Analysis Of Artemisinin Inhibiting The Flaviviruses Replication And Its Mechanism

Flaviviruses are a class of viruses that seriously endanger human or animal health.Most flaviviruses can be transmitted by blood-sucking insects such as mosquitoes.Japanese encephalitis virus(JEV),West Nile virus(WNV),Dengue virus(DENV),and Zika virus(ZIKV)are typical flaviviruses.JEV and WNV could cause human viral encephalitis.JEV also has a bad impact on pig and other breeding industries.DENV mainly induces acute and bleeding.ZIKV would lead to Guillain-Barre syndrome in adults and fetal microcephaly.However,effective anti-flavivirus treatments are still lacking currently.So there is an urgent need for broad-spectrum drugs for existing and emerging flaviviruses.Artemisinin is well-known for anti-malarial function.In recent years,artemisinin has been found to show therapeutic effects on many viruses.However,its antiviral effect against flaviviruses remains unexplored yet.In this study,JEV,DENV,and ZIKV were taken as examples to analysis the therapeutic effect of artemisinin and its antiviral mechanism in vitro and in vivo.The specific contents are as follows:1. Artemisinin could inhibit JEV,DENV and ZIKV replicationIn vitro experiments,the host cells infected with JEV,DENV,and ZIKV were divided into artemisinin-treated group and control group.Artemisinin-treated group added non-toxic concentration of artemisinin to the fresh medium.While the control group added equal amount of DMSO to the changed medium.Cell and supernatant samples were collected at different time points.Quantitative Real-time PCR was used to detect the effect of artemisinin on viral RNA levels.The plaque assay was used to detect the effect of artemisinin on virus proliferation titers.The results showed that the artemisinin-treated group significantly inhibited the expression of viral RNA levels compared to the control group after the host cells infected with JEV,DENV or ZIKV.The results of plaque assay also showed that the artemisinin-treated group obviously inhibited the virus titers.In the24h samples,artemisinin treatment could reduce about 100 times the infectious JEV virus particles in the cell supernatant.The inhibition of DENV or ZIKV replication is about10-fold.In the 36h samples,the artemisinin-treated group can inhibit the JEV,DENV or ZIKV virus titers by about 10-fold compared with the control group.The results showed that artemisinin could significantly inhibit virus replication of JEV,DENV,and ZIKV.Which indicated that artemisinin might have a broad-spectrum antiviral effect on flaviviruses.2. Artemisinin enhanced flavivirus-induced type I interferon expression in host cellsThe mechanism of artemisinin against flavivirus was further explored.Quantitative Real-time PCR and ELISA detections showed that artemisinin promoted the expression and secretion of IFN-?in JEV,DENV or ZIKV-infected cells.The expression of downstream ISGs molecules:ISG56,Mx A,OAS-1,were also higher after artemisinin treatment.Besides,Western blot results showed that artemisinin promoted the phosphorylation and activation of p-IRF3,P-STAT1,and p-STAT2 in the IFN-I signaling pathway.Next,when IFNAR polyclonal antibody were used to block IFN signaling,artemisinin no longer showed anti-flavivirus effect significantly,which proved that artemisinin could exert anti-viral mechanism by promoting the expression of IFN-I.Our further research suggested that this enhanced IFN-I expression might be related to the increased expression of pattern recognition receptors RIG-I and TLR3 in flavivirus-infected host cells.3. Artemisinin reduced mortality and encephalitis symptoms of JEV-infected miceA mouse model of artemisinin to protect against JEV infected mice was designed to verify that artemisinin could reduce the mortality and relieve their brain inflammation.Six-week-old C57BL6J female mice were randomly divided into six groups for experiments:DMEM+DMSO,DMEM+Artemisinin,DMEM+Artesunate,JEV+DMSO,JEV+Artemisinin,JEV+Artesunate,15 mice per group.On day 0,mice were intraperitoneally injected with 10~5PFU JEV P3 or equal volume of DMEM.On days 3and 4,mice were intravenously injected with 10 mg/kg artemisinin,artesunate or an equal volume of DMSO.Observation lasted 23 days.On day 5,samples of 5 mice from each group were collected for following testing.The data of the remaining 10 mice was used to draw body weight,behavior,and survival rate curves.Besides,on day 23 tissue samples of the mice were also taken for the following pathology and molecular testing.The results showed that in JEV+DMSO group mice behaved typical neurological symptoms with a mortality rate of 100%.In JEV+Artemisinin group the mortality was 50%,and in JEV+Artesunate group the mortality was 40%.Artemisinin or artesunate increased the survival rate of mice,inhibited the weight loss of mice,improved the neurological symptoms of mice,and significantly reduced the viral load in the organs and blood of the whole body tissues of mice.No obvious virus was detected in the survived mice.Severe inflammation occurred in mouse brain of JEV+DMSO group.While in JEV+Artemisinin and JEV+Artesunate groups,artemisinin or artesunate alleviated the glial cell proliferation,neuronal cell death and production of inflammatory cytokine indused by JEV.Animal experiments proved that artemisinin has a good therapeutic effect on JEV-infected mice.Considering the results at the cellular level,we discovered that artemisinin has potential therapeutic value for flavivirus and potential application as a broad-spectrum anti-flavivirus drug.The study presented herein demonstrates for the first time the antiviral activity of artemisinin against flaviviruses JEV,DENV,and ZIKV with a novel antiviral mechanism.The type-1 interferon pathway plays a key role in the process of anti-flavivirus by aryemisinin.Artemisinin inhibits flavivirus replication by promoting the expression of IFN-I in host cells.Therapeutic application of artemisinin may constitute an intriguing broad-spectrum approach to rein in the replication of flaviviruses and to curb the neuroinflammatory response caused by neurotropic flaviviruses,and provide new thoughts for other molecular mechanisms of artemisinin.