase polymerase chain reaction