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Clin Microbiol Infect. 2013 Jul;19(7):668-73. doi: 10.1111/j.1469-0691.2012.03984.x. Epub 2012 Aug 3.

Detection of haemagglutinin D222 polymorphisms in influenza A(H1N1)pdm09-infected patients by ultra-deep pyrosequencing.

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  • 1Laboratory of Virology, National Institute for Infectious Diseases INMI IRCCS L. Spallanzani, Rome, Italy.


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.

© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

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