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Title: Inhibition of Foot-and-Mouth Disease Virus in Cell Culture with Antisense Morpholino Oligomers

Author
item VAGNOZZI, ARIEL - ORISE, ARS PIADC FELLOW
item STEIN, DAVID - AVI BIOPHARMA, OR
item IVERSEN, PATRICK - AVI BIOPHARMA, OR
item Rieder, Aida - Elizabeth

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/13/2007
Publication Date: 11/1/2007
Citation: Vagnozzi, A., Stein, D.A., Iversen, P.L., Rieder, A.E. 2007. Inhibition of Foot-and-Mouth Disease Virus in Cell Culture with Antisense Morpholino Oligomers. Journal of Virology. 81:11669-11680.

Interpretive Summary: Foot-and-mouth disease (FMD) is a highly contagious disease of farm animals leading to severe losses to the livestock production and export industries, along with a consequent social impact. To control FMD in endemic regions a systematic immunization with inactivated vaccines has been applied. However, recent outbreaks in FMD-free countries have emphasized the need to develop new strategies for animal protection in these areas. The purpose of this study was to evaluate the efficacy and specificity of phosphorodiamidate morpholino oligomers (PMO) at inhibiting the productive replication of FMDV. PMO are nucleic acid molecules that are water-soluble and resist degradation by nucleases. Six PPMO compounds were designed to base-pair with sequence elements thought to be important in FMDV RNA synthesis and translation initiation. Three of the PPMO had potent antiviral activity. Two of them, AUG1 and AUG2, complementary to the first and second functional AUG initiation codons of the FMDV polyprotein generated a 6-7-log specific decrease in viral titer and provided protection from two different strains within FMDV serotype A (A ^ 24 and A ^ 12). One of these PPMO (targeting the IRES) was highly active against a multiplicity of FMDV serotypes. Interestingly, serial exposure of cells to low concentrations of the AUG 1- targeting PPMO eliminated all traces of virus. Considering the agricultural and socio-economic importance of FMD and need for early disease interventions, the results herein provide optimism that PPMO can be considered a candidate technology for therapeutic development against FMD.

Technical Abstract: Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals that can lead to severe losses in the livestock production and export industries. Although vaccines have been extensively applied to control FMD, there is no antiviral therapy available to treat ongoing infections with FMD virus (FMDV). Six peptide-conjugated morpholino oligomers (PPMO), with sequences complementary to various segments of the 5 prime and 3 prime nontranslated regions of the FMDV genome, were evaluated in cell cultures. Three of the PPMO, targeting domain 5 of the IRES (5D), and the two translation start codon regions (AUG1 and AUG2), showed high antiviral activity. A dose-dependent and sequence-specific reduction in viral titer of greater than 5 logs, with a concomitant reduction of viral protein and RNA expression, was achieved by low micromolar concentrations of these three PPMO. Under identical conditions, three PPMO, targeting the 5 prime - terminal region of the genome, the cis-acting replication element (cre) and 3 prime stem-loop ab, showed less dramatic titer reductions of 1.5 to 2 logs. Treatment with the 5D PPMO reduced the titer of FMDV strains representing 5 different serotypes by 2-4 log ^ 10 compared to controls. BHK-21 cells infected with FMDV (strain A ^ 24 Cru) repeatedly treated with two of the more active PPMO generated resistant virus for which phenotypic and genotypic properties were defined. Three passages with low concentrations of the AUG1 PPMO extinguished all traces of detectable virus. The results indicate that PPMO have potential for treating FMDV infection, and also represent useful tools for study of picornaviral translation.