title: Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells.
abstract: The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA1 D130E, HA2 I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production.
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Xenia
authors.initials: X
authors.lastname: Wörmann
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Lesch
authors.affiliation: Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin, Germany.
authors.forename: Robert-William
authors.initials: RW
authors.lastname: Welke
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Okonechnikov
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Mirshat
authors.initials: M
authors.lastname: Abdurishid
authors.affiliation: Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Sieben
authors.affiliation: Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Institute of Chemistry and Biochemistry, Free University, Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Geissner
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Brinkmann
authors.affiliation: Institute for Biophysics, Johannes Kepler University, Linz, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Kastner
authors.affiliation: Center for Advanced Bioanalysis GmbH (CBL), Linz, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Karner
authors.affiliation: Institute for Biophysics, Johannes Kepler University, Linz, Austria.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.affiliation: Institute for Biophysics, Johannes Kepler University, Linz, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.affiliation: Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany.
authors.forename: Chakkumkal
authors.initials: C
authors.lastname: Anish
authors.affiliation: Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, Potsdam, Germany; Institute of Chemistry and Biochemistry, Free University, Berlin, Germany.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Seeberger
authors.affiliation: Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee, Germany. Electronic address: meyer@mpiib-berlin.mpg.de.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Meyer
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee, Germany. Electronic address: karlas@mpiib-berlin.mpg.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Karlas
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics*; D000818:Animals; D002645:Chickens; D004285:Dogs; D004847:Epithelial Cells / Q000821:virology; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000254:growth & development; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D059386:Reverse Genetics; D012692:Serial Passage; D014779:Virus Replication / Q000235:genetics
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D011991:Receptors, Virus
keywords: A/Hamburg/04/2009; Hemagglutinin; High-throughput sequencing; Reverse genetics; Viral adaptation