The Mechanism Of Antiviral Activities Of Melatonin And Berberine Against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED)is an acute,highly contagious intestinal disease caused by porcine epidemic diarrhea virus(PEDV),which infects pigs of all ages.Clinical symptoms associated with PEDV may include watery diarrhea,vomiting and dehydration,anorexia and depression.Newborn piglets are most sensitive to PEDV infection with an incidence rate up to 100%,and a mortality rate as high as 80%～100%.Symptomatic treatment is not effective and eturn feeding may cause mixed infection.Current available inactived or attenuated PEDV vaccines cannot provide complete protection against PED.Therefore,it is important to develop effective antiviral drugs,which is of great significance for PED prevention and control.1.Melatonin inhibits PEDV infection by down-regulating ROSMelatonin(N-acetyl-5-methoxytryptamine),a guanamine present in various bacteria,fungi,plants and animals,plays a role in viral infection through different mechanisms,such as anti-apoptotic,anti-inflammatory,and antioxidant properties.Reactive oxygen species(ROS)are involved in many biological processes and organelles functions,such as lipid degradation,electron leakage of the mitochondrial electron transport chain,and protein folding in the endoplasmic reticulum.Environmental stress and inflammatory responses also induce ROS production,and thus affect the relationship between viruses and hosts.Therefore,in this part of the study,the relationship between melatonin,ROS and PEDV was explored.It showed that melatonin reduce the expression level of viral N protein and viral titers in PEDV-infected cells.In addition,PEDV infection promotes ROS production,and in turn ROS scavengers and NADPH oxidase inhibitors attenuate ROS caused by PEDV infection and inhibit proliferation of PEDV,indicating that ROS is beneficial for PEDV infection.Interestingly,melatonin also reduces ROS levels in PEDV-infected cells.Overall,this study demonstrates that melatonin is a potential anti-PEDV compound that inhibits PEDV proliferation by down-regulating ROS production in infected Vero cells.2.Melatonin inhibits PEDV dependent on the SIRT1-ROS pathwaySIRT1,a member of class III histone deacetylase,is a NAD+dependent deacetylase and a mammalian homolog of yeast silencing regulator 2(Sir2).SIRT1 plays a vital role in the process of programmed cell death,lipid and glucose metabolism,and inflammatory signaling,and also has a certain antioxidant activity.Therefore,we explored the mechanism of SIRT1 in PEDV infection.It was found that both PEDV infection and ROS treatment down-regulate SIRT1 protein levels.Overexpression of SIRT1 not only inhibits PEDV infection,but also down-regulates ROS caused by PEDV infection.Importantly,the results showed that melatonin is capable of up-regulating the expression level of SIRT1,while silencing SIRT1 eliminates the inhibitory effect of melatonin on PEDV proliferation.In summary,melatonin inhibits PEDV infection dependent on the SIRT1-ROS pathway.3.Berberine inhibits PEDV infectionBerberine chloride(C20H19NO4.Cl),a natural isoquinoline alkaloid extracted from traditional Chinese medicine.Berberine has various biological functions and exhibits antiviral activity during various viral infections.It has been reported that PEDV can induce apoptosis to support viral replication.Therefore,in this study,we explored the antiviral activity of Berberine on PEDV.The results showed that Berberine treatment significantly inhibited the cytopathic effect caused by PEDV and reduced the amount of intracellular and extracellular virions in PEDV-infected cells.In addition,by detecting the effects of Berberine on different stages of virus life cycle,it was found that Berberine inhibited the replication and assembly of PEDV without affecting the process of virus adsorption,entry and release.Finally,the apoptosis marker proteins PARP1 and caspase7 were examined,and the results showed that the cleaveage of PARP1 and caspase7 proteins in virus-infected cells were significantly attenuated.Together,it is likely that Berberine is another potential natural bioactive compound against PEDV infection.