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, 16 (2), e1008102
eCollection

"Submergence" of Western Equine Encephalitis Virus: Evidence of Positive Selection Argues Against Genetic Drift and Fitness Reductions

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"Submergence" of Western Equine Encephalitis Virus: Evidence of Positive Selection Argues Against Genetic Drift and Fitness Reductions

Nicholas A Bergren et al. PLoS Pathog.

Abstract

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
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.
Fig 2
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
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
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
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
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
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.

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Grant support

The research reported herein was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (https://www.niaid.nih.gov/) under award numbers 5T32AI060549-12 and R24AI120942. NAB received the T32 award and SCW received the R24 award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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