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. 2017 Oct 25;7:13983. doi: 10.1038/s41598-017-14522-6

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© The Author(s) 2017

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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Effect of the temperature on the efficiency of virus rescue and on the efficacy of transfection in C6/36 cells. (a) West Nile Virus (WNV) infectious clone (2.5ng, 5ng, 10ng and 50ng) under the control of human cytomegalovirus promoter (pCMV) was transfected in C6/36 cells at 28 °C or 37 °C in quadruplicate. Viral RNA copies were quantified by RT-qPCR in cell supernatant at the first passage (upper panel). The % of success to recover a virus is reported in the lower panel. (b) ISA procedure was applied to WNV by transfecting 3 overlapping DNA amplicons (100ng, 500ng and 1000ng) covering the entire viral genome under the control of human cytomegalovirus promoter (pCMV) in C6/36 cells at 28 °C or 37 °C in quadruplicate. Viral RNA copies were quantified by RT-qPCR in cell supernatant at the first passage (upper panel). The % of success to recover a virus is reported in the lower panel. (c) Expression of eGFP in C6/36 cells measured 2 days after transfection of the eGFP gene under the control of the D. melanogaster actin 5C promoter at 28 and 37 °C (left panel). Bar graph representing the relative fluorescence intensity (right panel).