Unraveling the Potential Antiviral Activity of Isoxazoline-Carbocyclic Monophosphate Nucleotides Against Echovirus 11

From July 2022, a novel Echovirus 11 (E11) variant has been associated with severe neonatal infections and liver failure. Currently, there are no vaccines or antiviral options for the targeted treatment of non-polio enterovirus (EV) infections; therefore, anti-EV drugs are urgently needed. In this study, the putative anti-E11 activity of two isoxzoline-carbocyclic monophosphate nucleotides (<b>4a</b> and <b>4b</b>) was evaluated in vitro by cytopathic effect (CPE) reduction in VERO 76 cells and qRT-PCR. Treatment with nucleotide <b>4a</b> at 25 and 50 μM successfully diminished the CPE caused by E11 by 90% and 75%, respectively, and induced a reduction in viral RNA in the supernatant by 72% and 89%. In contrast, the treatment with 25 and 50 μM of <b>4b</b> caused a minor inhibition of CPE (58 and 38%), and no significant E11 RNA level changes were observed. A time course viral progeny production assay was performed to assess the inhibitory effect of nucleotide <b>4a</b> on E11 infection progression. Compared to the control, the treated group showed a significant drop in viral RNA levels, with reductions of 43% at 10 h, 65% at 24 h, and 96% at 48 h post-infection. The results showed the extensive antiviral properties of the monophosphate nucleotide <b>4a</b> in vitro. Moreover, a retrospective molecular docking study strongly supports that nucleotide <b>4a</b> is an RdRp inhibitor capable of decreasing E11 genome replication and virus particle formation in VERO 76 cells.