Inhibitory Effects Of Small Interfering Rnas On Replication Of Porcine Epidemic Diarrhea Virus In Vero Cells

Porcine epidemic diarrhea?PED?is a highly contagious intestinal infectious disease of swine,caused by the porcine epidemic diarrhea virus?PEDV?.PEDV can cause enteric disease with clinical signs including diarrhea,vomiting and dehydration in neonatal piglets.At present,the outbreak of the disease in pig farms has caused huge economic losses to the pig industry.Therefore,the study of new preparations for the prevention and treatment of PED has important scientific significance for the prevention and control of PED.The phenomenon of RNA interference?RNAi?is a defense mechanism in natural.This is a powerful technology of gene silencing.RNAi is a post transcriptional gene silencing mechanism mediated by exogenous or endogenous dsRNA.RTSC degrades the target mRNA and silences it.As a gene blocking technology,RNAi is widely used for gene function research and human disease treatment due to its sensitivity and specificity.In this study,inhibition of porcine epidemic diarrhea virus replication in Vero cells using RNA interference technology.The PEDV M and N genes are highly conserved among different strains.M protein is the main component of the viral envelope and is a transmembrane glycoprotein.The M protein plays an important role in the viral assembly process.The N protein forms a helical nucleocapsid with genomic RNA.N protein plays a very important role in the process of virus entering the cell,and N protein also participates in the assembly and release of virus particles.Both M and N genes are important genes in the PEDV genome and are involved in the replication of the virus.In this study,two siRNA sequences were screened from the M and N genes of PEDV respectively.Then,a siRNA sequence that was non-specific to the PEDV genome was designed as a negative control.After that,three pairs of shRNA-type nucleotide chains were synthesized.Single-stranded nucleotides were annealed to make the double-strands,respectively.Three pairs of double-stranded DNA strands were cloned into the downstream of the promoter of the pGenesil-1 plasmid.After sequencing,two PEDV-specific siRNA expression plasmids?pGenesil-M and pGenesil-N?and one negative control plasmid?pGenesil-NC?were successfully constructed.The constructed three plasmids were respectively transfected into Vero cells by liposome.After selection with G418 and PCR identification,three cell lines of correctly recombinant was screened.Different cell lines were inoculated with PEDV for 48 h.The viral infection titers were determined by TCID₅₀.Expression of PEDV mRNA and protein were detected by TaqMan Real-time quantitative PCR and Western-blotting,respectively.As a result,in the Vero cells of the PEDV-specific pGenesil-M and pGenesil-N,the virus titer,mRNA,and protein levels were all lower than those of the negative control?pGenesil-NC?and positive control of the non-transfected plasmid.The PEDV-specific pGenesil-M and pGenesil-N expression plasmids could inhibit the synthesis of PEDV mRNA and the M and N protein in Vero cell in various degrees.Especially,the inhibitory effect of pGenesil-N plasmid was significantly higher than pGenesil-M plasmids.In conclusion,this study demonstrated that siRNA targeting the M and N genes of PEDV can inhibit the replication of PEDV in Vero cells.Different siRNAs have different inhibitory effects.These results provide scientific basis for the development of new biologics for the prevention and treatment of PEDV.