title: Viral subpopulation diversity in influenza virus isolates compared to clinical specimens.
abstract: BACKGROUND
  Influenza virus (IFV) isolates obtained from mammalian cell cultures are valuable reagents used for vaccine production, antigenic characterization, laboratory assays, and epidemiological and evolutionary studies. Complete genomic comparison of IFV isolates with their original clinical specimens provides insight into cell culture-driven genomic changes which may sequentially alter the virus phenotype.
  
  
  OBJECTIVE
  The genome of the viral isolates and of the viruses in the clinical specimens was examined by deep sequencing in order to determine nucleotide heterogeneity (measured number of variances or numbers of mixed bases) as a marker for IFV population diversity.
  
  
  METHODS
  Clinical respiratory specimens were collected between July and October 2012 and identified by RT-PCR as positive for influenza A H3N2 or H1N1, or influenza B. The viruses in the clinical specimens were amplified using mammalian cell culture. Next generation sequencing (NGS) was used to investigate genomic differences between IFV isolates and their corresponding clinical specimens.
  
  
  RESULTS
  There was less nucleotide heterogeneity in 5 of 6 viral isolates compared to the corresponding clinical specimens, especially for influenza B. A phylogenetic analysis of the hemagglutinin (HA) gene consensus sequences obtained from deep and Sanger sequencing showed that the viral isolates and their corresponding clinical specimens contained the same IFV strains with less than 5% pair-wise genetic distance.
  
  
  CONCLUSIONS
  The IFV sequence data analysis detected a substantial decrease in nucleotide heterogeneity from clinical specimens to viral cultures in 5 out of 6 investigated cases.
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: Wiriyar@afrims.org.
authors.forename: W
authors.initials: W
authors.lastname: Rutvisuttinunt
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: PiyawanC@afrims.org.
authors.forename: P
authors.initials: P
authors.lastname: Chinnawirotpisan
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: ButsayaT@afrims.org.
authors.forename: B
authors.initials: B
authors.lastname: Thaisomboonsuk
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: PrinyadaR@afrims.org.
authors.forename: P
authors.initials: P
authors.lastname: Rodpradit
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: ChuanpisA@afrims.org.
authors.forename: C
authors.initials: C
authors.lastname: Ajariyakhajorn
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: WudtichaiM@afrims.org.
authors.forename: W
authors.initials: W
authors.lastname: Manasatienkij
authors.affiliation: Phramongkutklao Hospital, Bangkok, Thailand. Electronic address: ssriluck@hotmail.com.
authors.forename: S
authors.initials: S
authors.lastname: Simasathien
authors.affiliation: Walter Reed/ AFRIMS Research Unit Nepal, Kathmandu, Nepal. Electronic address: ShresthaSK@afrims.org.
authors.forename: S K
authors.initials: SK
authors.lastname: Shrestha
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: YoonI@afrims.org.
authors.forename: I K
authors.initials: IK
authors.lastname: Yoon
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: ChontichaK@afrims.org.
authors.forename: C
authors.initials: C
authors.lastname: Klungthong
authors.affiliation: Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: Fernandezs@afrims.org.
authors.forename: S
authors.initials: S
authors.lastname: Fernandez
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D062665:Epidemiological Monitoring; D005260:Female; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009981:Influenza B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D008297:Male; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D055815:Young Adult
chemical_list: D012367:RNA, Viral
keywords: Clinical specimens; Influenza; Next generation Sequencing; Variances; Viral isolates; Viral subpopulations