PMC

Survival curves for the TC-83 mutants in 6-day-old CD-1 mice following intracranial injection (A) and subcutaneous injection (B) of 10⁵ PFU. Statistical differences were determined using Kaplan-Meier tests, and significance is detailed in the text.

Survival curves for the TC-83 mutants in 6-day old CD-1 mice following intracranial injection (A) and subcutaneous injection (B) of 10⁵ PFU from novel RdRp mutant virus rescues. R1 denotes replicate 1. R2 denotes replicate 2. Statistical differences were determined using Kaplan-Meier tests and are detailed in the text.

Illumina sequencing virus mutation frequency analysis of TC-83, 3x and 4x after 1 passage on Vero, HEK-293, C7/10 or U4.4 cells. Overall mutation frequency for each cell type (A). Number of genomic positions with minority variants present above a frequency of 0.001 (B). Mutation frequency of different mutations (C). TC-83 3x: blue; TC-83 4x: green; TC-83: red. Repeated measures ANOVA was used to determine statistical significance, P < 0.05 is represented by *, P < 0.01 is represented by **, P < 0.001 is represented by *** and P < 0.0001 is represented by ****.

Illumina sequencing virus diversity hotspots for the coding regions of TC-83, 3x and 4x genomes after 1 passage on Vero, HEK-293, C7/10 or U4.4 cells. A representative replicate is pictured for each virus isolate from each cell type. For full results, see . TC-83 (A, D, G, J), TC-83 3x (B, E, H, K) and TC-83 4x (C, F, I, L). Vero (A, B, C), HEK-293 (D, E, F), C7/10 (G, H, I) and U4.4 (J, K, L). Genome organization is color-coded using the following: nsP1, blue; nsP2, red; nsP3, green; nsP4, purple; capsid, orange; E3, black; E2, gold; 6k, navy blue; and E1, maroon.

Weight change, immunogenicity and survival following vaccination and challenge. Five-week-old mice were injected with 10⁵ PFU of TC-83, 3x, 4x or mock in a 50ul volume (n = 8 per vaccine treatment). Mice were monitored daily for percent weight change from the original weight at D0 (A). The effectiveness of the vaccine was determined by neutralizing antibody titer using serum from 6-week post-vaccination (B), and statistical differences were determined by repeated measures ANOVA, where P < 0.01 is represented by **. Mice were challenged with 10⁵ PFU of VEEV strain 3908 and were monitored for percent weight change from the original weight at D0 (C) and survival (D).

Survival of 6-day old CD-1 mice following subcutaneous injection with 10⁵ PFU in a 50 µl volume of Passages 0, 5 or 10 TC-83, 3x or 4x from infant mouse intracranial passages (A). Statistical differences were determined using Kaplan-Meier tests, and significance is detailed in the text. Illumina-sequencing diversity analysis was used to determine changes in virus diversity of passage 5 TC-83, 3x or 4x in infant mice (B). Mutation frequency of the E2 8922 mutation required for TC-83 attenuation (C). Illumina sequencing virus diversity hotspots for the coding regions of TC-83 (D), 3x (E) and 4x (F) genomes after five intracranial passages. Genome organization is color-coded using the following: nsP1, blue; nsP2, red; nsP3, green; nsP4, purple; capsid, orange; E3, black; E2, gold; 6k, navy blue; and E1, maroon.

A schematic of the TC-83 genome showing the placement of the four mutations in the RdRp gene (A). Growth curves of TC-83 and the RdRp mutants in interferon negative Vero cells (B) or interferon competent HEK-293 cells (C). Experiments were performed in triplicate and titers were determined by standard plaque assay. Repeated measures ANOVA was used to determine statistical significance, and bars indicate standard deviation. Resistance of TC-83 mutants to treatment by 5FU (D). Cells were treated with 0, 10, 25, 50, 100, 200 or 300 µg/ml of 5FU and infected using an MOI of 0.01; all experiments were performed in triplicate. Samples were titered by standard plaque assay. Statistical differences were determined by repeated measures ANOVA, and significance is detailed in the text. Resistance of TC-83 mutants to treatment by Ribavirin (E). Cells were treated with 0, 50, 100, 200, 300 or 400 µM Ribavirin and infected using an MOI of 0.01; all experiments were performed in triplicate. Samples were titered by standard plaque assay. Statistical differences were determined by repeated measures ANOVA, and significance is detailed in the text.