title: Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing.
abstract: This study was aimed at establishing the genetic heterogeneity of influenza virus haemagglutinin (HA) gene quasi-species and the polymorphisms at codon 222, by application of ultra-deep pyrosequencing (UDPS) to respiratory samples from patients hospitalized for influenza A(H1N1)pdm09 infection, presenting with severe or moderate-mild disease. HA diversity was significantly higher in samples collected from patients with severe manifestations than in those from patients with moderate-mild manifestations (p 0.02). D222 polymorphism was detected in 40.7% of patients by UDPS, and in only 7.1% by Sanger sequencing. D222E, D222G, D222N and D222A were observed in 37.0%, 11.1%, 7.4% and 3.7% of patients, respectively; 10.7% of samples harboured more than two variants. The relative frequency of each single variant showed a wide range of intrapatient variation. D222G/N/A were detected, as either minor or predominant variants, only in severe cases, whereas D222E was equally represented in severe and moderate-mild infections. Other amino acid variants were observed at different positions within the analysed HA fragment. Consistent with higher heterogeneity, non-D222 variants were more frequently detected in severe cases than in moderate-mild cases. In addition, seven non-D222 mutations carried by minority variants, not previously described, were observed.
authors.affiliation: Laboratory of Virology, National Institute for Infectious Diseases INMI IRCCS L. Spallanzani, Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Selleri
authors.forename: A
authors.initials: A
authors.lastname: Piralla
authors.forename: G
authors.initials: G
authors.lastname: Rozera
authors.forename: E
authors.initials: E
authors.lastname: Giombini
authors.forename: B
authors.initials: B
authors.lastname: Bartolini
authors.forename: I
authors.initials: I
authors.lastname: Abbate
authors.forename: G
authors.initials: G
authors.lastname: Campanini
authors.forename: F
authors.initials: F
authors.lastname: Rovida
authors.forename: L
authors.initials: L
authors.lastname: Dossena
authors.forename: M R
authors.initials: MR
authors.lastname: Capobianchi
authors.forename: F
authors.initials: F
authors.lastname: Baldanti
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000473:pathology / Q000821:virology*; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000235:genetics; D012137:Respiratory System / Q000821:virology
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral