ShRNA Transgenic Swine Display Resistance To The Infection Of Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is a highly contagious disease of more than 70 species of domestic and wide cloven-hoofed animals including swine, cattle, goat and sheep. Foot-and-mouth disease virus (FMDV) is the pathogen responsible for FMD. FMDV is a RNA virus belonging to the Aphthovirus genus of the Picornaviridae family, and its genome is a single coding, positive-sense RNA, about 8500 nt in length. FMDV is one of the most important animal pathogens in the world, causes enormous global economic losses on livestock production and trade. Due to the viral high mutant rate vaccines against FMDV do not work effectively. The mechanism of RNA interference (RNAi) as a functional antiviral pathway in mammalian cells was just confirmed. The potential of RNAi as an antiviral strategy against FMDV in relevant animal systems is of great interest, and the generation of a novel swine breed harboring the shRNAtransgene could be an alternative approach for controlling FMD.Here, we generated FMDV-specific shRNAtransgenic cells targeting against either nonstructural protein 2B or polymerase 3D of FMDV, and these transgenic cells had stable shRNA expression and remarkable anti-FMDV ability. The shRNAtransgenic positive cells had milder Cytopathic Effect (CPE) and displayed significant lower viral production than that of the control cells after infected with 100 and 1000 TCID50 dose FMDV (P≤0.05).Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). TGCS had detectable shRNA expression in five tested tissues. TGCS and Non-TGCS were transported to the National P3 Lab in Lanzhou Veterinarian Research Institute for the challenge experiments. The FMDV strain Guangdong/CHA/86 of Serotype O was used to challenge these swine to investigate their anti-FMDV capability. At the challenge dose of the 100 SID50, one TGCS was completely protected while all the normal control swine (NS) developed clinical signs. Mean onset of lesions in the other four TGCS swine was delayed by 2.65 days,-101.9% later than NS (P≤0.05). The mean time for developing severe lesions in the other infected TGCS was 1.35 days longer than that of NS, delayed 96.4%(P≤0.05). At the dose of 10 SID50, one TGCS swine recovered after showing clinical signs for three days, while all Non-TGCS developed FMD and one swine died by 5 d.p.c. Viral RNA load in blood and tissues of TGCS was reduced comparing to that of Non-TGCS or NS in both challenge dose study.Vaccinated swine (VS) was also used as positive control in the challenge experiments. VS did not show obvious clinical signs, but one VS died by 6 d.p.c, and one VS was found viral RNA in two types of tissues. These results indicated that TGCS displayed resistance to the infection of FMDV and the shRNA might help the animal clear the virus.Immune cells including CD3+, CD4+, CD8+, CD21+, and CD172+ type cells and the production of IFN-y were analyzed and there were no significant difference observed between TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may mainly derived from the RNAi-based antiviral pathway.These results showed the anti-FMDV capability of RNAi both in cells and in transgenic cloned swine. And the viral in blood and tissues of TGCS were reduced. This work provided a foundation for the breeding approach of anti-infectious disease in swine.