Transgenic Mice Model Of Foot And Mouth Disease Virus Interference RNA

Object: Foot-and-mouth disease (FMD),whose etiological agent is foot-and-mouth disease virus (FMDV), is an acute and highly contagious disease occurring in cloven-hoofed animals. FMDV has 7 serotypes and over 70 subtypes. Owing to the absence of reciprocal protection among all the serotypes, it is difficult to control FMD through vaccination and impossible to eliminate FMD by conservative natural breeding. A recent occurrence of a large epidemiogenesis has made the development of emergency antiviral strategies essential for preventing outbreaks of FMD.RNA interference(RNAi)refers to the process of sequence-specific, posttranscriptional gene silencing (PTGS) conserved in a wide range of eukaryotic organisms from plants to mammals,which is induced by 21- to 23-nucleotide (nt) siRNA that demonstrates sequence homology to the silenced gene. It is an important mechanism of cellular defense and differentiation regulation and plays an important role in the regulation of gene expression,the prevention of viral infection and the control of gene transposition. siRNA has showed its potential antiviral abilities in cultured mammalian cells and animals,and has been used as a new powerful tool for gene-specific therapeutics for viral disease. With the progressions of the research about RNAi,some investigations have been performed successfully to inhibit the replication of viruses. To develop new methods for controlling FMD, In this work, we describe the use of RNAi in inhibiting virus replication in BHK-21cells and suckling mice. On the foundation,FMDV siRNAs transgenic mice model were prepared and the resistance to replication of FMDV was evaluated, which offers an insight into the use of RNAi in animal breeding for disease resistance and necessary experimental data for study on gene function of FMDV and prevention of FMD by RNAi.Methods: Firstly,7 target sequences aimed at the FMDV mRNAs of the construction proteins were designed,the insert DNA sequences were synthesized respectively and their expression vector were constructed based on the plasmid pSilencer 5.1-H1 Hygro(Ambion).Two recombinant plasmids targetted to conservative 2B,3D regions were transfected to BHK-21 cell monolayer.The inhibition of siRNAs to FMDV replication was detected on transfected cell ,which were inoculated with O,A,AsiaI FMDV; Suckling mice were infected with 5 LD50 and 20 LD50 O,A FMDV after injected with two plasmids and the death numbers were observed; On the foundation,transgenic mice of FMDV siRNAs were prepared by microinjection. To test the anti-FMDV activity of the siRNAs, we challenged F1 positive transgenic mice with 0.5 ml 100 LD50 AsiaI FMDV by a peritoneal injection. All saline-treated mice were control group. 72h after the viral challenge,the mice were executed by exanguinating from fossa orbitalis and the heart,liver,spleen,lung,kidney were cut and fixed by 10% formaline.Routine pathological section were prepared and the pathologic change was observed by microscope. The resistance to FMDV infection of transgenic mice was evaluated by immunohistochemistry assay.Results: By identification, FMDV siRNA expression vectors were constructed and they were named: pSi-FMDV1,pSi-FMDV2,pSi-FMDV3,pSi-FMDV4,pSi-FMDV5,pSi-FMDV6,pSi-FMDV7. It was demonstrated that transfection of BHK-21 cells with the 2 siRNA-expressing plasmids --pSi-FMD2,pSi-FMD3-- could induce a lower CPE compared with the controls. Further, the TCID50 of the FMDV serotypes A, O, and Asia 1 detected in supernatants collected from cells transfected with FMDV-specific siRNA-expressing plasmids was lower than that of control cells. In addition, when challenged by 5 LD50 or 20 LD50 of the FMDV serotypes A, O, or Asia 1 after injecting FMDV-specific siRNA-expressing plasmids, 10–40% suckling mice could resist virus infection. On the foundation,transgenic mice of FMDV siRNAs were prepared by microinjection.The positive integration rate of F0 was low,while that of F1,F2,F3,F4 were 10%-20%.At present F6 transgenic mice were passaged and the phenotype of offspring had no any abnormality. From the results of H.E staining of different pathological section,compared with control,the pathologic change of transgenic mice was light or almost normal.Especially, the splenic corpuscle of transgenic mice augmented and macrophage,epithelioid cell node in junction of white pulp and red pulp increased obviously.The results of immunohistochemistry showed that FMDV could be detected in liver,spleen,kidney of control mice and the number of virus in spleen was large.However,FMDV only could be detected in spleen and liver of transgenic mice and the number was little,compared with control.Conclusion: The results showed that the siRNAs targetted to FMDV can inhibit virus replication in vitro and in vivo at some degree,which offered an insight into the use of RNAi in large livestock breeding for disease resistance.