| Porcine pseudorabies(PR)is an economically significant infectious disease caused by porcine pseudorabies virus(PRV),which not only seriously endangers the pig breeding industry,but also poses a potential threat to human health.Vaccination is an effective strategy in preventing and controlling PR.However,severe PR outbreaks have occurred in pig herds vaccinated with Bartha-K61 vaccine in many areas of China since 2011,implying that the current PR vaccines can not protect pigs from variant PRV infection,and posing a great challenge to pig industry in China.In addtion,the recent studies alarmed that PRV can accidentally cause acute encephalitis in humans.Therefore,it is urgent to find out drugs aimed to prevent and/or treat PR.Epigallocatechin gallate is one of the main active ingredients in green tea.Its antiviral activity has been confirmed in many virus families,such as Retroviridae,Orthomyxoviridae and Flaviviridae,presenting an antiviral effect on HIV,IAV,HCV and other important pathogens.Whether EGCG has antiviral effect on PRV is still unclear,and the molecular mechanism of potential effect of EGCG on PRV infection has not been declareed.This study aims to explore the antiviral effect of EGCG on PRV and its potential mechanism.1.The antiviral activities of EGCG on PRVIn this study,we investigated the anti-PRV activity of EGCG in vitro and in vivo.EGCG significantly inhibited infectivity of PRV strain Ra and variant PRV strain XJ5 in both PK15 B6 cells and Vero cells with a manner of dose-dependent,and 50 μM EGCG could completely block viral infection at different multiple of infections(MOIs).Furtherly,EGCG blocked adsorption and entry of PRV to PK15 B6 cells in a dose-dependent manner,with an adsorption inhibition superior to entry inhibition.PRV replication was suppressed in PK15 B6 cells treated with EGCG.However,EGCG did not affect PRV assembly but promote PRV release.Furthermore,40 mg/kg EGCG completely protected BALB/c mice from challenge with variant PRV XJ5 strain,in case of treatment either pre-or post-challenge.These results revealed that EGCG exhibits anti-PRV activity mainly by inhibiting its adsorption,entry and replication in vitro.Meanwhile,EGCG ameliorated the survival of mice challenged with PRV.Therefore,EGCG might be a potential natural agent against PRV infection.2.Potential molecular mechanism of antiviral effect of EGCG on PRV replicationAutophagy and apoptosis play important roles in virus replication.In this study,we founded that classic PRV RA strain and variant PRV XJ5 strain could induce PK15 and PK15 B6 cells incomplete autophagy and apoptosis through the Caspase 3 pathway,and 50 μM EGCG can impair this effect.Furtherly,we observed that PK15 and PK15 B6 cells infected with PRV promoted the expression of BNIP3.By overexpression or knockdown of BNIP3 protein in above cells,expression of BNIP3 was found to enhance PRV induced incomplete autophagy and apoptosis mediated by Caspase 3,and promote PRV replication.In addition,PK15 and PK15 B6 cells inoculated with either PRV RA or variant PRV XJ5 strain significantly up-regulated the expression of JAK1 and the phosphorylation level of STAT1,STAT5 and STAT6,and abated the phosphorylation level of STAT3.PRV replication was inhibited in PK 15 cells treated with Ruxolitinib,an inhibitor of JAK1,and also reduced the phosphorylation of STAT1,STAT3,STAT5,and STAT6,inhibited the expression of BNIP3 togather with incomplete autophagy and apoptosis mediated by Caspase 3.Knock down of STAT1 in PK 15 cells infected with PRV promoted both the expression of BNIP3 protein and the incomplete autophagy and apoptosis mediated by Caspase 3.While knock down of STAT3,STAT5,STAT6 in PK 15 cells infected with PRV,either the expression of BNIP3 protein or ther incomplete autophagy and apoptosis mediated by Caspase 3 was decreased.In conclusion,STAT1 negatively regulates PRV induced cells incomplete autophagy,apoptosis mediated by Caspase 3 and viral replication,while STAT3,STAT5,and STAT6 positively regulate the biological processes mentioned above.In this study,we found that EGCG inhibited the expression of JAK1 protein,decreased phosphorylation level of STAT5 and STAT6,and down regulated the expression of BNIP3 protein,thus alliviating PRV-induced incomplete autophagy and Caspase 3-mediated apoptosis.Meanwhile EGCG promotes the phosphorylation level of STAT3,and STAT3 positively regulates PRV replication,indicating that EGCG does not exert antiviral effects through STAT3.In summary,EGCG inhibits PRV-induce autophagy and apoptosis through the JAK1/STAT5(STAT6)/BNIP3 pathway,and then inhibite the replication of PRV. |
