title: Sequence amplification via cell passaging creates spurious signals of positive adaptation in influenza virus H3N2 hemagglutinin.
abstract: Clinical influenza A virus isolates are frequently not sequenced directly. Instead, a majority of these isolates (~70% in 2015) are first subjected to passaging for amplification, most commonly in non-human cell culture. Here, we find that this passaging leaves distinct signals of adaptation, which can confound evolutionary analyses of the viral sequences. We find distinct patterns of adaptation to Madin-Darby (MDCK) and monkey cell culture absent from unpassaged hemagglutinin sequences. These patterns also dominate pooled datasets not separated by passaging type, and they increase in proportion to the number of passages performed. By contrast, MDCK-SIAT1 passaged sequences seem mostly (but not entirely) free of passaging adaptations. Contrary to previous studies, we find that using only internal branches of influenza virus phylogenetic trees is insufficient to correct for passaging artifacts. These artifacts can only be safely avoided by excluding passaged sequences entirely from subsequent analysis. We conclude that future influenza virus evolutionary analyses should appropriately control for potentially confounding effects of passaging adaptations.
authors.affiliation: Center for Systems and Synthetic Biology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712.
authors.forename: Claire D
authors.initials: CD
authors.lastname: McWhite
authors.affiliation: Center for Systems and Synthetic Biology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; Center for Computational Biology and Bioinformatics, The University of Texas at Austin, Austin, TX 78712; Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712.
authors.forename: Austin G
authors.initials: AG
authors.lastname: Meyer
authors.affiliation: Center for Systems and Synthetic Biology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712; Center for Computational Biology and Bioinformatics, The University of Texas at Austin, Austin, TX 78712; Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712.
authors.forename: Claus O
authors.initials: CO
authors.lastname: Wilke