1. Fig. 2 BinJV and BinJ/VIF-prME viruses do not replicate in vertebrate cells.
BinJV and BinJ/VIF-prME viruses do not replicate in vertebrate cells. (A) IFA analysis by confocal microscopy of WNVKUN, BinJV, and BinJ/WNVKUN-prME virus–infected C6/36 cells and mammalian cells. Cells were fixed and immunolabeled 5 days after infection and culture at 37°C (top) or 34°C (bottom). Mammalian cells: BSR (baby hamster kidney), WT MEFs (wild-type murine embryonic fibroblasts), IFNAR−/− MEFs, and Vero cells (African green monkey kidney). Viruses were immunolabeled with mAb 4G4 (green), and cell nuclei were stained with Hoechst (blue). (B) IFA analysis of C6/36 and Vero cells transfected with RNA derived from WNVKUN, BinJV, and BinJ/WNVKUN-prME viruses. Cells were fixed and immunolabeled 5 days after transfection after culture at the indicated temperatures as described for (A). (C) RNA from BinJ/WNVKUN-prME virus, WNVKUN, or BinJV was used to transfect the indicated cell lines (x axis) and cultured for 5 days. Virus titers in the supernatants (three biological replicates) were then determined by CCID50 assays using C6/36 cells and mAb 4G4. The limit of detection was 2 log10CCID50/ml. (D) BinJ/VIF-prME viruses and WNVKUN were used to infect C6/36 cells and a panel of vertebrate cells. Cells were fixed and immunolabeled as in (A) 5 days after infection and cultured at 28°C (C6/36) or 37°C for the vertebrate cell lines: VSW (viper), 3CPL (crocodile), DF-1 (chicken), Vero (monkey), and IFNAR−/− MEFs (mouse). As a positive control, all the vertebrate cell lines were shown to be replication competent for WNVKUN (bottom row). Note that the IFA images for the infection of Vero cells and IFNAR−/− MEFs with BinJ/WNVKUN-prME appear in (A).
Jody Hobson-Peters et al., Sci Transl Med 2019;11:eaax7888
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works