Fig 1. Chimeric EIL/VEEV viruses do not replicate in vertebrate cells, and package subgenomic RNAs into viral particles in mosquito cells.

(A) The schematic representation of EILV, VEEV TC-83 and recombinant viral genomes. (B) Replication of EIL/VEEV and EIL/nLuc/VEEV in C7/10 and NIH 3T3 cells. Cells were infected at an MOI of 20 PFU/cell. Media were harvested at the indicated times post infection, and titers were determined by plaque assay on C7/10 cells. (C) Concentrations of genomic RNA, SG RNA 1 and SG RNA 2 in the sample of EIL/GFP/VEEV harvested at 48 h PI of C7/10 cells. The number of copies per ml of virus were determined by RT-qPCR using primers specific to EILV nsP2, GFP and VEEV E2 genes as described in Materials and Methods. (D) C7/10 cells were infected with EIL/GFP/VEEV at an MOI of 20 PFU/cell, and viral RNA were labeled with [³H]uridine between 16 and 24 h post infection. RNAs were isolated from both the cells and the released viral particles and analyzed by agarose gel electrophoresis in denaturing conditions.