| Objective: The present study aims to explore the inhibitory effect of DMF on enterovirus EVD68 in different cell lines and the antiviral stage of Dimethylformamide(DMF),so as to provide new ideas for the development and application of antiviral drugs in clinical research.Methods:(1)Observe the cytopathic effect(CPEs): the same MOI virus was used to infect the cells treated with different doses of DMF(25mM,50 mM,75 mM,100 mM,150 mM and 125 mM)and the untreated control group,and the cytopathic effect was observed 48 hours after infection.(2)Effect on virus replication: the cells treated with the same concentration of DMF were infected with the same MOI virus.The cells were collected at 0h,4h,6h,8h and 10 h.The total RNA was extracted and the viral RNA replication level was detected by Real-Time PCR.(3)Effect on the expression of viral protein VP1: the same MOI virus was used to infect the experimental group treated with DMF and the control group.The expression level of viral protein VP1 was detected by Western blot.(4)Effect on EV-D68 progeny virus particles production in cells: the experimental group cells treated with different concentrations of DMF and the control cells without treatment were infected with the virus of the same MOI,and the supernatant was collected 48 hours later.After PBS cleaning,the virus was diluted 10 times with serum-free DMEM,and then the cells were infected with 96-well plate with four multiple holes in each dilution gradient.The cytopathic effect was observed every day.After 72 hours,the virus titer was calculated by Reed-Muench method.(5)DMF cytotoxicity test: the cells were laid on a 96-well plate,and then the cell viability was detected by MTS method after adding different concentrations of DMF,48h(25mM,50 mM,75 mM,100 mM,125 mM,150 mM,175 mM,200 mM,250 mM).(6)Effect of DMF on virus adsorption and entry stage: A549 cells,BEAS-2B cells and RD cells were treated with DMF and untreated cells as control,and then incubated with EV-D68 at 4 ? or 37 ? for 2 hours to promote virus attachment or entry.The level of virus RNA was detected by Real Time-PCR.(7)Effects on different cell lines: RD and A54948 h were infected with EV-D68 in DMF group or control group without DMF.The expression of CPE,viral protein VP1 and virus titer in supernatant were observed.(8)Detect the expression of VP1 in cells and supernatants after MO/KY infection with other EV-D68 strains.Results: 1.DMF inhibited the replication of EV-D68: 1)DMF inhibited the cytopathic effect of EV-D68 in A549 cells,and significantly decreased the virus titer of offspring in the cell supernatant,and inhibited the expression of EV-D68 viral protein VP1 in a dose-dependent manner.MTS was used to detect whether the concentration of DMF used in the three kinds of cells was toxic to the cells.The results showed that DMF had a significant inhibitory effect on EV-D68-infected cells within the safe concentration range.2)In the experiment of virus adsorption and invasion to cells,compared with the untreated group,it was found that compared with the control group,the viral RNA of the treated group was not significantly inhibited.3)After EV-D68 infection,the cells and supernatant were collected at 0h,6h,8h,12 h,24h and 48 h.It was found that compared with the control group,the expression of VP1 in DMF-treated cell lysate and supernatant was significantly inhibited at the detectable time point.At the time point of one-round replication of the virus,RNA replication was also significantly inhibited in the treatment group.2.The DMF also had significant inhibitory effect on enterovirus epidemic strain MO/KY,the expression of VP1 in cell lysate and supernatant was inhibited,and the production of virion in offspring was also inhibited.Conclusion: 1.Compared with the control group,DMF can inhibit the expression of virus protein and the production of offspring virus in cells infected with EV-D68 virus.2.Compared with the control group,DMF can inhibit the expression of RNA of EV-D68.3.Compared with the control group,DMF has no significant inhibition on the attachment and entry of EV-D68 virus.4.The DMF has significant inhibitory effect on enterovirus D68 prototype strain Fermon and epidemic strain(MO/KY)in cells. |
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