Figure 2. Changes in E1 RNA sequence alters viral replication fitness in macrophages, but not other cell types.

(A). Schematic representation of the TC83 and TC83 E1 RNA mutant (TC83/E1_ID-syn) genomes. Synonymous SNPs from E1 of enzootic VEEV (strain 307537) were introduced into the vaccine epizootic VEEV (strain TC83) to generate an RNA mutant (TC83/E1_ID-syn). The single coding change present in E1 (Fig 1C, asterisk) was omitted from the mutant. Red denotes sequences from the parent epizootic strain (TC83), and blue denotes sequences from the enzootic strain (307537). (B-E). Replication kinetics of VEEV TC83 and TC83/E1_ID-syn in (B) Raw264.7, (C) primary bone marrow-derived macrophages (BMDMs), (D) primary bone marrow-derived dendritic cells (BMDCs), and (E) immortalized mouse embryonic fibroblasts (iMEF). Cells were infected with indicated viruses at a MOI of 0.1 (Raw264.5, BMDMs, iMEF) or MOI 0.01 (BMDCs). Cell culture supernatant was serially harvested at 1, 6, 12, 24, 36, and 48 hpi and infectious virus was titered using focus forming assay (FFA). Each experiment was performed in triplicate three to four times independently and the mean and SD are graphed. Statistical analysis was performed by calculating the area under the curve (AUC) for each replicate, and the AUC values from WT and mutant viruses were analyzed by unpaired t-test.