Molnupiravir and Its Active Form, EIDD-1931, Show Potent Antiviral Activity against Enterovirus Infections In Vitro and In Vivo

Figures

• 

  Figure 1. The molecular formula of EIDD−1931 and EIDD−2801 and the in vitro antiviral effects against EV−A71. (A) The molecular formula of EIDD−1931 and EIDD−2801. (B–G) The antiviral activities of EIDD−1931 (B–D) and EIDD2801 (E–G) against EV−A71 in different cells lines. RD cells, Vero cells, and Huh7 cells were infected with the EV−A71 H strain at 100 × TCID50. Different doses of the test compounds were then added. At 72 h.p.i, the antiviral parameters were measured. The antiviral effects and cytotoxicity of EIDD−1931 and EIDD−2801 were measured using a CellTiter−Glo cell viability assay kit. The EC50 and CC50 were calculated using Origin 9.0 software. SI = CC50/IC50.

• 

  Figure 2. Antiviral activities of EIDD−1931 and EIDD−2801 against EV−A71 in vitro. (A–D) EV−A71 virus particle yields and viral RNA reduction assay. RD cells were infected with EV71 at an MOI of 0.1 PFU/mL in the presence of different concentrations of EIDD−1931 or EIDD−2801. After 30 h, the virus particle yields in the supernatant were measured by TCID50. Total cellular RNA was extracted and subjected to qRT−PCR to measure viral RNA expression. All data are shown as means ± standard deviation from three independent experiments. Statistical significance was calculated using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (E–G) Inhibitory effects of EIDD-1931 and EIDD−2801 on viral VP1 protein. (E) RD cells were infected with EV−A71 at an MOI of 1 PFU/mL in the presence of EIDD−1931 or EIDD−2801. At 16 h.p.i., the cells were fixed for immunofluorescence assays. EV−A71 VP1 proteins were stained using mouse anti−EV−A71 antibody (12D7) (red), and cell nuclei were stained using Hoechst 33,342 (blue), scale bar: 50 µm. (F,G) RD cells infected with EV−A71 (MOI = 1) were treated with the test compounds at the indicated concentrations. At 24 h post infection, the cells…

• 

  Figure 3. EIDD−1931 acts at the post-entry stage of EV−A71 virus infection. (A) Schematic of the time-of-drug-addition assay. (B) Levels of intracellular viral RNA. RD cells were treated with EV−A71 and the test compounds according to the timeline shown in panel A. After 30 h, the viral RNA was extracted and determined via qRT−PCR. The data collected from three independent experiments are represented as means ± standard deviations. Statistical significance was analyzed using one-way ANOVA. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

• 

  Figure 4. EIDD-1931 and EIDD-2801 exert in vivo antiviral effects against EV-A71 infection. Four groups of 1-day-old ICR suckling mice were i.p. infected with 106 PFU of EV-A71 virus (H strain), followed by i.p. treatment with EIDD-1931, EIDD-2801, or vehicle at the indicated dosages for 7 consecutive days. Survival (A,C) and body weight (B,D) were recorded every day until 21 d.p.i. Data for B and D are presented as means ± standard deviations. Survival data were analyzed with a log-rank test. ** p < 0.01, **** p < 0.0001.

• 

  Figure 5. EIDD-1931 and EIDD-2801 reduce the viral loads in various tissues of 1-day-old ICR suckling mice. Three groups of 1-day-old ICR suckling mice were i.p. infected with 106 PFU of EV-A71 virus (H strain), followed by i.p. treatment with EIDD-1931 and EIDD-2801 at a dosage of 200 mg/kg for 4 consecutive days. Then, the mice were euthanized, and brain (A), heart (B), intestine (C), liver (D), limb muscles (E) and lung (F) were separately harvested to determine the viral RNA loads using qRT-PCR. Data are presented as means ± standard deviations, and Student’s unpaired t test was performed for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.