| Porcine Transmissible Gastroenteritis virus (TGEV) belongs to Coronavirus genus, Coronaviridae, it is the main cause of transmissible gastroenteritis (TGE) of swine. TGE is acute and hyperinfectious disease, which usually outbreaks in winter and spring. Infected animals are characterized by vomiting, diarrhea and severe dehydration. There are no differences among ages and strains of swine in susceptibility to the disease, the mortality of piglets aging less than 2 weeks can be high up to 100%. Generally, the causes of death are dehydration and disorder of metabolism of electrolyte. The mortality decrease as the animals grow, but the survived animals show growth retardation and low rate of reward to feeding. TGE covers a wide range all over the world. Most countries with porcine industry suffer from this disease, resulting in enormous economic loss. Since the first report in 1950s in our country, the disease has been found in many places. TGE is one of the list B infectious diseases, and should be inspected in market according to the law in our country.No conventional medicine shows efficacy for TGE and vaccination is the commonly used method so far. But the widely used vaccines themselves contain various disadvantages. The inactive vaccine is of both high cost and low protection. The attenuated one has potential risk of virus spread, reverting to a virulent form, and perhaps interfering with the diagnosis of wild strain of TGEV. As the replacements, genetically engineering vaccine and nucleic acid vaccine still need to be improved due to their costs and efficacy in immunoprotection, etc. All these obstacles in the course of treated and prevented pigs from TGE result in less profit in swine industries. Therefore, researching on new, safe and effective vaccines or other measurements for treatment and prophylaxis of TGEV is necessary and urgent.As an ancient defense mechanism of cells against hostile genes, RNA interference (RNAi) cleaves mRNA in a sequence-specific manner. As an efficient, economic and convenient research tool, RNAi has exceeded traditional antisense technology on the level of gene knockdown, becoming a powerful tool in scientific reseach both in vitro and in vivo. RNAi have been shown especially tremendous development potential in anti-cancer and anti-virus diseases, and a lot of cancer-associated genes and viral replications have been effectively silenced.TGEV, the pathogen of TGE, has a clear etiological classification, known well of biological characteristics, general genomic structure and function. In addition, TGEV has a single, positive-strand RNA, which can function as an mRNA itself. Taken together, there are good technical backgrounds and biological bases for employing RNAi technique as an anti-TGEV strategy.In sum, the goal of this study is to protect ST cells and minipigs from TGEV infection by using specific and potent RNAi technology, which advances fast and costs decreasingly, also to break through the bottleneck of therapy of TGE and to find some new therapeutics. In addition, we hope to provide scientific references for researching and manufacturing agents against other viruses and for large animal-associated RNAi experiments.This study contains the following three parts:Part IThe 5’sequence of S gene from domestic isolates TGEs-1, including a part of polymerase gene, was successful amplified with designed primers covering a mutable district of TGEs-1 gene. Sequencing results were blasted with the homologous isolates in GenBank, and the homology is high up to 98%. Then, seven siRNA targeting TGEs-1 genome were generated by combining our identified sequence of TGEs-1 S gene with the other conserved genes among different strains existing in domestic SC-Y strain. After that, 8 shRNA-expressing vectors, pEGFP-U6/P1, pEGFP-U6/P2, pEGFP-U6/S1, pEGFP-U6/S2, pEGFP-U6/M, pEGFP-U6/N1, pEGFP-U6/N2 and a non-specific interference control, pEGFP-U6/T were successfully constructed, all of which contained an EGFP-expressing gene. The above works established the foundation of the following work aiming to efficiently interfer the replication of TGEs-1 in ST cells and minipigs.Part IIThis part focused on shRNAs-expressing plasmids against TGEV on cellular level. ST cells were respectively transfected with different shRNA-expressing plasmids with 0.4μg per well when they reached a confluence of 70-80%. 