Autopahgy Mechanism Of Pcv2 Replication Promoted By Ota And Regulation Effects Of Selenium And Yeast Polysaccharide

Porcine circovirus type 2(PCV2)is the etiological agent of porcine circovirus-associated diseases(PCVAD),which includins post-weaning multi-systemic wasting syndrome,porcine respiratory disease complex,enteric disease,reproductive disease,which is arguably one of the most economically important diseases affecting the swine industry worldwide.However,not all pigs infected with PCV2 will develop PCVAD,other factors,such as animal management,coinfection and immunostimulation,have been reported to be associated with the disease,but the pathogenic mechanism of PCV2 remains uncertain.Ochratoxin A(OTA)is a mycotoxin produced principally by ubiquitous strains of Aspergillus and Penicillium.Owing to its natural origin,pervasiveness at low levels and chemical stability,OTA contamination persists throughout the feed supply and the food chain.Autophagy is a highly conserved mechanism that damaged organelles and misfolded proteins are degradated through the lysosomal pathway,which is widely present in eukaryotes.Normaly,autophagy acts as a defense mechanism against viral infections.However,during the long-term evolution,viruses have evolved a variety of strategies to escape or inhibit host cell autophagy,even ulitlize autophagy mechanism to promote their own replication.Our previous work suggests that OTA may be an important trigger of PCV2 replication and that the varying levels of OTA in pig feed may partially explain why the morbidity and severity of PCVAD vary among PCV2-infected pig farms.However,the underlying mechanism through which OTA promotes PCV2 replication requires further investigation.Despite much progress in the PCV2 vaccine,vaccine efficacy remains uncertain due to variations in concentration of antigen,adjuvant type and the administration doses.Nutrition supplementation in trace elements is suggested to be an effective prophylactic protection against viral infections.Selenium-enriched yeast is a new type of organic selenium preparation developed by our laboratory,which could be used as a nutritional supplement for selenium in diatery feed for animals.Selenium,an essential micronutrient for both humans and animals,which has many important biological functions,including enhancing the antioxidant function and immunity,anti-viral infections,and so on.Yeast polysaccharide is the main component of the yeast cell wall and also has biological functions such as enhancing immunity,promoting growth,and anti-viral infections,which has a wide range of sources and low cost.The present study was conducted to investigate the autophagy mechanism involved in OTA promoted PCV2 replication and selenium affecting PCV2 replication,and the effects of yeast polysaccharide on PCV2 infections in mice,which is helpful for the study of pathogenesis of porcine circovirus disease,and could provide scientific basis for the application of selenium,yeast polysaccharide and selenium-enriched yeast in animal husbandry production.Experiment Ⅰ,ochratoxin a-induced autophagy in vitro and in vivo promotes PCV2 replicationOchratoxin A(OTA)is a mycotoxin produced by Aspergillus and Penicillium.Porcine circovirus type 2(PCV2)is recognized as the causative agent of porcine circovirus-associated diseases.Autophagy is a metabolic mechanism that long-lived proteins and damaged organelles are degradated through the lysosomal pathway.Many studies have found that many viruses including PCV2 could ulitilize autophagy to promote their own replication.Recently,we reported that low doses of OTA promoted PCV2 replication in vitro and in vivo,but the underlying mechanism needed further investigation.The present studies further confirmed OTA-induced PCV2 replication promotion as measured by cap protein expression,viral titer,viral DNA copies and the number of infected cells.Our studies also showed that OTA induced autophagy in PK-15 cells,as assessed by the markedly increased expression of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ,autophagy-related protein 5(ATG5),and Beclin-1 and the accumulation of green fluorescent protein(GFP)-LC3 dots.OTA induced complete autophagic flux,which was detected by monitoring p62 degradation and LC3-Ⅱ turnover using immunoblotting.Inhibition of autophagy by 3-methylademine(3-MA)and chloroquine(CQ)significantly attenuated OTA-induced PCV2 replication promotion.The observed phenomenon was further confirmed by the knock-down of ATG5 or Beclin-1 by specific siRNA.Further studies showed that N-acetyl-L-cysteine(NAC),an ROS scavenger could block autophagy induced by OTA,indicating that ROS may be involved in the regulation of OTA-induced autophagy.Furthermore,we observed significant increases in OTA concentrations in lung,spleen,kidney,liver and inguinal lymph nodes(ILN)and bronchial lymph nodes(BLN)of pigs fed 75 and 150μg/kg OTA compared with controls in vivo.Administration of 75 μg/kg OTA significantly increased PCV2 replication and autophagy in the