| Research background and objective:EV-D68 is an important human pathogen,which spreads through respiratory tract and causes a variety of respiratory and nervous system-related diseases,posing a serious threat to human life.At present,effective vaccines and drugs have not yet been developed to prevent and treat EV-D68 infection.The host’s innate immune system is the first line of defense against viral infection.TLR7 is an important immune defense component that induces the production of type I IFN and proinflammatory cytokines by recognizing viral RNA.On the contrary,many viruses have developed unique mechanisms to evade recognition by the host immune system.The relationship between EV-D68 and TLR7 has not been reported so far.Vesatolimod is a TLR7 agonist that can enhance the host innate immune response to resist the replication and transmission of HBV,HIV and other viruses.The purpose of this study is to explore the interaction between TLR7 and EV-D68,and to provide a new target for drug development of EV-D68.The value of TLR7 agonist Vesatolimod as a therapeutic agent for EV-D68 was also explored.Methods:1.Effect of EV-D68 infection on TLR7:Using the EV-D68 prototype strain Fermon and isolated circulating strains MO and KY to infect cells,the effect of virus infection on TLR7 was detected by immunoblotting.2.Effects of Vesatolimod on the replication of EV-D68:(1)EV-D68 infected Vesatolimod or DMSO-treated cells at different concentrations to observe the cytopathic effect caused by viral infection,the production of progeny virus in supernatant was detected by TCID50,and the expression of viral protein VP1 was detected by immunoblotting.(2)Toxicity of Vesatolimod to cells and detection of its half effective concentration:Cells were treated with Vesatolimod of different concentrations,and cell viability was detected by CCK8.EVD68 infected Vesatolimod-treated cells of different concentrations.qRT-PCR was used to detect viral RNA levels,and curves were drawn to calculate EC50.(3)Effects of Vesatolimod on inhibition of EV-D68 replication:Vesatolimod cells were added at specific time points before and after EV-D68 infection,and the expression of VP1 protein was detected at 24 h post-infection(hpi).(4)In order to explore the broad spectrum of inhibition effect of Vestolimod on EV-D68,BEAS-2B,SH-SY5Y and T98G cells were infected with EV-D68,and the expression of VP1 protein and generation of progeny virus were detected at 48 hpi.Next,we tested the inhibitory effect of Vesatolimod on isolated circulating strains.MO and KY were used to infect A549 cells and SH-SY5Y cells,respectively.The expression of VP1 protein and the production of progeny viruses were detected by TCID50.3.Study on the stage of inhibition of EV-D68 replication by Vesatolimod:(1)Effect of Vesatolimod treatment on virus attachment and entry stage:Vesatolimod or DMSO-treated A549 cells were infected with EV-D68 at 4℃ or 37℃ for 2 h,respectively.The level of viral RNA after infection was detected by qRT-PCR.(2)Vesatolimod-treated cells were infected with EV-D68 of the same MOI,and cells and supernatants were collected at specific time points to detect the expression levels of viral RNA and viral protein VP1 in cells and supernatants at different time points.4.Preliminary study on the mechanism of Vesatolimod against EV-D68:(1)Construct a stable TLR7 knockdown A549 cell line,and Vesatolimod or DMSO-treated shTLR7 cell line were infected with EV-D68,and observe CPE、detect the level of VP1 protein at 24 hpi.(2)A549 cells were treated with Vesatolimod or DMSO,and cells were collected at 0 hpi,12 hpi and 24 hpi,respectively.The expression level of type I IFN RNA was detected by qRT-PCR.Result:1.EV-D68 infection induces the expression of TLR7.2.Vesatolimod inhibits EVD68 infection,significantly.Compared with cells were treated with DMSO,the CPE phenomenon in the cell treated with Vesatolimod was inhibited.Through experiments such as immunoblotting and the determination of viral titer in progeny,it was found that Vesatolimod treatment of cells reduced the replication level of EV-D68 RNA and VP1 protein synthesis,and had a significant inhibitory effect on the replication of both the Fermon and MO,KY.In addition,Vesatolimod inhibited EV-D68 replication in A549 cells at an EC50 value of approximately 0.16 μM.The results of CCK8 test showed that no significant cytotoxicity or proliferation reduction was observed in A549 cells even when Vesatolimod concentration was as high as 100 μM.For BEAS-2B,SHSY5Y and T98G cells,Vesatolimod treatment with concentration of 1-50 μM did not have obvious toxicity.Vesatolimod treatment had the most significant inhibitory effect on EV-D68 replication from 2 h before virus infection to virus infection.3.Study on the stage of inhibition of EV-D68 replication by Vesatolimod:In the attachment and entry experiments,we detected the level of viral RNA expression in drug and DMSOtreated cells,respectively.We found that there was no significant difference between Vesatolimod and DMSO-treated cells.After Vesatolimod or DMSO-treated cells were infected with EV-D68,we used immunoblotting to detect the expression of EV-D68 virus protein VP1 at 0 h,4 h,6 h,8 h,12 h,and 24 h,and found that Vesatolimod inhibited the replication of the virus during the replication phase after EV-D68 entered the host cell.4.The inhibitory effect of Vesatolimod on EV-D68 replication depends on the activation of the TLR7 signaling pathway,but does not depend on the production of type Ⅰ IFN.We constructed TLR7 knockdown cell line and found that the inhibitory effect of Vesatolimod on EV-D68 replication was significantly reduced in A549 cell line of shTLR7.Next,we treated the cells with Vesatolimod or DMSO and detected the expression level of type Ⅰ IFN RNA after EV-D68 infection.We found that Vesatolimod did not induce the expression of type Ⅰ IFN RNA.Conclusion:1.EV-D68 infection induces the expression of TLR7.2.Vesatolimod inhibits the replication of both the prototype strains and isolated circulating strains of EV-D68 in different cells.3.Vesatolimod does not affect the attachment and entry of EV-D68 into cells,and its anti-enterovirus effect plays a role in the replication phase of EV-D68.4.Vesatolimod inhibits EV-D68 infection through the antiviral signal mediated by TLR7,but its antiviral effect does not depend on the production of type Ⅰ IFN. |
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