28 hours later, the cells were infected with TGEs-1(200CCID50), and 40 h after infection they were analyzed by CPE and MTS methods. CPE analysis shows that pEGFP-U6/P2 and pEGFP-U6/P1 transfected cells didn’t emerge disease or only showing weak suspected "lesions" in the margin of well, meanwhile other wells displayed different degree of characteristic lesions. The shRNA-expressing vectors pEGFP-U6/S1 and pEGFP-U6/N1 have more strongly anti-infection, only showing weak lesions in the well margin, than pEGFP-U6/S2, pEGFP-U6/M and pEGFP-U6/N2, which showing lesions almost in the whole well. Among them, the inhibitory effects of pEGFP-U6/S2 and pEGFP-U6/N2 are similar with that of non-specific plasmid pEGFP-U6/T. In sum, the data above show that the ability of shRNA-expressing vectors pEGFP-U6/P1、pEGFP-U6/P2、pEGFP-U6/S1 and pEGFP-U6/N1 to protect cells from TGEV destruction is fairly strong, especially pEGFP-U6/P1 and pEGFP-U6/P2. In addition, the analysis of MTS shows that the number of viable cells in wells transfected with pEGFP-U6/P1、pEGFP-U6/P2、pEGFP-U6/S1 or pEGFP-U6/N1 is obviously bigger than that transfected with the other plasmids, and the data further confirmed the conclusion drawn from CPE analysis. Taken together, we chose pEGFP-U6/P1 and pEGFP-U6/P2 as the interference vectors for the next experiment on minipigs. Besides, before doing experiments in vivo, we also analyzed the ability of decreasing the quantity of TGEs-1 RNA by RT-qPCR method, and the data indicated that comparing with non-specific plasmid pEGFP-U6/T, the plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) could reduce the number of virus RNA in cell culture in a large scale, especially the pEGFP-U6/P2 nearly reach a 100% inhibition, which suggested that the virus inhibition triggered by the plasmids was caused by sequence-specific RNAi.Part IIIIn this part, we studied the effect of shRNA-expressing plasmids on prophylaxis of TGEV in vivo. Piglets aging 25 days were randomly divided into 3 interference groups (pEGFP-U6/P1, pEGFP-U6/P2 and a combination of the two) and 3 control groups (just transfected with pEGFP-U6/T, just infected with TGEs-1 and normal), and each group contained 2 piglets. Then piglets were injected with shRNA-expressing vectors (3mg/20ml/pig) through the precaval vein, and inoculated TGEs-1 24h later. They were sacrificed 3 days after infection, and the liver, lung, kidney, jejunum, ileum, caecum, spleen and mesenteric lymph node of each piglet were collected. Each sample was divided into 3 parts for analysis of histopathological changes, immunohistochemistry and RT-PCR, respectively.Twenty six hours after infection, one of the piglets just infected with TGEs-1 began to diarrhea, flabbiness, dehydration and thinness, and this situation lasted till it was sampled. Meanwhile, piglets in other groups were normal with a good appetite. Histopathological changes analysis showed that there were granular degeneration of hepatocytes and renal tubular swelling in some minipigs transfected with pEGFP-U6/P1、pEGFP-U6/P2 or the combination of them, but all intestinal villi in them were in good shape. Besides the histopathological changes in livers and kidneys above, minipigs just infected with TGEs-1 showed by severe glomerular swelling as well as shorten and dropped intestinal villi. The results of IFA and RT-PCR indicated that virions and virus RNA couldnot be found in all the samples from minipigs transfected with shRNA-expressing plasmids, which suggested that pEGFP-U6/P1 and pEGFP-U6/P2 could reduce TGEs-1 virion efficiently in vivo and thus lessen the histopathological changes triggered by TGEs-1.In short, through complete analyses of the anti-TGEs-1 ability of shRNA-expressing plasmids in vitro and in vivo, we have found that shRNA-expressing plasmids could effectively protect ST cells as well as minipigs from pathological changes triggered by TGEs-1. So, we believe that undergoing further optimization, the pEGFP-U6/P1 and pEGFP-U6/P2 could be utilized in clinic as anti-TGEV agents in the future. |
PDF Full Text Request