lung,spleen,kidney and BLN of pigs.Taken together,it could be concluded that OTA-induced autophagy in vitro and in vivo promotes PCV2 replication.Experiment Ⅱ,OTA induces autophagy via the AKT/mTOR and ERK1/2 signaling pathways in PCV2-infected PK-15 cellsThe previous study showed that ochratoxin a(OTA)could induce oxidative stress-mediated autophagy to promote porcine circovirus type 2(PCV2)replication.However,the underlying mechanism of OTA-induced autophagy remains unclear.In the present study,we investigated the roles of different kinases in OTA-induced autophagy by using specific inhibitors.The results showed that OTA could inhibit the activities of AKT and mTOR in PCV2-infected PK-15 cells,which leads to enhanced autophagy.Insulin could reverse the decrease of AKT and mTOR,autophagy and PCV2 replication promotion induced by OTA,which further demonstrating the critical role of AKT/mTOR signaling pathway in OTA-induced autophagy and PCV2 replication promotion.In the meantime,OTA could activate ERK1/2 signaling pathway;U0126 could inhibit ERK1/2 phosphorylation,reduce OTA-induced autophagy and PCV2 replication promotion,indicating that OTA induces autophagy and PCV2 replication promotion through the activation of ERK 1/2.Moreover,U0126 could upregulate OTA induced mTOR phosphorylation inhibition,indicating that OTA induces autophagy through the inhibition of mTOR by the activation of ERK1/2.Experiment Ⅲ,SeMet attenuates OTA-induced PCV2 replication promotion by inhibiting autophagy through activating the AKT/mTOR signaling pathwayPorcine circovirus type 2(PCV2)is recognized as the causative agent of porcine circovirus-associated diseases.PCV2 replication could be promoted by low doses of ochratoxin A(OTA)as in our previous study and selenium has been shown to attenuate PCV2 replication.However,the underlying mechanism remains unclear.The aim of the study was to investigate the effects of selenomethionine(SeMet),the major component of organic selenium,on OTA-induced PCV2 replication promotion and its potential mechanism.The present study demonstrates that OTA could promote PCV2 replication as measured by cap protein expression,viral titer,viral DNA copies and the number of infected cells.SeMet at 2,4 and 6 μM had significant inhibiting effects against OTA-induced PCV2 replication promotion.In addition,OTA could activate autophagy as indicated by up-regulated light chain 3(LC3)-Ⅱ and autophagy-related protein 5 expressions and autophagosome formation.Furthermore,SeMet could attenuate OTA-induced autophagy and up-regulate OTA-induced p-AKT and p-mTOR expression inhibition.Rapamycin,an inhibitor of AKT/mTOR,could reverse the effects of SeMet on OTA-induced autophagy and the PCV2 replication promotion.In conclusion,SeMet could block OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR pathway.Therefore,SeMet supplementation could be an effective prophylactic strategy against PCV2 infections and autophagy may be a potential marker to develop novel anti-PCV2 drugs.Experiment Ⅳ:Protective effect of yeast polysaccharides on PCV2 infections in micePorcine circovirus type 2(PCV2)is recognized as the causative agent of porcine circovirus-related disease(PCVAD).Previous studies have shown that autophagy plays an important role for PCV2 replication.Meantime,inhibition of autophagy could reduce PCV2 replication.Yeast polysaccharide(YSP),an important component of yeast cell wall,has been shown to inhibit the replication of many viruses.However,the effect of YSP on the PCV2 replication remains unstudied.The aim of this study was to investigate the effect of YSP on the replication of porcine circovirus type 2(PCV2)in mice and the related mechanisms of autophagy.100 ICR mice were randomly divided into 5 groups:Control group,PCV2 infection group,YSP-L group(100 mg/kg daily),YSP-M group(200 mg/kg daily)and YSP-H group(400 mg/kg daily).After continuous administration of YSP for 14 days,4 groups of mice except the negative control group were given intraperitoneal injection of 2000 TCID50 PCV2,and continued to be administered intragastrically for another 28 days.At 7 d,14 d,21 d,and 28 d after infection,PCV2 DNA copy number and Cap protein expression in different tissues were detected by qRT-PCR and immunohistochemistry;histopathological changes were determined by H&E staining,expressions of LC3 and ATG5 were determined by Western blotting,SOD activities,MDA contents,IL-2 and TNF-α contents in the serum were determined.The experimental results showed that different doses of YSP could significantly reduce the PCV2 DNA copy number and Cap protein expression in lungs and spleens of PCV2-infected mice;improve the lung and spleen pathological damage in PCV2-infected mice;enhance the antioxidant properties of mice by increasing SOD activities and reducing MDA contents in the serum,and increase the levels of IL-2 and TNF-α.Meantime,compared with the PCV2 infection group,different doses of YSP could significantly decrease the expression levels of LC3-Ⅱ and ATG5 in lungs and spleens.