Evaluation of potential in vitro recombination events in codon deoptimized FMDV strains

Authors: Spinard Iii, Edward; FISH, IAN - Orise Fellow; Azzinaro, Paul; RODRIGUEZ-CALZADA, MONICA - Orise Fellow; Hartwig, Ethan; Smoliga, George; Arzt, Jonathan; De Los Santos, Teresa; Medina, Gisselle

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2023
Publication Date: 9/13/2022
Citation: Spinard III, E.J., Fish, I., Azzinaro, P.A., Rodriguez-Calzada, M., Hartwig, E.J., Smoliga, G.R., Arzt, J., De Los Santos, T.B., Medina, G.N. 2022. Evaluation of potential in vitro recombination events in codon deoptimized FMDV strains. Viruses. 15(3). https://doi.org/10.3390/v15030670.
DOI: https://doi.org/10.3390/v15030670

Interpretive Summary: Foot-and-mouth disease (FMD) is the most contagious animal viral disease known. Use of live attenuated vaccines (LAVs) has been proposed as a strategy to control FMD because such vaccines can stimulate a strong and long-lasting immune response. Recoding the genome of FMDV by codon deoptimization has resulted in viable attenuated viral strains that offer various distinct levels of protection. However, LAVs have the potential to revert to virulent phenotypes by various mechanisms including the recombination with circulating wild type strains. In this study, we evaluate the ability of recoded FMDV attenuated strains to recombine with other FMDV strains using an in vitro system. This information could be used for development of improved LAV candidates against FMD.

Technical Abstract: Use of live attenuated vaccines has been proposed as an alternative strategy to control foot and mouth disease (FMD). However, among others, reversion to virulence or possible loss of pre-designed DIVA markers caused by recombination with circulating wild type strains pose intrinsic safety risks and may limit their utility. Recently we derived viable attenuated FMDV strains with codon deoptimized P2 and P3 genomic regions, including DIVA markers (A24-P2P3Deopt). To test the ability of A24-P2P3Deopt virus to recombine with other FMDV strains, an in vitro cell recombination assay was developed. Briefly, two non-infectious in vitro synthesized RNA templates containing either deletion of P1 capsid coding region ('P1), or deletion of three amino acids in 3Dpol ('GDD), were co-transfected in cells followed by standard incubation. Under these conditions, viable virus could only be generated if recombination between the two templates occurred. Our analyses show that 'P1-P2P3Deopt and 'GDD RNA co-transfection produced the same number of viable infectious particles as co-transfection of 'P1 and 'GDD RNAs at 24 hours post transfection (hpt), but lower virus titers at 48 hpt. Characterization by next generation sequencing of single plaques isolated from the 'P1-P2P3Deopt × 'GDD co-transfected cells revealed that at 24 and 48 hpt, most of the isolated recombinant viruses were composed of WT sequence at the consensus level. Presumably, non-deoptimized sequences (185nt) in P3 region upstream of the 'GDD marker, facilitated template switching resulting in a WT phenotype. All isolated plaques contained at least sub-consensus levels of deoptimized sequence, while a minority of plaques contained genomes composed of deoptimized sequence at the consensus level towards the 3’ end of the 3C-3D region. When eight such mixed recombinants were passaged and sequenced, two recombinant viruses were detected displaying WT sequence at the consensus level. Taken together these data indicate that partial codon deoptimization in the P2 and P3 regions of the FMDV genome did not prevent the generation of recombinant viruses, however, no recombinants with DIVA markers at the consensus level could be detected.