Establishment Of Multiplex RT-PCR For Porcine Diarrhea Pathogen And Relationship Investigation Between PEDV And Autophagy

Porcine viral diarrhea is a com,on infectious disease in pigs,which is seriously endangering the development of pig industry.The pathogens of porcine viral diarrhea mainly include porcine epidemic diarrhea virus,transmissible gastroenteritis virus and porcine rotavirus.The clinical symptoms of these three diseases are very similar,with vomiting,watery diarrhea and dehydration as the main characteristics.There is an urgent need for a rapid and sensitive method for the identification and detection of the three viruses.Since 2010,porcine epidemic diarrhea has erupted in a large number of pig farms in China,and characterized by high mortality rate.Compared with the classical epidemic strains,the pathogenic characteristics of the epidemic strains have changed,and the pathogenic mechanism needs to be further studied.1.Establishment of multiplex rt-pcr for three porcine diarrhea pathogensIn this study,three pairs of primers were designed according to the E gene of PEDV,S gene of TGEV and VP3 gene of PoRV.A Multiplex RT-PCR method was established for the detection of three porcine viral diarrhea pathogens,PEDV,TGEV and PORV.The method has good specificity and sensitivity,and can detect the minimum amount of 100 pg RNA template.It can be used in clinical diagnosis and identification of these three viruses.Of 133 clinical samples,45 were PEDV positive,1 was TGEV positive and 6 were PoRV positive.The results showed that the clinical infection rate of PEDV was the highest and PEDV is the most harmful pathogen to pig industry.2.Effect of PEDV on autophagyThe distribution of fluorescent spotting location was observed by laser confocal microscopy.The autophagy-related gene protein LC3 was detected by Western Blot.The results show that PEDV infected IPEC-J2 cells can induce the formation of autophagy bilayer structure in cells,and make LC3-GFP-mRFP in the cell spotting localization,up-regulation of LC3-II expression level.The similar results were obtained in IPEC-J2 cells,indicating that PEDV infection induced cell autophagy.To investigate the effect of autophagy on viral replication,it was found that the PEDV titer in the autophagy inducer Rapamycin treated IPEC-J2 cells was increased,while the PEDV titer in the autophagy inhibitor 3-MA treated IPEC-J2 cells was decreased.The studies have shown that autophagy promotes PEDV replication.The recombinant plasmid pCAGGS-nsp6-HA was constructed and transfected into IPEC-J2 cells.The relationship between nsp6 protein and autophagy was observed by the above experiments.The results showed that nsp6 protein could induce autophagy in IPEC-J2 cells.These studies provide a new perspective for exploring the interaction between PEDV and host cells and the pathogenesis of PEDV.