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

Hongmei Wang,Wenhao Liu,Guangpeng Li,Jifeng Zhong,Hongbin He,Li Yu,Lei Cheng,Jianbin Li,Hongjun Yang,Fangrong Ding,Xiao Liu,Chengqiang He,Jianming Wu,Yunping Dai,Jianming Qiu

doi:10.1371/journal.pone.0042356

Abstract

BackgroundAlthough it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge.Principal FindingHere we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h.ConclusionRNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

Highlights

Foot-and-mouth disease (FMD) is a severe, clinically acute, vesicular disease of cloven-hoofed animals, including cattle, swine, and sheep, as well as more than 70 species of wild animals, its outbreaks have occurred in every livestock-containing region of the world with the exception of New Zealand [1]
RNA interference (RNAi)-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from Foot-and-mouth disease virus (FMDV) infection, and it could be a candidate Short hairpin RNA (shRNA) used for cultivation of transgenic cattle against FMDV
RNAi-VP4 almost completely eliminated expression of VP4. These results strongly suggested a direct role of shRNAs in blocking transient expression of viral proteins in 293T cells

Summary

Introduction

Foot-and-mouth disease (FMD) is a severe, clinically acute, vesicular disease of cloven-hoofed animals, including cattle, swine, and sheep, as well as more than 70 species of wild animals, its outbreaks have occurred in every livestock-containing region of the world with the exception of New Zealand [1]. The etiological agent of FMD is foot-and-mouth disease virus (FMDV), which is the type species of the Aphthovirus genus of the Picornaviridae family. It is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h

Methods

Results

Discussion

Conclusion