| Japanese encephalitis （JE） is one of the important insect-borne zoonotic diseases in the worldwide especially in most parts of Asia. JE is caused by the Japanese encephalitis virus （JEV）. The pigs are an important reservoir host and the source of infection in the spread and proliferation process of JEV. JE is very serious harm to swinery; it can cause abortion, stillbirth and herds of pregnant sows’reproductive disorders. Pseudorabies is a acute animal disease that caused by the pseudorabies virus （PRV）, it can cause a variety of animal to fall ill, it main cause the animal’s neurological symptoms, respiratory symptoms and sows miscarriage, stillbirth, and infertility, causing huge losses in the economic of pig industry.In recent years, with the rapid development of transgenic biotechnology, molecular breeding for disease resistance in animals has been considerable progress, the occurrence of the disease, especially viral diseases can be controlled from the source. The key of molecular breeding for disease resistance is the discovery, functional verification and molecular mechanisms of a broad-spectrum, highly effective antivirus gene. In recent years, many articles pointed out that the IFITM proteins have good inhibition of virus proliferative effect for a variety of enveloped RNA viruses.In view of this, pig source IFITM3 antiviral effect and related mechanism of the enveloped RNA viruses JEV and DNA viruses PRV were researched in this study, including the following:1.sIFITM3 antiviral effect and mechanism of JEVUsing 100 PFU or 50 PFU of JEV （SA14-14-2 strain, P3 strain and SX09S-01 strain） to infect sIFITM3 over-expression cells BHK-21-sIFITM3 and control cells BHK-21-eGFP, plaque assay results show that compared with the control group, the proliferative ability of different strains of JEV on BHK-21-sIFITM3 cell lines significantly have been inhibited, the size of plaques decreases, the number of plaques was significantly decreased （P<0.01）, that shows the replication of genotype I and genotype Ⅲ of JEV were significantly inhibited by sIFITM3. The sIFITM3 silence tests further illustrate that sIFITM3 can inhibit the replication of JEV.Based on the the protein characteristics of sIFITM3 and the characteristics or effects of other virus inhibited the replication by sIFITM3 that has been reported, this study is focusing on exploring wether the sIFITM3 can inhibit JEV replication at the adsorption and entering process of the virus to the host cell. Real-time PCR results showed that the adsorption of JEV on BHK-21-sIFITM3 cells and BHK-21-EGFP cells were no significant difference （P> 0.05）, indicating that the over-expression of sIFITM3 did not affect the adsorption process of JEV infected cells; JEV adsorption and entry test results showed that:compared with control BHK-21-eGFP cells, the JEV virus particles entry into the BHK-of-sIFITM321 cells was significantly decreased （P<0.05）, indicating that the overexpression of sIFITM3 has a significant inhibitory effect to the entry step of the process of JEV infection of BHK-21 cells, while no significant effect on JEV infection adsorption process of BHK-21 cells.2. sIFITM3 antiviral effect and mechanism of the PRVUsing 30 PFU of PRV Ea strain and PRV TK7gE7LacZ+ strain infected BHK-21-sIFITM3/eGFP, plaque assay results show that, relative to BHK-21-eGFP cells, the proliferation of PRV Ea strain and the PRV TK7gE7LacZ+ strain were suppressed in BHK-21-sIFITM3 cell line, plaques size decreases, the number of plaques decreased （P <0.05）, indicating that sIFITM3 has a certain inhibitory effect of the PRV virus. The sIFITM3 silence test also confirmed the sIFITM3 having an inhibitory effect on the proliferation of PRV.Next we did PRV adsorption test to further explore the mechanism which sIFITM3 inhibits PRV proliferation and using plaque formation assay to detect viral replication. The results show thatthe amount of adsorption of the PRV virus particles in BHK-21-sIFITM3 cells significantly lower than BHK-21-eGFP control cells （P<0.01）, and the over-expression of sIFITM3 has a significantly inhibitory effect at the adsorption step of the process of PRV infected host cells. It shows that the sIFITM3 can play its antiviral mechanism of PRV in the adsorption process of PRV infects the host cells.3. the construction of transgenic mice over-expression sIFITM3In order to research the ability of over-expression of sIFITM3 against JEV and PRV infection in the mice body level, the sIFITM3 fragment was ligated into the eukaryotic plasmid pCA to abtain the plasmid PCA-sIFITM3, using microinjection method the PCA-sIFITM3was injected into C57BL/6 mice embryonic cells. After mice birth, detecting in the of DNA and mRNA levels that sIFITM3 gene was integrated into the mouse genome and can be transcribed; semi-quantitative PCR and real-time quantitative PCR results display sIFITM3 gene expressed efficiently in mice tissues.Then through propagation, we obtain the number of different generations of transgenic mice 30.4 the inhibitory effect of JEV infection on sIFITM3 transgenic miceThrough the tail vein injecting 105PFU JEV P3 strain into 7-weeks-old transgenic mices,the results showed that compared with the the negative mices challenged group, transgenic mices challenged group mices in body weight and mortality was not significant difference; Real-time PCR detection of viral particles copy number results show that the copy number of virus particles in the blood and brain tissue of transgenic mice after challenge was significantly lower than the negative challenge group. Indicated in the body horizontal over-expression of sIFITM3 has some inhibitory effect to JEV proliferation.5 The inhibitory effect of PRV infection on sIFITM3 transgenic miceBy foot injection,20LD50/50μl PRV （Ea strain） injecting of 4-week-old transgenic mices, the results showed that compared with the negative challenge mices, transgenic challenge mices body weight change and mortality difference was not significant; Real-time PCR detection of viral particles in the copy number of the results showed that 4d after challenge the copy number of virus particles in the brain tissue of transgenic mices higher than the negative challenge group. Indicated in the body horizontal, over-expression of sIFITM3 can promote the proliferation of PRV, but do not have any inhibitory effect. |
