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Abstract
Foot-and-mouth disease (FMD) is an economically significant animal disease because of the speed of its transmission. Routine vaccination may not be effective for early protection in an outbreak situation. Small interfering RNA (siRNA) can be used as a rapid, effective, and an alternative antiviral approach. In this study, we screened 15 synthetic siRNAs to inhibit FMD virus replication in IBRS-2 cells and selected 10 siRNA sequences. Furthermore, we produced 7 adenoviruses expressing shRNA targeting conserved regions of FMDV, such as a leader sequence and nonstructural protein regions, and showed their antiviral effects. We compared the antiviral effects among them and compared between synthetic siRNAs and adenovirus-delivered siRNAs. In particular, the most efficient siRNA, 3C2, was the conserved sequence in the O, A, Asia 1, and C serotypes of FMDV and was located in the predicted loop structure. The pool of sequences including 3C2 and recombinant adenoviruses could be applied for multiple siRNAs and protection in a broad range of cells and animals.
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Figure 1.
Schematic representation of siRNA generation. (A) Schematic representation of annealed siRNA. (B) Schematic representation of recombinant adenovirus DNA. (C) Diagram of the FMDV full genome. The black arrows indicate the target region of shRNA.
Figure 2.
Inhibition of FMDV replication in IBRS-2 cells by synthesized siRNA. IBRS-2 cells were transfected with 10 nM of each siRNA including negative control siRNA. After 12 h of transfection, the IBRS-2 cells were infected immediately with 100 TCID50 of FMDV (O/SKR/2002). The relative viral level was measured at 48 h p.i. using antigen ELISA (∗p < 0.05, t-test, a statistically significant differences between negative control (con) and others). Optical density (OD) was changed to relative antigen level (%) which was percentage of sample OD to negative control OD. Error bars indicate standard deviation (SD).
Figure 3.
Inhibition of FMDV replication in IBRS-2 cells by recombinant adenovirus. IBRS-2 cells were infected with AdshRNA at 3 × 106 TCID50. After 12 h of infection, the IBRS-2 cells were infected immediately with 100 TCID50 of FMDV (O/SKR/2002). The relative viral protein level measured at 48 h p.i. using antigen ELISA (∗p < 0.05 and ∗∗∗p < 0.0001, t-test, a statistically significant differences between control (Ad-Lamin A/C) and others. Optical density (OD) was changed to the relative antigen level (%), which was percentage of sample OD to negative control OD. Error bars indicate standard deviation (SD).
Figure 4.
Nucleotide sequence alignment of 3C2 siRNA region for seven serotypes of FMDV and RNA structure including 3C2 siRNA region. (A) Sequence alignment and editing were performed using CLUSTAL W (16) and BioEdit program (17). (B) RNA secondary structure of the partial 3C region including 3C2 siRNA sequences was obtained from the Mfold web server (http://bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html). siRNA 3C2 sequences are included in the magnified figure and nucleotide T (thymine) represents U (uracil) in the RNA structure. Red lines represent G-C pairs and blue and green lines represent A-U and G-U pairs.
Figure 5.
Cross-inhibition of Ad-3C2 against O, A, and Asia 1 type of FMDV in IBRS-2 cells. IBRS-2 cells were infected with Ad-shRNA at 3 × 106 TCID50. After 12 h of infection, the IBRS-2 cells were infected immediately with 100 TCID50 of FMDV (O/SKR/2002, A22/IRQ, and Asia 1/MOG/05). The relative viral protein level measured at 24 and 48 h p.i. using antigen ELISA. Optical density (OD) was changed to the relative antigen level (%), which was percentage of sample OD to negative control OD. Error bars indicate standard deviation (SD).
Table 1.
Target sequences of siRNAs used in this study
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