Studies On Anti-porcine Deltacoronavirus Infection By ShRNA Expressing Plasmids

Porcine Deltacoronavirus(PDCoV)belongs to the order Nidovirales,genus Deltacoronavirus,family Coronavidae,which was found in recent years.It can result in severe diarrhea,vomiting,and dehydration in pigs,especially in newborn piglets with high morbidity and mortality,which is one of the main causative agent of diarrhea in piglets.PDCoV can cause serious widespread damage in most countries.It was first reported in Hong Kong,China in 2010,detected and isolated in pigs in the United States in 2014,since then it has been discovered by the other countries had reported it.There are no effective vaccines and drugs for treatment and prevention at present,so it is imperative to develop effective,new and safe drug or vaccines.RNA interference(RNAi)is the induction of mRNA degradation of the target genes by ds RNA in cells,so the expression of the target gene is decreased or silenced.RNA interference has been shown tremendous development potential in anti-viral and anti-cancer diseases.Using RNA interference technology to reduce the pathological phenomenon caused by PDCoV infection,and has a deep technical background.The following studies have been carried out:1.Focusing on the effect of shRNA expressing plasmid against PDCoV infection in vitro.The different shRNA expressing plasmids were respectively transfected into LLC-PK1 cells,then the cells subjected to limiting dilution to obtain the cell line.The cells were infected with PDCoV(100 TCID50),and 36 h post-infection they were analyzed by cell damage,viral titers and target gene expression levels.The results of CPE,Western Blot,q RT-PCR,MTS and TCID50 showed that the amounts of PDCoV RNAs in samples transfected with the pcDNA3.1-Double-shRNA-N1 expressing plasmids were reduced by92.7 %.2.We studied the effect of shRNA expressing plasmids on prophylaxis of PDCoV in vivo.Newborn piglets were randomly divided into 3 experimental groups(transfected with pcDNA3.1-Double-shRNA-N1,pcDNA3.1-shRNA-NC,and normal)and control groups(transfected with pcDNA3.1-Double-shRNA-N1,pcDNA3.1-shRNA-NC and normal),and each group contained 2 piglets.After 24 h the piglets injected with the shRNA expressing plasmid through the precaval vein twice,PDCoV(106 TCID50)was orally administered.The feces were collected every 4 h from the 12 h before infection,and the viral genome was determined by q RT-PCR.Euthanasia of all experimental animals were performed after48 h infection,and the small intestine of each piglet were collected to observe the pathological changes.From the dissection of piglets,pathological section of small intestine and q RT-PCR analysis,the pcDNA3.1-Double-shRNA-N1 expressing plasmid can effectively reduce PDCoV virion in vivo and thus lessen the histopathological changes triggered by PDCoV.Through complete analyses of the anti-PDCoV ability of shRNA expressing plasmid in vitro and in vivo.We conclude that shRNA expressing plasmid can effective protect LLC-PK1 cells and piglets from pathological changes triggered by PDCoV.