Inhibit The Replication And Infection Of Foot-and-mouth Disease Virus. RNAi Research

RNA interference, also termed RNAi, is a molecular mechanism of post-transcriptional gene silencing in eukaryotes that is discovered at the end of the last decade of twentieth century. It had been selected by Science journal as the greatest breakthrough of scientific technologies in 2001. RNAi process is initiated by double-stranded short interfering RNAs (siRNAs) (19-27 bp long) and mediated by a series of protein complexes, resulting in a sequence-specific downregulation of homologous gene expression at transcriptional or post-transcritptional or translational level. SiRNAs can be generated endogenously or exogenously when cells areinvaded by viruses and transposons, or transfected with plasmids expressing double-stranded RNAs. RNAi mechanism is highly conserved in eukaryotes from yeasts to mammals and proves to play a key role in developmental regulation and antiviral defense. At present, although there is more to RNAi than we can yet fathom, it has been extensively used as a reverse genetic tool to explore gene function as well as a therapeutic strategy against important human genetic diseases or viral diseases. Here we wish to review the mechanism of RNAi and its use as an antiviral agent.Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. The etiological agent of FMD is FMD virus (FMDV), which belongs to the genus Aphthovirus of the family Picornaviridae. VP1, one of four structural proteins of FMDV, is essential during the life cycle of the virus and plays a key role in virus attachment to susceptible cells.Here, we construct two plasmids (pNT21 and pNT63) expressing shRNAs targeting VP1 of FMDV. First, we evaluate the gene silencing efficiency of both plasmids using an enhanced green fluorescent protein (EGFP) reporter system in BHK-21 cells. Then, the antiviral potentials of both plasmids in BHK-21 cells and suckling mice are investigated. The results indicated that cotransfection of either pNT21 or pNT63 with a GFP plasmid resulted in an 80 to 90% reduction in EGFP signal relative to the control. Moreover, the antiviral potential induced by either pNT21 or pNT63 was evident in the face of challenge with a homologous FMDV, and the inhibition extended to almost 48 h. In suckling mice experiments, around 80% animals subcutaneously inoculated with 100 μg of plasmids in the neck survived a challenge with 20 LD₅₀ of homologous FMDV at 6 h post-inoculation. In addition, we found that pre-adiministration of a plasmid expressing VP1 mRNA FMDV significantly promoted survival of the animals. Our data suggest that RNAi may provide a viable therapeutic approach to treat FMDV infection.For RNAi to be a viable anti-FMDV agent, a crucial issue that needs to be addressed is the high genetic polymorphism exhibited by this group of virus. In this study, we identified five conserved regions of FMDV genome (namely 5' NCR, VP4, VPg, POL and 3' NCR) by sequence analysis. Using a cocktail siRNA generation kit, siRNAs directed against these regions of FMDV genome were produced by human recombinant dicer enzyme. Our results showed that cotransfection of BHK-21 cells with these siRNAs significantly reduced the level of EGFP reporter gene expression.Moreover, these siRNAs gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype 0 at 48 h post-infection (hpi). The inhibition extended to at least 6 days postinfection. For serotype Asial, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by around 10-fold when the cells had been pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. In addition, we demonstrated that an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.It has been clearly demonstrated that RNAi is an effective means of suppressing virus replication in vitro, but the antiviral potential of RNAi in animal systems needs to be further evaluated. Here we show thattreatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural proteins 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, we used guinea pigs as an animal system to evaluate the inhibitory effects of recombinant Ad5 on FMDV infection. The results showed that three of five guinea pigs inoculated with 10⁶ PFU of Ad5-POL and challenged 24 h later with 50 ID₅₀ of homologous FMDV were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. However, a simple combination of treatments such as double inoculation, a high-dose inoculation, or inoculation with an Ad5-NT21-Ad5-POL mixture cannot improve the protection of animals. Surprisingly, a group inoculated once with an Ad5-NT21-Ad5-POL mixture and challenged immediately after inoculation had reduced susceptibility to virus infection, suggesting that recombinant Ad5 expressing FMDV-specific shRNAs can rapidly prevent viral disease in animals. Based on the preliminary experiments in guinea pigs, we designed one part of the study to measure the antiviral activity of recombinant Ad5 in swine. The rusults indicated that two of three swine inoculated with 4×10⁹ PFU of an Ad5-NT21-Ad5-P0L mixture and challenged 24 h later with 100 ID₅₀ of homologous FMDV were fully protected from the major clinical disease. The inhibition was specific because a control recombinant Ad5 expressing E. coli LacZ-specific shRNA showed no marked antiviral potential.Sequence-specific RNA silencing by double-strand RNA has been observed in many eukaryotes. Accumulating data suggest that it is the major antiviral defense mechanism in plants and invertebrates. The discovery that this cellular mechanism is also highly conserved though somewhat impaired in mammals has stimulated debate about the evolution of antiviral systems. Here we suggest that the existence of the interferon response as an evolutionary intermediate could account for both the relative decline of RNA silencing and the development of protein-based immunesystems in vertebrates. In addition, we emphasize the opportunities presented by RNA silencing and the deeper understanding of vertebrate antiviral systems that is needed.