A Study On Antiviral Activity Of Dihydroartemisinin Against Porcine Reproductive And Respiratory Syndrome Virus In Marc-145 Cells

Porcine reproductive and respiratory syndrome virus(PRRSV)the infection of this virus could lead clinically anorexia and fever on mature pigs,and miscarriage,premature delivery,stillbirth,mummified fetus and scrawny pig let on pregnant sows,and infected pig lets mainly are characterized with respiratory disturbance.PRRS is one of the most highly contagious pandemics,and this virus is difficut to be cleared once pigs are infected.PRRSV widely spreads around the world.In 2006,PRRS broken out in East C hina and South C hina and leaded to severe loss in C hinese swine industry and economic development.C urrently,the major strategy to prevent and treat PRRS is vaccination.However,on one thing,vaccination has the risk of virus returning,and on the other thing,PRRSV has evolved into various mechanisms to escape the immunity of hosts,which results in a limited effect of vaccination.Antiviral pharmaceutics are the second line of defense against viral infections.However,until today,there is no approved pharmaceutic for preventing and treating PRRS.In recent years,many studies have shown that natural and low-poisonous C hinese herbal medicines have significant prevention and treatment effects on viral infections.Therefore,the discovery of effective antiviral medicine s from natural compounds is becoming one of research hotspots.In this dissert,the effect of dihydroartemisinin(DHA)against PRRSV in vitro was evaluated systematically.Using the indirect immunofluorescence assa y(IFA),two compounds dihydroartemisinin(DHA)and artesunate(ART)showed significant inhibitory activities against PRRSV infection from artemisinin derivates.DHA was selected for further studies of antiviral effects and involved mechanisms due to it’s better solubility and low toxicity compared to other similar compounds.Firstly,a model for evaluating antiviral activity of compounds against PRRSV infection in Marc-145 cells was established.MTT assay was used for determining non-toxic concentration and IFA was used for determining optimal antiviral concentration of DHA.The antiviral effect of DHA against PRRSV post 48 h infection was affirmed by real-time PCR(RT-PCR)and Western Blot analysis,respectively.Three different treatment modes,including the direct incubation of DHA and the virus,the pretreatment of DHA on cells and adding DHA at different time point post infection,were used to investigate which stage of viral infection was intervened by DHA treatment through detecting the expression level of NSP-9 m RNA with RT-PCR assay.Our results showed that DHA could considerably inhibit PRRSV infection at the concentration of 0.6-2.5 μg/m L in a dose-dependent manner without harmful effects on cells’ survive.The results of RT-PCR and Western Blot indicated that DHA notably inhibited the expression of NSP-9 m RNA and N protein in a dose-dependent manner,respectively.In addition,DHA exhibited inhibitions on PRRSV in multiple stages of infection,including adsorption,internalization,synthesis and release of PRRSV to Marc-145 cells,while DHA did not exhibit inhibiton on PRRSV in the experiments of direct incubation of DHA with viruses and pretreatment of DHA with cells.These results suggested that DHA showed antiviral effects with a multiple mechanisms except for direct weakening viruses and blocking the receptors of Marc-145 cells to PRRSV.Our research initially proved that DHA has a potent antiviral activity against PRRSV infection in vitro,which has provided an experimental evidence for the development of novel agent for the prevention and treatment of PRRSV epidemics.