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Open AccessArticle

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles

Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA
Centre for Neglected Tropical Diseases, Departments of Vector Biology and Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
Department of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, USA
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA
Author to whom correspondence should be addressed.
Current address: Glenn Biggs Institute for Alzheimer’s and Neurodegenerative Diseases, UT Health San Antonio, San Antonio, TX 78229, USA.
Current address: Department of Microbiology and Immunology, SUNY Center for Environmental Health and Medicine, Institute for Global Health and Translational Science, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Current address: School of Life Sciences, University of Keele, Keele ST5 5BG, UK.
Viruses 2020, 12(5), 546;
Received: 3 April 2020 / Revised: 6 May 2020 / Accepted: 13 May 2020 / Published: 15 May 2020
(This article belongs to the Special Issue Transmission Dynamics of Insect Viruses)
Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3Δ56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3Δ56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance. View Full-Text
Keywords: alphavirus; arbovirus; fidelity mutants; mutation frequency alphavirus; arbovirus; fidelity mutants; mutation frequency
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Patterson, E.I.; Khanipov, K.; Swetnam, D.M.; Walsdorf, S.; Kautz, T.F.; Thangamani, S.; Fofanov, Y.; Forrester, N.L. Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles. Viruses 2020, 12, 546.

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