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

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.

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation

“…Recombination events could lead to the emergence of revertants, potentially restoring the virulence of modified attenuated FMDV strains. While this particular study did not delve into recombination events, it is worth noting a recent investigation by Spinard et al [ 49 ] that explored the potential for recombination between WT and FMDV CPD strains. Their study found that although recombination could occur between the FMDV CPD and WT strains, the resulting recombinant viruses exhibited much lower fitness as compared to the WT strains, and consistently became extinct.…”

The Application of the Gibson Assembly Method in the Production of Two pKLS3 Vector-Derived Infectious Clones of Foot-and-Mouth Disease Virus

“…Most viral populations were mixtures of defective viruses; however, 1 out of 42 plaques contained viruses with complete genomic RNAs. The complementation most likely occurs from genetic recombination among the wild-type and the defective genomes because of strand switching during negative-strand synthesis [ 35 ]. In our study, transfection with pKLS3_O189 or pKLS3_NP05 might produce both RT-PCR-detectable defective and complete genomic RNAs.…”