Viral RNA-Dependent RNA Polymerase Inhibitor 7-Deaza-2′- C -Methyladenosine Prevents Death in a Mouse Model of West Nile Virus Infection

Abstract

West Nile virus (WNV) is a medically important emerging arbovirus causing serious neuroinfections in humans and against which no approved antiviral therapy is currently available. In this study, we demonstrate that 2′-C-methyl- or 4′-azido-modified nucleosides are highly effective inhibitors of WNV replication, showing nanomolar or low micromolar anti-WNV activity and negligible cytotoxicity in cell culture. One representative of C2′-methylated nucleosides, 7-deaza-2′-C-methyladenosine, significantly protected WNV-infected mice from disease progression and mortality. Twice daily treatment at 25 mg/kg starting at the time of infection resulted in 100% survival of the mice. This compound was highly effective, even if the treatment was initiated 3 days postinfection, at the time of a peak of viremia, which resulted in a 90% survival rate. However, the antiviral effect of 7-deaza-2′-C-methyladenosine was absent or negligible when the treatment was started 8 days postinfection (i.e., at the time of extensive brain infection). The 4′-azido moiety appears to be another important determinant for highly efficient inhibition of WNV replication in vitro. However, the strong anti-WNV effect of 4′-azidocytidine and 4′-azido-aracytidine was cell type dependent and observed predominantly in porcine kidney stable (PS) cells. The effect was much less pronounced in Vero cells. Our results indicate that 2′-C-methylated or 4′-azidated nucleosides merit further investigation as potential therapeutic agents for treating WNV infections as well as infections caused by other medically important flaviviruses.

FDA-Approved Drugs Efavirenz, Tipranavir, and Dasabuvir Inhibit Replication of Multiple Flaviviruses in Vero Cells

“…In the HCV subgenomic replicon system, dasabuvir inhibits genotype 1a and 1b replicons with EC 50 values of 7.7 and 1.8 nM, respectively [30]. In our in vitro assays, dasabuvir had a micromolar EC 50 , which was comparable to other small molecule inhibitors that showed effectiveness in laboratory animals infected with VBFs [25][26][27]39,40]. The antiviral activity of dasabuvir was demonstrated regardless if applied before infection, at the time of infection, or post-infection ( Figures 2B and 4, Supplementary Figure S1).…”

“…Plaque assays were performed in Vero cells (for ZIKV and WNV titers) or in the PS cells (to determine TBEV titers) as described previously [23][24][25]. The obtained viral titer values were recalculated to percentages of viral titer inhibition, applied to constructing the dose-response and inhibition curves, and used to calculate the 50% effective concentration (EC 50 ).…”

“…The results obtained from antiviral assays were confirmed using a cell-based flavivirus immunostaining assay with a mouse monoclonal antibody that specifically recognizes the flavivirus group surface antigen, as described previously [25]. Briefly, Vero cells seeded onto 96-well plates were treated with the test compound (0, 40, or 50 µM dasabuvir or efavirenz; 0, 75, or 100 µM tipranavir) and infected with the individual viruses at a multiplicity of infection of 0.1.…”

A Library of Nucleotide Analogues Terminate RNA Synthesis Catalyzed by Polymerases of Coronaviruses Causing SARS and COVID-19

“…S1. Some of the uridine analogues listed above have been investigated as inhibitors of viral polymerases (Kumaki et al 2011;Eyer et al 2019;Arup et al 1992;Lauridsen et al 2012). The 2'-O-methyluridine triphosphate is of particular interest, since it has been demonstrated that 2'-O-methyl nucleotides are resistant to removal by the 3'-exonuclease found in coronaviruses (Minskaia et al 2006).…”

West Nile Virus Induced Cell Death in the Central Nervous System

“…The limitations of targeting cell death in reversing WNV disease, include the timing, infection severity, and treatment dosage. For example, timing of such treatments is very critical-if too late, the damage cannot be prevented or reversed [66].…”