It was well-established that RNAi targeting viral genes could protect experimental animals from FMDV challenge, and the protection occurs rapidly and was specific. For example, neck subcutaneous injection of a multiple RNAi plasmid targeting VP1 gene rendered the suckling mice much less susceptible to FMDV serotype O and Asia I 
. Likewise, shRNA targeting 3D gene protected guinea pigs 
and swine 
from challenge virus. Moreover, an attenuated Salmonella choleraesuis-mediated siRNA targeting 3D gene protected guinea pigs and swine against FMDV 
, respectively. Although RNAi has been used as an effective antiviral strategy due to its specific silencing of viral gene expression, thus effectively controlling the severity of FMD infection and spread 
, the pivotal issues of RNAi based anti-viral strategy are the delivery and stability of the RNAi reagents with delivery being the major hindence 
. At the present time, there is no clinical application of RNAi targeting approach in farm. Given the fact that transgenic mice expressing FMDV targeting shRNA became much more resistant to the challenge of FMDV serotype Asia 1 
, transgenic cloning may be a useful tool for RNAi anti-viral strategy. Currently, no studies of transgenic shRNA function in cloven-hoofed animals have been reported.
FMDV consists of a single-stranded RNA genome of approximately 8.5 kb, the RNA is translated as a single long open reading frame into a long poly-peptide, which is undergone a series of post-translational proteolytic cleavages to generate different viral proteins, so RNAi may be screened based upon its ability to degrade the viral RNA. In fact, VP1, VP4, 3D, 2B, 5′NCR, VPg, or 3′NCR have been previously selected as RNAi target genes 
. If there are conserved regions of viral genes among different serotypes of FMDV, the viral genes could be screen as RNAi targets, so we choose RNAi-VP2, RNAi-VP3, RNAi-VP4 as target site in this study. We observed that shRNA targeting VP2, VP3 or VP4 gene of FMDV could silence expression of viral genes in 293T cells (), and inhibit over 91%, 94% and 98%, respectively, of viral replication in BHK-21 cells (). We achieved a very high level of inhibition of virus replication, likely due to selection of stable clones in which most cells expressed targeting shRNAs. The transient transfection of RNAi expressing plasmids/siRNA into BHK-21 cells was a common delivery method 
, with limitation of transfection efficiency, shRNA/siRNA was not expressed in most cells, resulting in an reduced inhibition of viral replication. In our study, stable BHK-21 cell lines expressing targeting shRNAs were selected via FACS sorting for eGFP, which is co-expressed in H1 Lenti-virus vector. Thus, to evaluate efficiency of any given candidate RNAi, the stable clones should be established in order to achieve a high level of anti-virus activity.
In this study, we transfected RNAi-VP4 into bovine fetal fibroblasts cells, followed by transfering the transgenic cells into enucleated oocyte cytoplasts, selection of reconstructed embryos were selected based on their expression of eGFP () and finally transferring the reconstructed embryos to synchronized recipient cows. Since the major focus of our study is to evaluate the efficiency of FMDV shRNA targeting after transgenic delivery, we used 4-month-old fetuses, insteading of adult animals, for the sake of saving time and money. We confirmed shRNA integration into chromosome of cloned fetuses by Southern Blotting and the expression of shRNA by Northern. Since four-month-old transgenic fetuses could not survive in vitro, for FMDV challenge assay, we used primary tongue epithelium cells established from small pieces of the mucosa collected from the tip of bovine tongue. Since the targeting sequence of RNAi-VP4 was conserved among O, A, and Aisa1 serotypes of FMDV, and ASIA1/YS/CHA/05 strain is able to grow well in BHK-21 cells and in primary tongue epithelium cells, we used ASIA1/YS/CHA/05 strain as challenge virus in this study. We found that shRNA expressed in transgenic fetuses could significantly degrade viral RNA after inoculation of FMDV at a titer of 100 TCID50 (), and inhibited viral replication (). Thus, primary transgenic bovine fetus tongue epithelium cells became much more resistant to FMDV challenge.
The most important threat caused by FMDV is the high speed of viral replication, short incubation time, and high contagiousness. Thus, although protective immune responses against FMDV can be efficacious, the rapidity of virus replication and spread can outpace the development of immune defenses and overrun the immune system 
. Our observation that shRNA inhibited over 91% of viral replication at 48 h after challenge () suggest that RNAi-based virus targeting is useful for transgenic cows to get more time to develop immune defense. Needless to say, whether transgenic cows indeed become resistant to FMDV infection will wait for the future study using adult transgenic cows upon FMDV challenge. In fact, we have so far obtained on six-month old male transgenic dairy cattle (data not shown). The investigations in this area will help to improve the design of transgenic genes and the development of RNAi-based strategies against FMD.
Due to their high degree of sequence specificity, shRNAs become ineffective in the presence of escape mutations within and outside the targeted regions 
, and effective silencing of a single viral gene does not always translate into antiviral effect due to genetic compensation or redundancy 
. Furthermore, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Thus, it may be important to use multiplex shRNAs 
if RNAi is to be developed for therapeutic use. In this study, we used shRNAs targeting of viral genes VP2, VP3, and VP4, and observed a significant inhibition of FMDV. Combination of these shRNAs may be necessary to avoid the evolution of escape variants.
In conclusion, we obtained three transgenic fetuses expressing RNAi-VP4 against FMDV. Study using primary tongue epithelium cells derived from these fetuses reveal that RNAi-VP4 degraded viral RNA and inhibited viral replication. This shRNA merits further investigation for cultivation of transgenic cattle against FMDV.