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. 2018 Mar 12;8:4374. doi: 10.1038/s41598-018-22307-8

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Relevance of amino acids 129 and 180 for the binding of KPF1 hmAb. (a) Binding of KPF1 hmAb to WT and mutant HA proteins. HEK293T cells were transiently transfected with the pCAGGS plasmids expressing WT or amino acid substitutions E129K, K180N, K180Q or E129K/K180N mutant HAs. Mock transfected cells were used as internal control. At 24 h post-transfection, cells were fixed and protein expression was evaluated by IFA using the hmAb KPF1, or a goat pH1N1 anti-HA polyclonal antibody as a control. DAPI was used for nuclear staining. Merge from representative images (10x magnification) are included. Scale bar, 50 nm. (b) Frequency of amino acid changes found in IAV H1N1 HA over time. Publicly available sequences of IAV H1N1 HA protein (Influenza Research Database) isolated between 2000–2009 (n = 8,586; black) or 2010–2018 (n = 8,417; grey) were analyzed and plotted according to the percentage of sequences containing the indicated amino acids at positions 129 (right) or 180 (left).