Study On Inhibition Of Foot-and-mouth Disease Virus Replication And Infection By RNAi

RNA interference(RNAi)is a molecular mechanism of post-transcriptional gene silencing in eukaryotes, which is a fascinating discovery in the last decade of the 20th century. The key factor to initiate RNAi process is the double-chain short interfering RNAs (sRNAs) with a length of 19~27 nt. RNAi process is also mediated by a series of protein complexes, and results in a sequence-specific downregulation of homologous gene expression at transcriptional or post-transcriptional or translational level. With the progression of the research on RNAi, people have found that siRNA can be able to inhibit the replication of viruses. In recent years, many researchers have performed many investigations on the interference of virus replication, such as Human immunodeficiency virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Dengue virus, Poliovirus, Influenza virus (IV), severe acute respiratory syndrome (SARS) coronavirus, and so on. And many advances in this respect have been obtained.Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals in the whole world. Because of many easily infected animals, the virus plasmodium and the very fast propagation, the disease often breakouts and results in a great loss in economy. The government devotes much attention to the preventing of this disease. However, many new virus plasmodiums result that the traditional vaccine is not able to avoid the breakout and propagation of the disease.The discovery of RNAi mechanism and the advance in the anti-virus research open a new field for the prevention and treatment of the FMD. In this study, we successfully performed RNA interference to the FMD virus in vitro and in vivo. From the analysis of the function of gene and it's conservative character, we discuss the relationship of the choice of siRNA targeting regions and the interference effect through the prediction of the second order structure of the interference target, the comparison of siRNA sequence homologous itself, G/C content and the second order structure of cDNAi. The necessary experimental data have been accumulated for the choice of siRNA and the application of RNAi technology to prevent and treat the FMD.The sequences of IERS, 2B, 2C and 3D genes from the seven serotypes of FMDV were compared with each other on the basis of the analysis of gene function , which included serotypes O, A, C, SATI, SAT2, SAT3 and Asia 1. According to the conserved sequence of these genes and the design principles recommend by Ambion Company (www.ambion.com), nineteen siRNA templates and a control were designed, synthesized, and then cloned into the downstream of the U6 promoter of the siRNA expression vector pSilencer1.0-U6, resulting in twenty recombinant plasmids.Firstly, the inhibiting role of the shRNA expressing plasmids targeting to 2B gene was investigated at the molecular level. The recombinant plasmid pEGFP+2B was constructed by in-frame fusing the 2B coding sequence to the 3'-end of the EGFP gene in the expression vector pEGFP-C1. The shRNA expressing plasmids targeting 2B and pEGFP+2B were separately co-transfected into BHK-21 cells. Fluorescence microscopic observation and flow cytometric and real-time RT-PCR analysis revealed that pSi-2B3, pSi-2B4, pSi-2B5 and pSi-2B16 could inhibit 2B expression. The most effective siRNAs are pSi-2B4 and pSi-2B16. The further analysis indicates that the inhibitory rate of the pSi-2B4 to 2B gene is 37.41%, 86.68% and 63.89% at protein level after co-transfected for 24h, 48h and 72h, respectively; and that the corresponding inhibitory rate of the pSi-2B16 is 46.99% , 73.18% and 58.77%, respectively. At mRNA level, the inhibitory rate of the pSi-2B4 to 2B gene is 6.04%, 69.34% and 46.57%, respectively; that of the pSi-2B16 is 55.41%, 99.58% and 68.98%, respectively. This indicates that the shRNA expressing plasmids can effectively inhibit 2B expression.Then, the inhibiting role of the shRNA expressing plasmids to the replication of FMDV was investigated at the cell level. FMDV WFL was cultured in the BHK-21, PK-15 and IBRS-2 cells, respectively. The velocity of reproduction was observed in the different cells, then shRNA expressing plasmids were transfected into the three kinds of cells. The experimental results indicate that pSi-2B16, pSi-2C56 and pSi-3D18 all exhibit an inhibiting role to the FMDV WFL in the BHK-21, PK-15 and IBRS-2 cells. And the inhibiting role in the IBRS-2 cells lasts the longest time extending to 144h. The inspecting to the 8h post-infected cell culture shows that the inhibiting rate of the pSi-2B16, pSi-2C56 and pSi-3D18 to FMDV WFL is 93.8%, 63.2% and 59.5%, respectively. ELISA result indicates that the inhibiting rate of the pSi-2B16, pSi-2C56 and pSi-3D18 is 89%, 79.6% and 71.8%, respectively.In suckling mice experiments, the three-day-old mouse was injected with plasmids of 100μg per mouse and injected 50 LD50 per mouse after 12 hours, the survival rate of the pSi-2B16 and pSi-2C56 is 25%. The shRNA-induced RNA interference plays a protective role after 24h. When the pSi-2B16/2C56/3D18 was injected together into a mouse, the survival rate is 50%. The TCID50 inspecting into the sample of the survival mouse indicates the highest survival rate is 98.2%. When suckling mice were interruptively treated for three times with 600μg per mouse of pSi-2B16 or pSi-2B16/2C-56/3D18, the survival rate could reach as high as 100%. There was not any abnormal phenomenon to be found in the 14d'observation. The relative quantitatively PCR measurement of RNA post-transcriptional level in the muscular tissue virus of the suckling mice, compared with the infected-contrast group, the inhibiting rate of the relative expression of the virus RNA reach as high as 99.95%. Section of the pathologic tissue exhibits that there is a slight pathologically change or not anyone in internal organs of the infected sulking mice.