A Study On Inhibition Of Platycodin D Against Porcine Reproductive And Respiratory Syndrome Virus In Vitro

Porcine reproductive and respiratory syndrome(PRRS),caused by PRRS virus(PRRSV)infection,is a major economically epidemic disease which has been heavily harmed the global swine industry for almost 30 years.PRRS is mainly charactered with porcine respiratory symptoms,high mortality of newborn piglets and reproductive disturbance of pregnant sows.The circulating PRRSV strains in China belong to North American type II.In spite that vaccination has been widely used for preventing PRRS epidemics clinically,it's efficacy was limited by high variation,circulation of multiple viral subtypes and antibody-dependent enhancement of PRRSV.Therefore,the development of novel agents against PRRSV has been highlighted.Platycodon grandiflorum(PDG)is one of frequently used traditional Chinese medicine materials.Clinically in TCM,roots of PDG,with efficacies of eliminating phlegm and abscess and freeing lung and relieving asthma,were used as one of important herb medicines to treat respiratory diseases.With a higher content(up to 0.6%),Platycodin D(PD)is a dominating saponin in PDG.It has been reported that PD has bioactivities of antitumour,reducing cholesterol,anti-inflammation and relieving asthma.In the present dissertations,the activity of PD against PRRSV infection was discoveried and investigated systematically,and mechanisms of action were also explored.In this study,activities of more than 50 pentacyclic triterpene compounds against PRRSV infections were screened using indirect immunofluorescence assays(IFA)and PD was proven to have a remarkable antiviral activities.A systematical and in-depth study for evaluating activities of PD against PRRSV infection in vitro was performed.MTT assay was employed to determine cytotoxicity of PD,and RT-PCR assay was used to evaluate effects of PD on PRRSV NSP-9 mRNA expression,and Western blot and IFA were used to determine expression of PRRSV N protein.These assays were employed in combination to evaluate activities of PD against PRRSV in Marc-145 cell and PAMs cultures at 48 hours post infection(hpi)and at different time-pionts during 72 hpi.Furthermore,directinactivation of PD on PRRSV,and effects of PD on adsorption,internalization,replication,and release of PRRSV,and effects of PD on TNF-a andI L-8 RNA expressions induced by PRRSV infection in PAMs were also studied.The results showed that PD significantly inhibited the proliferation of PRRSV in Marc-145 cell and PAMs cell cultures at the concentration range of 1.25-5 ?g/mL in a dose-dependent manner.At all examined time points within 72 hpi,PD showed a unremitting inhibition on the replication of PRRSV.The results of time-of addition tests indicated that PD was able to block the stages of adsorption,internalization,replication and release of PRRSV in a dose-dependent manner,and PD is also able to directly combine to PRRSV and weaken the activity of virions.Pretreatement on Marc-145 cells with PD did not affect the susceptibility of cells to PRRSV.Moreover,PD treatment reduced the expression of TNF-a and IL-8 RNA induced by PRRSV infection in PAMs cell cultures in a dose-dependent manner.The present study showed that PD has a considerable activity against PRRSV infection in vitro.Mechanisms of action of PD against PRRSV include weakening activity of virions by direct combination with virus,inhibiting the stages of attachment,internalization,replication and release of PRRSV,and reducing the expression of TNF-a and IL-8 RNA induced by PRRSV infection.