The Mechanism Of Cinchonine Inhibiting The Replication Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea is a kind of intestinal disease of swine.It is caused by porcine epidemic diarrhea virus.The mortality rate of newborn piglets infected with PEDV is up to 100%.Although there are commercially available PEDV vaccines in China and other countries,highly virulent PEDV has been prevalent due to the high variability of PEDV genome.Antiviral drug therapy is considered an effective way to treat coronavirus infection.Therefore,it is necessary to develop effective drugs for the prevention,treatment and control of PEDV.Cinchonine,a natural compound that can inhibit PEDV replication,was screened by targeting endoplasmic reticulum stress in this study.Cinchonine is an extract of Cinchona bark,which can activate the apoptosis of human liver cells induced by endoplasmic stress and has many biological activities such as antimalaria,antiobesity and antimultidrug resistance.In this study,we found that Cinchonine inhibited the proliferation of PEDV,and its anti-PEDV activity was verified in vitro.The expression of autophagy and endoplasmic stress related proteins were detected,and the effect of SQSTM1/P62 on PEDV replication was also verified,which revealed the molecular mechanism of inhibiting PEDV proliferation by Cinchonine.The detailed researches are as follows:1.Study on the effect of Cinchonine against Porcine Epidemic Diarrhea Virus ReplicationTo determine the optimal concentrations of Cinchonine on different cells,we first used the CCK8 assay to evaluate the toxicity of Cinchonine on Vero cells,LLC-PK1 cells and ST cells.The results of CCK8 assay showed that the cell survival rate was greater than 90% at a concentration of less than or equal to 200 μM,so the maximum concentration of Cinchonine was determined to be 100 μM in the following study.Based on the results of indirect immunofluorescence assay(IFA),Western blot and the quantitative real-time PCR(RT-q PCR),Cinchonine was found to effectively inhibit the infection of PEDV GⅠ group strains.In order to investigate the broad-spectrum inhibition of PEDV replication by Cinchonine,Vero cells were also infected with PEDV GⅡ strains(YN15,GDU and JSXC)in this study to verify the anti-PEDV of Cinchonine.The results of Western blot and RT-q PCR indicated that Cinchonine could significantly reduce the transcription of PEDV RNA and proteins in different strains of PEDV-infected cells.In addition,the main action time of Cinchonine was determined by administrating Cinchonine at different time periods after infection,and it was found that Cinchonine exhibited the strongest anti-PEDV activity when it was added at 2 h,thereby suggesting that early treatment was more effective against PEDV infection.2.Study on the mechanism of Cinchonine exerting anti-PEDV by regulating SQSTM1/p62More and more studies have been proved that autophagy is related to the replication of viruses.It has been reported that autophagy can be induced after PEDV infection,which increased the conversion rate of endogenous LC3 and gradually decreased the expression of SQSTM1/p62.However,the role of exogenous changes in SQSTM1/p62 expression in PEDV infection remains unclear.In this study,we first evaluated the effect of Cinchonine on autophagy of Vero cells and LLC-PK1.The results showed that the conversion of LC3-Ⅰ to LC3-Ⅱ was markedly increased and the expression level of SQSTM1/p62 was significantly up-regulated,demonstrating that Cinchonine could regulate intracellular autophagy.Therefore,we speculated that Cinchonine could effectively inhibit PEDV replication,which is related to autophagy or SQSTM1/p62.We observed that when autophagy was inhibited by 3MA,the conversion of LC3 induced by Cinchonine was significantly decreased,and the antiviral of Cinchonine was greatly weakened.We further confirmed that the overexpression of SQSTM1/p62 could reduce the expression of viral protein and the transcription of viral RNA,indicating that Cinchonine may exert its anti-PEDV effect through SQSTM1/p62-dependent pathway.Further study showed that SQSTM1/p62 could mediate PEDV N protein degradation via its UBA domain,thus inhibiting virus replication.In conclusion,this study systematically identified a new natural compound Cinchonine that can effectively inhibit PEDV infection,and further confirmed that the inhibition of PEDV replication by this compound may be related to autophagy.This study provides a theoretical basis for the research and development of novel anti-PEDV drugs.