title: Rapid strategy for screening by pyrosequencing of influenza virus reassortants--candidates for live attenuated vaccines.
abstract: BACKGROUND
  Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses.
  
  
  RESULTS
  This manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures.
  
  
  CONCLUSIONS
  The present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.
authors.affiliation: Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America; Battelle, Atlanta, Georgia, United States of America.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Shcherbik
authors.affiliation: Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America; Battelle, Atlanta, Georgia, United States of America.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Pearce
authors.affiliation: Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America; Battelle, Atlanta, Georgia, United States of America.
authors.forename: Marnie L
authors.initials: ML
authors.lastname: Levine
authors.affiliation: Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Klimov
authors.affiliation: Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Villanueva
authors.affiliation: Virology, Surveillance and Diagnosis Branch, Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Tatiana L
authors.initials: TL
authors.lastname: Bousse
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000276:immunology; D007252:Influenza Vaccines / Q000235:genetics / Q000276:immunology; D016865:Reassortant Viruses / Q000235:genetics* / Q000276:immunology*; D014613:Vaccines, Attenuated / Q000276:immunology*
chemical_list: D007252:Influenza Vaccines; D014613:Vaccines, Attenuated