Transient inhibition of foot-and-mouth disease virus replication by siRNAs silencing VP1 protein coding region

Abstract

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a severe, clinically acute, vesicular disease of cloven-hoofed animals. RNA interference (RNAi) is a mechanism for silencing gene expression post-transcriptionally that is being exploited as a rapid antiviral strategy. To identify efficacious small interfering RNAs (siRNAs) to inhibit the replication of FMDV, candidate siRNAs corresponding to FMDV VP1 gene were designed and synthesized in vitro using T7 RNA polymerase. In reporter assays, five siRNAs showed significant sequence-specific silencing effects on the expression of VP1-EGFP fusion protein from plasmid pVP1-EGFP-N1, which was cotransfected with siRNA into 293T cells. Furthermore, using RT-qPCR, viral titration and viability assay, we identified VP1-siRNA517, VP1-siRNA113 and VP1-siRNA519 that transiently acted as potent inhibitors of FMDV replication when BHK-21 cells were infected with FMDV. In addition, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, and a single nucleotide substitution was identified as the main factor in resistance to RNAi. Our data demonstrated that the three siRNA molecules synthesized with T7 RNA polymerase could have transient inhibitory effects on the replication of FMDV.

Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

“…In fact, VP1, VP4, 3D, 2B, 5′NCR, VPg, or 3′NCR have been previously selected as RNAi target genes [6], [12], [13], [14], [15], [16], [17], [18]. If there are conserved regions of viral genes among different serotypes of FMDV, the viral genes could be screen as RNAi targets, so we choose RNAi-VP2, RNAi-VP3, RNAi-VP4 as target site in this study.…”

“…This approach is a highly specific tool to down-regulate gene expression [11] and has been extensively utilized to inhibit FMDV in vitro and/or in vivo [6], [12], [13], [14], [15], [16], [17], [18]. Furthermore, transgenic mice expressing FMDV targeting shRNA was much more resistant to viral infection, as evidenced by minor abnormal pathology, as compared to the control mice after challenge with FMDV [19].…”

Secondary RNA Structure and its Role in RNA Interference to Silence the Respiratory Syncytial Virus Fusion Protein Gene

“…Likewise, when cells were treated with siRNAs mixtures, in addition to individual siRNAs the results demonstrated that the inhibitory effects of the mixtures on foot and mouth disease virus (FMDV) RNA replication and viral titer were approximately same as that of individual component siR-NA [39]. They suggested that mixtures did not exert significant inhibitory effect because each siRNA of the mixture might possess equal efficacy to inhibit replication of FMDV.…”

Development of sheep kidney cells with increased resistance to different subgenotypes of BVDV-1 by RNA interference

“…Specific gene silencing also can be triggered in mammalian cells by using synthetic short interfering RNA (siRNA), and plasmid or virus-mediated short hairpin RNA (shRNA). These RNAi strategies have been used successfully for suppressing the replication of human and animal viruses, including hepatitis B virus (HBV) (Kayhan et al, 2007), human immunodeficiency virus type (HIV) (Coburn and Cullen, 2002), porcine reproductive and respiratory syndrome virus (PRRSV) (Luo et al, 2013) and foot-and-mouth disease virus (FMDV) (De los Santos et al, 2005;Lv et al, 2009;Xu et al, 2012). Furthermore, we and others reported that transgenic animals expressing shRNA against virus genes showed a significant resistance to viral challenge (Pengyan et al, 2010;Li et al, 2014;Daniel-Carlier et al, 2013;Du et al, 2014).…”