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. 2018 May 10;8:7481. doi: 10.1038/s41598-018-25596-1

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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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Characterization of the GI JEV VLPs produced from the stable 51-10 clone. (A) The yield of VLPs was determined in the supernatant of the 51-10 clone cultured in F12 medium supplemented with 2% or 10% FBS or in serum-free medium at 37 °C or 28 °C for 3 days by Ag-ELISA. (B) The secreted GI JEV VLP antigens collected from the supernatant of the 51-10 clone were analyzed by Western blot assay with anti-JEV HIAF and are indicated as GI VLPs. The supernatant collected from GI JE YL2009–4 virus-infected or the non-infected cells is presented as JEV or NC (negative control). Full-length Western blots are presented in Supplementary Fig. S2. (C) The GI YL2009-4 virus (JEV PC) and 51-10 clone-derived VLPs underwent ultracentrifugation in a gradient of 5% to 25% sucrose. The distribution of JEV protein (Δ) and infectious activity () of JEV PC in the gradient was examined by Ag-ELISA and micro-plaque assay, respectively. The distribution of GI VLP particles (■) was detected by Ag-ELISA. (D) The morphology of purified GI JEV YL2009-4 virions (JEV PC) and GI VLP by staining with 2% uranyl acetate was analyzed by transmission electron microscopy. (E) The antigenicity of GI VLP was analyzed by Ag-ELISA with mouse anti-JEV HIAF (MHIAF) and flavivirus-reactive mAbs (4G2, 6B3B-3, 23-2, T16, 7A6C-5, 2F2, and 2H4). The supernatant collected from non-transfected CHO-HS(-) cells was used as the NC (negative control). The endpoint titers between GI virus and GI VLPs were run in duplicates and calculated by Student t test. Data are mean ± SD.

Inline graphic — Characterization of the GI JEV VLPs produced from the stable 51-10 clone. (A) The yield of VLPs was determined in the supernatant of the 51-10 clone cultured in F12 medium supplemented with 2% or 10% FBS or in serum-free medium at 37 °C or 28 °C for 3 days by Ag-ELISA. (B) The secreted GI JEV VLP antigens collected from the supernatant of the 51-10 clone were analyzed by Western blot assay with anti-JEV HIAF and are indicated as GI VLPs. The supernatant collected from GI JE YL2009–4 virus-infected or the non-infected cells is presented as JEV or NC (negative control). Full-length Western blots are presented in Supplementary Fig. S2. (C) The GI YL2009-4 virus (JEV PC) and 51-10 clone-derived VLPs underwent ultracentrifugation in a gradient of 5% to 25% sucrose. The distribution of JEV protein (Δ) and infectious activity () of JEV PC in the gradient was examined by Ag-ELISA and micro-plaque assay, respectively. The distribution of GI VLP particles (■) was detected by Ag-ELISA. (D) The morphology of purified GI JEV YL2009-4 virions (JEV PC) and GI VLP by staining with 2% uranyl acetate was analyzed by transmission electron microscopy. (E) The antigenicity of GI VLP was analyzed by Ag-ELISA with mouse anti-JEV HIAF (MHIAF) and flavivirus-reactive mAbs (4G2, 6B3B-3, 23-2, T16, 7A6C-5, 2F2, and 2H4). The supernatant collected from non-transfected CHO-HS(-) cells was used as the NC (negative control). The endpoint titers between GI virus and GI VLPs were run in duplicates and calculated by Student t test. Data are mean ± SD.