Antiviral Activities Of Artesunate, Aloperine And Pogostone Against Porcine Reproductive And Respiratory Syndrom Virus In Vitro

Porcine reproductive and respiratory syndrome(PRRS),which usually causessevere reproductive disorder,and respiratory disease,and growth retardation in the pigs,is a worldwide epidemic causing huge economicly losses in swine industry.PRRSV was first founded in the late 1980 's in United States,and nowadays this disease has been widely spread around the world including Europe,North America and Southeast Asia.PRRSV was first isolated from infected pigs in C hina in 1996.In recent years,vaccinationwas used to control PRRSV,such as inactivated vaccine,attenuated vaccine,DNA vaccine,subunit vaccine and live vector based vaccines.The effect of vaccination is affirmed,but there are certainlimitations such as failure of vaccination,antibody dependent enhancement and lower immunogenicity.Thus the development of novel antiviral agents against PRRSV is highlighted.Traditonal C hinese medcines(TCM)have been played a vital role in the prevention and treatment of virus infectious diseases in China.TCM provide a valuable resource for the funding of antivirals as TCM contain rich compounds with diverse chemical structures and bioactivities.It has been demonstrated that many compunds isolated from TCM,such as glycyrrhizic acid and sakosaponin,have extensive antiviral activities.However,documented studies on screening and effective components against PRRSV from TCM are very limited.In this dissertation,we reported that three compounds form TCM,artesunate,aloperine and pogostone,have potent antiviral activities against PRRSVin Marc-145 cells.The antiviral activities of the three compounds against PRRSV were systematically evaluated and their antiviral mechanisms of action were also investigated.Firstly,we established a model of PRRSV infection in Marc-145 cells.Based on this model,ten compounds including artesunate,aloperine and pogostone,out of 80 compounds from TCM,showed considerable antiviral activities against PRRSV infection b y immunofluorescence assay.The cytotoxity of the three compounds was investigated by MTT assay.qPCR and Western Blot analysis were used for evaluations of the inhibition on the production of PRRSV NSP-9 mRNA and the N protein of three compounds,respectively.The results showed thatartesunate,aloperine and pogostone,in the concentration ranged 0.6?g/mL~2.5?g/mL,10?g/mL~40?g/mL and 3?g/mL~12?g/mL,respectively,significantly inhibited PRRSV replication with a dose-dependent model.The three compounds did not show inhibition on PRRSV infection whilethe cells were pre-treatedwith tested compounds.In the experiment of direct inactivation of compounds on viruses,artesunate and aloperine,but not pogostone,showed direct inactivation on viruses.In the experiment for investigating that which stage of PRRSV replication was inhibited by the tested compounds,results showed that the three compounds can inhibit processes of PRRSV attachment and interlization to the cells.Moreover,pogostone can inhibit RNA replication of progeny viruses and aloperine can inhibit release of progeny viruses.Taken together,artesunate,aloperine and pogostone have significant antiviral activities against PRRSV with not completely the same mechanisms.We identified for the first timethat three compounds,artesunate,aloperine and pogostone,have considerable antiviral activities against PRRSV infection in vitro with mechanisms of suppressing multi-stages of PRRSV replication.Artesunate is one of clinically extensively used antimalarial agents,and aloperine and pogostone is the main bioactive ingredient of clinical commonly used TCMSophora alopecuroide and Pogostemon cablin,respectively.The three compounds have high biological safety.The results of this dissertation hasprovided experimental basis of further studies on antiviral effects of the three compounds against PRRSV in vivo.