Fig 1.
Maximum clade credibility tree based on thirty-three WEEV genomes.
Numbers at nodes indicate posterior probabilities of ≥ 0.9. Bars at nodes indicate 95% confidence intervals of divergence dates, and the x-axis represents time in years. The four distinct lineages, groups A and B1 through B3, are indicated. Nonsynonymous synapomorphic mutations are indicated on the tree based on their identified node of occurrence. Taxon/tip labels include year of isolation, strain name, and state where the virus was isolated. Figure originally published by Bergren et al. (2014) Western Equine Encephalitis Virus: Evolutionary Analysis of a Declining Alphavirus Based on Complete Genome Sequences. Journal of Virology. 2014;88(16):9260–7. doi: 10.1128/jvi.01463-14. https://jvi.asm.org/content/88/16/9260.
Table 1.
Nonsynonymous synapomorphic mutations that contribute to the definition of WEEV lineages.
Fig 2.
Diagram of viruses used throughout the study and their relevant mutations.
Amino acid residues listed on WEEV/IMP181 and WEEV/BFS932 reflect unaltered amino acids at sites of interest. Amino acids with an asterisk reflect changes made to derive the specific construct. †Note that WEEV/BFS932 is not derived from an infectious clone but is a plaque purified isolate.
Fig 3.
Competition assays in C. tarsalis showing the ratios of virus in the blood meal and salivary glands on day 10 post-blood meal.
Mean and standard error of replicates (3 replicates with n = 5 per replicate) in assays competing A) IMP181 v. IMP181-6X (p-value 0.0005318); B) IMP181 v. IMP181-3X-NonConservative (p-value 0.0005303); C) IMP181 v. IMP181-3X-Conservative (p-value 0.01245); and D) IMP181 v. BFS932 (p-value 0.08105). *Indicates significant (p ≤ 0.05) change in virus ratio as determined by Wilcoxon test.
Fig 4.
Competition assays in HOSPs showing the ratios of virus in the inoculum and serum on days 1 and 2 post-infection.
Mean and standard error in assays competing A) IMP181 v. IMP181-6X (p-value 0.02201); B) IMP181 v. IMP181-3X-NonConservative (p-value 0.01991); C) IMP181 v. IMP181-3X-Conservative (p-value 0.25); and D) IMP181 v. BFS932 (p-value 1.0) (n = 7 per group). * Indicates significant (p ≤ 0.05) change in virus ratio as determined by Wilcoxon test.
Fig 5.
Weight, survival, and viremia in 5–6 week old Syrian golden hamsters following infection with IMP181, IMP181-6X, and BFS932.
Panels show A) weight; B) survival; and C) viremia. *Indicates statistical significance (p ≤ 0.05).
Fig 6.
Viral burden in 5–6 week old Syrian golden hamsters that show significant differences following infection with IMP181, IMP181-6X, and BFS932.
Panels show viral burden in the (A) brain, (B) heart; (C) muscle; (D) kidney. *Indicates statistical significance (p ≤ 0.05).
Fig 7.
Differences in histopathology during peak disease of 5–6 week old Syrian golden hamsters infected with IMP181, IMP181-6X, and BFS932.
Brain and muscle images at 10X; perivascular cuffing, hemorrhage, and mononuclear infiltration marked with blue circles, red arrows, and green arrows, respectively. Yellow arrows on muscle slides indicate myositis. Liver and Lung images taken at 20X; yellow arrows on liver slides indicate foci of necrosis. All BFS932 images are from day 4 post-infection. IMP181-6X, IMP181, and MOCK were taken at day 5 post-infection.
Table 2.
Primers and viral sequence analyzed in pyrosequencing assay.