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. 2024 Feb 26;9:45. doi: 10.1038/s41541-024-00827-x

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic of chimeric IGIP-HA segment 4 of FLUBV. The IGIP sequence is cloned downstream the HA signal peptide sequence and upstream of peptide sequences that allow the release of IGIP from the mature HA protein. b Gene segment stability. Rearranged PB1 (left panel) and IGIP-HA (middle panel) in FluB-RAM/IGIP and FluB-RANS/IGIP analyzed by RT-PCR with specific primers (sequences available upon request). Differences in migration patterns of E1 and E7 PCR products and corresponding non-IGIP plasmids used as controls. MS-RT-PCR (right panel) of non-IGIP FLUBV strain control and FluB-att/IGIP strain. Migration pattern of HA between the two strains is shown and compared to a molecular weight marker ladder on the left. c Growth kinetics analyses at different temperatures of recombinant FLUBV constructs and FLUBV wild type strain. Yellow symbols correspond to FluB-RAM/IGIP (triangle) and FluB-RANS/IGIP (squares). The FLUBV wild type (FLUBV wt, red symbols) and non-IGIP FLUBV (gray symbols) growth kinetics curves were previously reported and shown in this graph for comparison since those were performed simultaneously^18,29. Virus growth kinetics were analyzed using the Gompertz growth non-linear regression model. Differences in growth rate between viruses were calculated by AUC analysis, followed by Brown–Forsythe and Welch ANOVA plus Dunnett’s T3 post hock analysis. Two-way ANOVA was employed to determine virus growth differences by timepoint. Statistically significant differences between viruses’ growth rates are denoted by different letters. d Detection of BHA and BNP expression in vitro via Western blot. MDCK cells were infected with FluB-RAM, FluB-RAM/IGIP, FluB-att, FluB-att/IGIP, and B/Bris WT. Protein extracts were used to detect FLUBV-HA and FLUBV-NP. As a loading control, GADPH was detected (additional details in Supplementary Fig. 1). e, f Absence of anti-IGIP antibodies after vaccination with FluB-RAM/IGIP. e Protein extracts of cells infected with FLUAV viruses (with and without IGIP), FluB-RAM, and FluB-RAM/IGIP were analyzed by Western blot using polyclonal antibody (pAb) from mice vaccinated with FluB-RAM/IGIP. GADPH was detected as a loading control (additional details in Supplementary Fig. 2). f HEK293T cells were transfected with 10 µg of pCAGGS GFP-IGIP, pCAGGS GFP, or pCAGGS empty. A mAb anti-IGIP, mAb anti-GFP, or pAb FluB-RAM/IGIP was used. GADPH was detected as a loading control (additional details in Supplementary Fig. 3). Numbers on the right in d–f indicate molecular weights from molecular weight markers. The predicted molecular weights of target antigens are denoted in italics.