Multiple microRNAs targeted to internal ribosome entry site against foot-and-mouth disease virus infection in vitro and in vivo

Yanyan Chang,Yongxi Dou,Huifang Bao,Xiangtao Liu,Xuenong Luo,Zaixin Liu,Kebin Mu,Xuepeng Cai,Xuerong Liu

doi:10.1186/1743-422x-11-1

Abstract

BackgroundFoot-and-mouth disease virus (FMDV) causes a severe vesicular disease in domestic and wild cloven-hoofed animals. Because of the limited early protection induced by current vaccines, emergency antiviral strategies to control the rapid spread of FMD outbreaks are needed.Here we constructed multiple microRNAs (miRNAs) targeting the internal ribosome entry site (IRES) element of FMDV and investigated the effect of IRES-specific miRNAs on FMDV replication in baby hamster kidney (BHK-21) cells and suckling mice.ResultsFour IRES-specific miRNAs significantly reduced enhanced green fluorescent protein (EGFP) expression from IRES-EGFP reporter plasmids, which were used with each miRNA expression plasmid in co-transfection of BHK-21 cells. Furthermore, treatment of BHK-21 cells with Bi-miRNA (a mixture of two miRNA expression plasmids) and Dual-miRNA (a co-cistronic expression plasmid containing two miRNA hairpin structures) induced more efficient and greater inhibition of EGFP expression than did plasmids carrying single miRNA sequences.Stably transformed BHK-21 cells and goat fibroblasts with an integrating IRES-specific Dual-miRNA were generated, and real-time quantitative RT-PCR showed that the Dual-miRNA was able to effectively inhibit the replication of FMDV (except for the Mya98 strain) in the stably transformed BHK-21 cells.The Dual-miRNA plasmid significantly delayed the deaths of suckling mice challenged with 50× and 100× the 50% lethal dose (LD50) of FMDV vaccine strains of three serotypes (O, A and Asia 1), and induced partial/complete protection against the prevalent PanAsia-1 and Mya98 strains of FMDV serotype O.ConclusionThese data demonstrate that IRES-specific miRNAs can significantly inhibit FMDV infection in vitro and in vivo.

Highlights

Foot-and-mouth disease virus (FMDV) causes a severe vesicular disease in domestic and wild cloven-hoofed animals
The pmiR-NC plasmid showed no visible changes in enhanced green fluorescent protein (EGFP) expression (Figure 2). pmiR242 and pmiR276 yielded more significant reductions of internal ribosome entry site (IRES)-EGFP expression, compared with pmiR153 and pmiR220 (Table 2)
Co-transfection of a mixture of these two IRES-specific miRNA expression plasmids with any of the three IRES-EGFP reporter plasmids resulted in a 78.4%–88.3% reduction in intensity of EGFP fluorescence, as compared with the individual plasmids of pmiR242 (44.3%–71.4%) and pmiR276 (60.5%–81.4%) (Figure 2, Table 2)

Summary

Introduction

Foot-and-mouth disease virus (FMDV) causes a severe vesicular disease in domestic and wild cloven-hoofed animals. The viral genome is composed of a positive-sense, singlestranded RNA that functions as an mRNA and contains a unique open reading frame (ORF) encoding a viral polyprotein This polyprotein is co-translationally processed, largely by virus-encoded proteases, to produce about 15 mature proteins plus many different precursors [7,8,9]. Initiation of FMDV RNA translation is directed by a large RNA cis-acting element of about 440 nucleotides (nts) termed the internal ribosome entry site (IRES) element [10,11]. This region is predicted to adopt a secondary structure that mediates RNA–protein interactions essential for ribosome recognition [12,13]. FMDV evolution is strongly influenced by high mutation rates and the dynamics of viral quasispecies, and results in everchanging targets for antiviral strategies, including vaccination [22,23]

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Discussion

Conclusion