Comparative genomics identifies small interfering RNA with activity against all five human betacoronaviruses

The Orthocoronavirinae virus subfamily (CoV) poses a continuous global health threat. Seven CoV species are known to infect humans and additional animal CoVs with potential for human spillover have been identified. Currently, antiviral drugs are available only against SARS-CoV-2, underscoring the need for broad-spectrum antivirals. Targeting the viral RNA genome with small interfering RNAs (siRNAs) induces its degradation by endogenous nucleases and represents an effective strategy to inhibit CoV replication. Previous studies have demonstrated the efficacy and feasibility of this approach against individual CoV species. However, its potential to serve as a broad-spectrum antiviral strategy effective across multiple members of the CoV subfamily has not been systematically explored. The conservation of all potential siRNA target sites within the CoV genome (n ≈ 30,000) was analyzed using a custom database comprising 250,000 full-length genome sequences from 44 CoV species isolated from diverse mammalian and avian hosts. Antiviral activity of selected siRNA candidates was screened using a replication-competent recombinant SARS-CoV-2 expressing green fluorescent protein. The effect of target site mismatches was assessed using luciferase reporters, and cross-species antiviral activity was evaluated against six representative wild-type CoVs. Cellular toxicity was investigated by monitoring cell death, confluence, metabolic activity, and in silico prediction of siRNA off-target activity. Conservation of potential siRNA target sites correlated with the evolutionary distance between CoV species. Several genomic regions were conserved across different CoV subgenera and genera, but no site was universally preserved across all 44 analyzed species. The antiviral activity and tolerability of 347 siRNAs targeting the most promising sites were evaluated in multiple screening rounds. Following optimization of siRNA design and chemistry, we identified a lead candidate, si117m, which displayed high tolerability and potent silencing activity at picomolar concentrations against target sites of SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43 and HCoV-HKU1. Our study demonstrates the potential of comparative genomics for developing broad-spectrum antiviral siRNAs. We identified a lead siRNA, si117m, which combines a favorable safety profile with cross-species activity against five human CoVs, supporting its potential for clinical translation and future pandemic preparedness.