Abstract
Foot-and-mouth disease (FMD) is a host-restricted disease of cloven-hoofed animals, such as cattle and pigs. There are seven major serotypes of FMD virus that exhibit high antigenic variation, making vaccine strain selection difficult. However, there is an internal ribosomal entry site (IRES) element within the 5′ untranslated region of the FMD virus (FMDV) RNA genome that is relatively conserved among FMDV serotypes and could be used as a pan-serotype target for disease interventions. To determine the potential for targeting the IRES as promising drug target, we designed a short interfering RNA (siRNA) targeting a relatively conserved region in the FMDV-IRES. The siRNA affected FMDV-IRES expression but not the expression of the encephalomyocarditis virus or hepatitis C virus IRES. To evaluate the effects of siRNA-mediated silencing, we established cell lines expressing a bicistronic luciferase reporter plasmid, which contained an FMDV-IRES element between the Renilla and firefly luciferase genes. The designed siRNA inhibited FMDV-IRES-mediated translation in a concentration-dependent manner. In order to sustain this inhibitory effect, we designed a short hairpin RNA (shRNA)-expressing lentiviral vector. The results showed that the lenti-shRNA vector significantly suppressed FMDV-IRES activity for up to 2 weeks in cell culture. Thus, our findings in this study provided a basis for the development of effective pan-serotype FMDV inhibitors.
Highlights
Foot-and-mouth disease (FMD) virus (FMDV; genus Aphthovirus, family Picornaviridae) is a positive-sense, singlestranded RNA virus that causes FMD, a highly contagious disease of cloven-hoofed animals
In order to evaluate the efficacy of short interfering RNA (siRNA)-mediated FMDVIRES silencing, we established cell lines containing a bicistronic reporter plasmid [16]
Using FMD virus (FMDV)-internal ribosomal entry site (IRES)-expressing cells, we examined the efficacy of the designed siRNA targeting the conserved region of the FMDV-IRES sequence (FMDV-con siRNA, Fig. 4)
Summary
Foot-and-mouth disease (FMD) virus (FMDV; genus Aphthovirus, family Picornaviridae) is a positive-sense, singlestranded RNA virus that causes FMD, a highly contagious disease of cloven-hoofed animals. There is an internal ribosomal entry site (IRES) element within the 5′ untranslated region (5′UTR) of the FMDV RNA genome, and this IRES mediates the translation of viral proteins [7, 8]. Because the IRES region is responsible for translational control functions, its nucleotide sequence is relatively conserved among FMDV serotypes. Short interfering RNA (siRNA) transfection siRNA targeting a region of FMDV-IRES conserved among the seven serotypes (FMDV-con siRNA; Fig. 1) was designed as follows using BLOCK-iT RNAi Designer (Thermo Fisher Scientific, Waltham, MA, USA): 5′- ACAGGCUAAGGAUGCCCUUCAGGUA-3′.
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