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

Yanyan Chang12, Yongxi Dou1, Huifang Bao1, Xuenong Luo1, Xuerong Liu12, Kebin Mu12, Zaixin Liu1, Xiangtao Liu1 and Xuepeng Cai13*

Author Affiliations

1 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Gansu, P. R of China

2 Research & Development Department, China Agricultural Veterinary Biological Science and Technology Co., LTD, Lanzhou 730046, Gansu, P. R. of China

3 China Animal Disease Prevention Control Center, Beijing 100125, P. R. of China

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Virology Journal 2014, 11:1  doi:10.1186/1743-422X-11-1

Published: 6 January 2014



Foot-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.


Four 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.


These data demonstrate that IRES-specific miRNAs can significantly inhibit FMDV infection in vitro and in vivo.

Foot-and-mouth disease virus; MicroRNA; Internal ribosome entry site; Transformed cell clones; Antiviral effect; Flow cytometry; Real-time quantitative RT-PCR