| Porcine epidemic diarrhea（PED）is an acute diarrheal disease of pigs caused by Porcine epidemic diarrhea virus（PEDV）.The main clinical manifestations are acute diarrhea and vomiting,dehydration and high mortality of newborn piglets.Porcine epidemic diarrhea is an important disease that endangers the pig industry in China and seriously affects the development of pig industry.At present,PEDV mutant strain has become an epidemic strain in China,and its prevalence cannot be ignored,and the diseases caused by it will be one of the most serious diarrhea diseases that harm piglets in the next few years.After decades of efforts,inactivated vaccines,genetically engineered vaccines and live vaccines have been developed,but there is no specific drug for porcine epidemic diarrhea on the market.Therefore,it is of great scientific significance and application value to fully understand the epidemic characteristics of the disease in Jiangsu province in the past two years and to find a new treatment strategy for PEDV infection.This study carried out an epidemiological investigation on the epidemic situation of porcine epidemic diarrhea in some areas of Jiangsu Province and conducted a preliminary study on the application of RNA interference technology to inhibit the replication of PEDV.The main results are reported as follows:1.Molecular epidemiology of porcine epidemic diarrhea in Winter and spring of 2020-2021 in Jiangsu ProvinceIn order to understand the prevalence of PEDV in Jiangsu in recent two years,777 samples include small intestine tissue,feces and anal swab were collected from 72 pig farms in 5 major breeding areas of Jiangsu Province（Yancheng,Suqian,Changzhou,Xuzhou and Yangzhou）during the winter and spring seasons of 2020-2021.The PEDV-COE conserved area was amplified by RT-PCR for PEDV detection.The results showed that 449 samples from 57 farms were positive,with the positive rate up to 79.2%（57/72）in pig farms and 57.8%（449/777）in samples.Among them,78.1%（107/137）in Yancheng,53.3%（218/409）in Suqian,48.2%（94/195）in Changzhou,80.0%（16/20）in Xuzhou and 87.5%（14/16）in Yangzhou,indicating that the PED epidemic situation in Jiangsu province is serious and needs to be paid attention to.The nucleotide sequence of S1 gene of representative samples from different regions was amplified and sequenced,and phylogenetic tree analysis was performed.The comparison of S1 gene sequence with corresponding sequences of representative strains in GenBank showed that the current epidemic strains in Jiangsu province had genetic diversity,which posed a great challenge to the prevention and control of PED in Jiangsu Province.According to the phylogenetic tree of S1 gene,CH-SQJS-3,CH-YCJS-1,CH-SQJS-1 and CH-SQJS-2 were all assigned to the GⅡ group,and the remaining 3 strains were in the GI group（CH-YZJS-1,CH-CZJS-1 and CH-XZJS-1）.It is in the same branch as the classic CV777 strain,but the attenuated DR13 and attenuated CV777 vaccines currently on the market only offer protection against the GI strain.A new strain of PEDV CH-SQJS-1 was isolated from the above positive samples by cell culture technique.To sum up,this study not only provides reliable reference data for the epidemic situation of PEDV in Jiangsu province,but also provides reference for the research and development of vaccines in Jiangsu province.Meanwhile,the isolation of the new strain provides material conditions for the follow-up research.2.AmiRNA inhibits PEDV replication in Vero cellsIn order to seek new treatment methods of PEDV,in this study,two pairs of amiRNA（amiRNA-349、amiRNA-1447）targeting the conserved sequences of PEDV N and S genes were designed and chemically synthesized according to the design principles of amiRNA.It was confirmed by enzyme digestion and sequencing analysis and then they were transfected into Vero cells using LipofectamineTM3000.After transfection,PEDV HN-13 strain was infected with a MOI of 0.01.After 48 h of transfection,CCK-8 was used to detect the difference in cell viability of transfected groups with different doses.Cells and supernatant were collected at 24h,48h and 72h after transfection,respectively,and the effect of amiRNA on inhibiting PEDV proliferation was detected by cytopathic effect（CPE）,TCID50,Real-time PCR and Western Blot.The results showed that the constructed amiRNA could effectively inhibit PEDV replication in Vero cells.The results of CPE and CCK-8 showed that after 48h,the cytopathic changes of amiRNA-349 and amiRNA-1447 transfected groups were significantly reduced,and the cell survival rate was higher than that of the virus-infected positive control group and the amiRNA-NC.The results of Real-time PCR,Western Blot and TCID50 showed that among the two kinds of amiRNA,The inhibition effect of amiRNA-1447 on PEDV was much stronger than that of amiRNA-349 at 48h and 72h,while the inhibition effect of amiRNA-1447 on PEDV was weaker than that of amiRNA-349 at 24h.To sum up,this study aimed at the pig epidemic diarrhea virus N and S gene is the most conservative regions to build out of the two kinds of effective amiRNA expression vector,which laid a foundation for prevention and control of RNAi pig epidemic diarrhea,but also for the development of the directional antiviral pig molecular breeding technology provides reference data,also use RNAi technology for prevention and control of other coronavirus laid a theoretical foundation.3.AmiRNA multi-target tandem expression vector inhibited PEDV replication in Vero cellsIn order to inhibit the replication of porcine epidemic diarrhea isolated strains of different genotypes and play a long-lasting antiviral effect,at the same time,in order to avoid the escape of virus mutation,In this study,the two single amiRNA sequences mentioned above were connected in tandem to construct amiRNA multi-target tandem expression vector（amiRNA-349+1447）targeting PEDV N and S genes simultaneously,enabling the simultaneous expression of a single amiRNA.CCK-8 results showed that amiRNA multi-target tandem expression vector had no significant cytotoxicity.TCID50 results showed that the virus titer in the amirNA-349+1447 group was significantly lower than that in the amiRNA-NC group and the positive control group（VC）at 24h,48h and 72h of virus infection,and the virus titer was reduced by 43.7,35.5 and 229.1 times,respectively.These results indicate that amiRNA multi-target tandem expression vector can effectively prevent virus replication in Vero cells.Real-time PCR and Western blot results showed that the inhibition effect was significantly enhanced in different time periods after series.There was no significant difference between the amiRNA-NC group and the virus-positive control group.In conclusion,the multi-target tandem miRNA expression vector was successfully constructed in this study,and its synergistic inhibitory effect on PEDV replication was proved at the cellular level,thus indicating that the tandem strategy of multiple miRNAs may resist selective mutation and mutation of viruses.4.Inhibitory effect of amiRNA on epidemic variation of PEDV strain CH-SQJS-1 in Jiangsu provinceTo verify the three constructed amiRNAs（amiRNA-349,amiRNA-1447 and amiRNA349+1447）have inhibitory effects on different PEDV strains.In this study,the three amiRNAs screened acted on different PEDV strains（PEDV CH-SQJS-1）to verify their inhibitory effects on different PEDV strains,so as to achieve extensive therapeutic effects on porcine epidemic diarrhea caused by different PEDV genotypes in clinical practice.TCID50 results showed that the virus titer in amiRNA-349+1447 transfected group was significantly lower than that in amiRNA-NC group and virus positive control group（VC）at 24h,48h and 72h of virus infection.Real-time PCR and Western blot results showed that amiRNA could effectively inhibit PEDV CH-SQJS-1 strain.The above results indicate that amiRNA constructed in this experiment can effectively prevent the replication of different PEDV strains in Vero cells,providing a new direction to solve the current dilemma of PEDV mutated strains and difficult to treat. |
