Study On The Antiviral Activity Of IFN-αRegulated By IRES

Foot-and-mouth Disease (FMD) is one of the most devastating viral diseases of cloven-hooved animal species. Each breakout of FMD caused great economical damage to stock raising and international trade, so it had been classified as an OIE List A disease. Foot-and-mouth Disease Virus (FMDV) is the causative agent of FMD, belongs to Aphthovirus genus of the Picornaviridae family, it contains a signal positive-strand which is about 8400nt. Like other RNA virus, its genome variability causes a big problew for its preventing and treating. Recent studies mostly focus on how to develop efficient anti-FMDV drugs.Alpha interferon (IFN-a) is a widely-used antiviral drug. Previous studies have shown that IFN-a was able to suppress FMDV replication and spread. FMDV can also inhibit IFN-a expression in infected cells by blocking cap-dependent translation. To overcome the blockade on IFN-a mRNA translation during FMDV infection, we generated an IRES-IFN construct named pc-IRES-IFN that carries FMDV's internal ribosome entry site (IERES) cDNA sequence between the promoter and porcine IFN-a gene. Mutant IRES was also introduced to replace the wild-type IRES as contral, there were called pc-IRES△T-IFN, pc-IRES△G-IFN. The data showed that IFN-a could express at normal situation, and had antiviral activity. We performed the infection experiments with VSV. After a 12 hour infection, PK cells transfected with pc-IRESAG-IFN and pc-IRES△T-IFN, as well as untransfected control cells, displayed no change in IFN-a production. In contrast, cells transfected with pc-IRES-IFN showed a 20% decrease in IFN-a productions. However, after 24 hours, IFN-a production in all transfected cells, as well as the untransfected control, increased to pre-infection level or above. Those demonstrate that the expression levels of IFN-αdisplayed the same trends at different timeframes. When infection by FMDV, the expression level of IFN-αunder the regulation of mutant IRES would decrease to-70% of pre-infection at 12h.p.i and -50% of pre-infection at 24h.p.i; but the expression level of IFN-αunder the regulation of wild-type IRES would decrease to-90% of pre-infection at 12h.p.i and increase to 125% at 24h.p.i. we also evaluated the antiviral activity of IFN-αby measuring the amount of viral RNA. The date of real-time RT-PCR demonstrated that the amount of viral RNA in cells transfected with pc-IRES-IFN was fivefold lower than that in cells transfected with pc-IRES△G-IFN and pc-IRES△T-IFN. Further, all of them are outclassed by the mock-transfected cells. Plaque assays were performed to confirm that cells transfected with pc-IRES-IFN were more resistant to FMDV infection than those transfected with pc-IRES△G-IFN and pc-IRES△T-IFN. It's easy to conclude that IFN-αexpression under the regulation of FMDV IRES could provide enhanced protection to transfected cells.Today's studies show that RNAi is a powerful antiviral tool. According to the mir-30 model, we designed a miRNA expression casstte which was named p3D-1250, targeted the 3D region of FMDV genome. BHK-21 cells were transfected with p3D-1250 and stable cells were selected by G418. Plaque assays showed that cells transfected with p3-D1250 could suppress FMDV replication. Real-time RT-PCR demonstrated that p3D-1250 could inhibite FMDV replication to 50%. All of the data indicated that designed miRNA could effetively inhitite FMDV replication.