title: Genetic diversity among pandemic 2009 influenza viruses isolated from a transmission chain.
abstract: BACKGROUND
  Influenza viruses such as swine-origin influenza A(H1N1) virus (A(H1N1)pdm09) generate genetic diversity due to the high error rate of their RNA polymerase, often resulting in mixed genotype populations (intra-host variants) within a single infection. This variation helps influenza to rapidly respond to selection pressures, such as those imposed by the immunological host response and antiviral therapy. We have applied deep sequencing to characterize influenza intra-host variation in a transmission chain consisting of three cases due to oseltamivir-sensitive viruses, and one derived oseltamivir-resistant case.
  
  
  METHODS
  Following detection of the A(H1N1)pdm09 infections, we deep-sequenced the complete NA gene from two of the oseltamivir-sensitive virus-infected cases, and all eight gene segments of the viruses causing the remaining two cases.
  
  
  RESULTS
  No evidence for the resistance-causing mutation (resulting in NA H275Y substitution) was observed in the oseltamivir-sensitive cases. Furthermore, deep sequencing revealed a subpopulation of oseltamivir-sensitive viruses in the case carrying resistant viruses. We detected higher levels of intra-host variation in the case carrying oseltamivir-resistant viruses than in those infected with oseltamivir-sensitive viruses.
  
  
  CONCLUSIONS
  Oseltamivir-resistance was only detected after prophylaxis with oseltamivir, suggesting that the mutation was selected for as a result of antiviral intervention. The persisting oseltamivir-sensitive virus population in the case carrying resistant viruses suggests either that a small proportion survive the treatment, or that the oseltamivir-sensitive virus rapidly re-establishes itself in the virus population after the bottleneck. Moreover, the increased intra-host variation in the oseltamivir-resistant case is consistent with the hypothesis that the population diversity of a RNA virus can increase rapidly following a population bottleneck.
authors.affiliation: Centre for GeoGenetics, Natural History Museum of Denmark, Øster Voldgade 5-7, Copenhagen K, 1350, Denmark. LPNielsen@snm.ku.dk
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Fordyce
authors.forename: Karoline
authors.initials: K
authors.lastname: Bragstad
authors.forename: Svend Stenvang
authors.initials: SS
authors.lastname: Pedersen
authors.forename: Thøger G
authors.initials: TG
authors.lastname: Jensen
authors.forename: Bente
authors.initials: B
authors.lastname: Gahrn-Hansen
authors.forename: Rod
authors.initials: R
authors.lastname: Daniels
authors.forename: Alan
authors.initials: A
authors.lastname: Hay
authors.forename: Marie-Louise
authors.initials: ML
authors.lastname: Kampmann
authors.forename: Christian A W
authors.initials: CA
authors.lastname: Bruhn
authors.forename: J Victor
authors.initials: JV
authors.lastname: Moreno-Mayar
authors.forename: María C
authors.initials: MC
authors.lastname: Ávila-Arcos
authors.forename: M Thomas P
authors.initials: MT
authors.lastname: Gilbert
authors.forename: Lars P
authors.initials: LP
authors.lastname: Nielsen
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D009439:Neuraminidase / Q000235:genetics*; D053139:Oseltamivir / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics; D012641:Selection, Genetic; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D014764:Viral Proteins; D053139:Oseltamivir; C487630:NA protein, influenza A virus; D009439:Neuraminidase