Inhibition Of Porcine Reproductive And Respiratory Syndrome Virus Replication By Silencing GP5Gene In MARC-145Cells

Porcine reproductive and respiratory syndrome virus （PRRSV） emerged inEurope and in the US in the last1980s and has since become a problem to the swineindustry worldwide. PRRSV causes a persistent and sometimes severe disease that ischaracterized by respiratory problems, weight loss and poor growth performance, aswell as reproductive failure in pregnant sows. Measures to regulate the disease havebeen complicated by the pattern of persistent, subclinical infection with occasionalepidemic outbreaks as well as the high heterogeneity of the virus and the failure ofthe antibody response to completely protect against viral re-infection andre-emergence. PRRSV is a member of the Arteriviridae family of enveloped viruseswith positive-sense （+） RNA genomes. PRRSV genome includes8open-readingframes （ORFs）. ORFla and ORFlb encode nonstructural proteins at the3’end of thegenome, ORFs2-5encode glycosylated membrane proteins GP2–GP5, ORF6encodes a non-glycosylated membrane protein （M）, and ORF7encodes thenucleocapsid （N） protein. The glycosylated200-residue GP5protein is the mostvariable protein of PRRSV. The major components of the PRRSV envelope are GP5and M, which together comprise at least half of the viral protein and formdisulfide-linked heterodimers in the virus. Deletion of either of these two ORFs froman infectious PRRSV clone led to a failure to produce viral particles, while deletionof the minor envelope proteins did not have an effect on viral production.The past decade has seen the rapid evolution of small-molecule gene-silencingstrategies, driven largely by the development of molecular biology andmolecular virology. The broad umbrella of gene-silencing therapeutics encompasses arange of agents that include DNA enzymes, short interfering RNA, antisense oligonucleotides, decoys, ribozymes, and aptamers.Three plasmids that expressing three shRNAs targeting the Porcine Reproductiveand Respiratory Syndrome Virus（PRRSV） protein GP5gene was designed. Marc145cells were infected with PRRSV after the shRNA-expressing plasmids were delivered6h, efficiency of RNA interfering was assayed by Real-time RT-PCR, TCID₅₀, CPEand indirect immunofluorescence assay （IFA）. This experiment successfully revealedthe shRNA expressing plasmids RNAi phenomenon of PRRSV replication procedurein MARC-145cells and showed that the three interference target site may benecessary for PRRSV replication. Four DNAzyme targeting the PRRSV protein GP5gene was designed. The active DNAzyme was derived by cleavage experiments invitro．Marc145cells were infected with PRRSV after the active DNAzyme weredelivered6h, efficiency of DNAzyme was assayed by Real-time RT-PCR, TCID₅₀,CPE and indirect immunofluorescence assay （IFA）. This experiment successfullyderived the active DNAzyme inhibition of PRRSV replication procedure inMARC-145cells and showed that the two interference target site may be necessaryfor PRRSV replication.In total, through the experiments presented above, Three plasmids expressingthree shRNAs that include RNAi-GP5-10、 RNAi-GP5-16、 RNAi-GP5-19wasdesigned and constructed. Two active DNAzyme that include Dz-GP5-10andDz-GP5-12was derived by cleavage experiments in vitro． This experimentsuccessfully derived the active that inhibition of PRRSV replication procedure inMARC-145cells and showed that the five interference target site may be necessaryfor PRRSV replication.In future studies, the combination may be used as a tool tostudy PRRSV replication, antiviral therapy and transgenic animals.