Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains

Edward Spinard; Ian Fish; Paul A Azzinaro; Monica Rodriguez-Calzada; Ethan J Hartwig; George R Smoliga; Aishwarya Mogulothu; Jonathan Arzt; Teresa de Los Santos; Gisselle N Medina

doi:10.3390/v15030670

Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains

Viruses. 2023 Mar 2;15(3):670. doi: 10.3390/v15030670.

Authors

Edward Spinard^{1

2}, Ian Fish^{1

2}, Paul A Azzinaro¹, Monica Rodriguez-Calzada^{1

3}, Ethan J Hartwig¹, George R Smoliga¹, Aishwarya Mogulothu^{1

4}, Jonathan Arzt¹, Teresa de Los Santos¹, Gisselle N Medina^{1

5}

Affiliations

¹ Plum Island Animal Disease Center (PIADC), ARS, USDA, P.O. Box 848, Greenport, NY 11944, USA.
² College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.
³ ORISE-PIADC Research Participation Program, Oak Ridge, TN 37831, USA.
⁴ Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269, USA.
⁵ National Bio and Agro-Defense Facility (NBAF), ARS, USDA, Manhattan, KS 66502, USA.

Abstract

Codon deoptimization (CD) has been recently used as a possible strategy to derive foot-and-mouth disease (FMD) live-attenuated vaccine (LAV) candidates containing DIVA markers. However, reversion to virulence, or loss of DIVA, from possible recombination with wild-type (WT) strains has yet to be analyzed. An in vitro assay was developed to quantitate the levels of recombination between WT and a prospective A24-P2P3 partially deoptimized LAV candidate. By using two genetically engineered non-infectious RNA templates, we demonstrate that recombination can occur within non-deoptimized viral genomic regions (i.e., 3'end of P3 region). The sequencing of single plaque recombinants revealed a variety of genome compositions, including full-length WT sequences at the consensus level and deoptimized sequences at the sub-consensus/consensus level within the 3'end of the P3 region. Notably, after further passage, two recombinants that contained deoptimized sequences evolved to WT. Overall, recombinants featuring large stretches of CD or DIVA markers were less fit than WT viruses. Our results indicate that the developed assay is a powerful tool to evaluate the recombination of FMDV genomes in vitro and should contribute to the improved design of FMDV codon deoptimized LAV candidates.

Keywords: FMDV; codon deoptimization; foot-and-mouth disease; recombination.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Codon
Foot-and-Mouth Disease Virus* / genetics
Foot-and-Mouth Disease* / genetics
Prospective Studies
Recombination, Genetic
Viral Vaccines* / genetics

Substances

Viral Vaccines
Codon

Grants and funding

This research was supported by the U.S. Department of Agriculture (USDA), Current Research Information System (CRIS) project number 1940-32000-061-00D, ARS, USDA; by the Plum Island Animal Disease Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between USDA and the U.S. Department of Energy; and by Non Assistance Cooperative Agreements (NACAs) number 58-8064-8-010 between ARS and the Department of Diagnostic Medicine/Pathobiology, of the College of Veterinary Medicine at Kansas State University, Manhattan, Kansas.