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Research Project: Intervention Strategies to Control and Eradicate Foreign Animal Diseases of Swine

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Title: Evaluation of potential recombination events in codon deoptimized FMDV strains

item SPINARD, EDWARD - Oak Ridge Institute For Science And Education (ORISE)
item FISH, IAN - Oak Ridge Institute For Science And Education (ORISE)
item Azzinaro, Paul
item Hartwig, Ethan
item Smoliga, George
item MOGULOTHU, AISHWARYA - University Of Connecticut
item RODRIGUEZ-CALZADA, MONICA - Oak Ridge Institute For Science And Education (ORISE)
item Arzt, Jonathan
item De Los Santos, Teresa
item Medina, Gisselle

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/25/2023
Publication Date: 3/2/2023
Citation: Spinard, E., Fish, I., Azzinaro, P.A., Hartwig, E.J., Smoliga, G.R., Mogulothu, A., Rodriguez-Calzada, M., Arzt, J., De Los Santos, T.B., Medina, G.N. 2023. Evaluation of potential recombination events in codon deoptimized FMDV strains. Meeting Abstract.

Interpretive Summary:

Technical Abstract: Use of live attenuated vaccines has been proposed as a provocative 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 with deoptimized P2 and P3 genomic regions, and 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 co-transfection produced the same number of viable particles, as co-transfection of 'P1 and 'GDD RNAs. Characterization of single plaques by next generation sequencing revealed that 8 out of 20 plaques isolated from 'P1-P2P3Deopt/'GDD co-transfected cells corresponded to wild type FMDV. These results indicated that recombination occurred within a 185 nt non-deoptimized 3D region upstream of the 'GDD marker. The remaining 12 plaques contained both, WT and deoptimized sequences. One such mix recombinant was passaged and sequenced, revealing only WT sequences. Taken together these data indicate that recombination can occur within deoptimized FMDV regions, however no recombinants with gain of function, or containing DIVA sequences that could outcompete WT virus are detected.