Abstract
Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.
Highlights
Foot-and-mouth disease (FMD) is a highly contagious disease that affects more than 33 species of clovenhoofed animals, such as swine, cattle and other livestock [1]
The short-hairpin RNAs (shRNAs)-coding vectors effectively suppressed target fusion GFP protein expression in BHK-21 cells To identify the interfering effect of the shRNA-coding vectors, plasmid PB-N3D2B was co-transfected with pEGFP-Target into BHK-21 cells at different mass ratios of 0:1, 1:1, 0.5:1, and 0.25:1
Establishment of shRNA-expressing monoclonal transgenic cell lines The PB-EN3D2B and PB-ENlacZNH21 plasmid were integrated into the genome of IBRS-2 cell using the piggyBac transposon system
Summary
Foot-and-mouth disease (FMD) is a highly contagious disease that affects more than 33 species of clovenhoofed animals, such as swine, cattle and other livestock [1]. FMD outbreaks often cause severe economic losses due to reduced productivity and the required slaughter of millions of infected or susceptible animals, and these outbreaks have even raised political disputes concerning trade embargos on animal products. The most commonly used strategies for controlling FMD outbreaks are routine vaccination and slaughtering of infected animals. Livestock slaughter results in substantial economic losses. Because of the rapidity and specificity of its effects, RNAi has been exploited as a promising technology for the control of important pathogens, including human immunodeficiency virus (HIV), herpes simplex virus (HSV) and hepatitis B virus (HBV) [7,8,9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
