Niclosamide inhibits the replication of highly pathogenic avian influenza A (H5Nx) viruses and antiviral-resistant mutants.

The recurrent spillover of highly pathogenic avian influenza (HPAI) H5 viruses into humans represents a major public health concern that is exacerbated by the emergence of drug-resistant viral variants. Host-targeting antiviral approaches, including drug repurposing, offer a promising alternative to conventional virus-directed therapeutics. Here, we evaluated the antiviral activity of niclosamide, an FDA-approved anthelmintic drug, against four HPAI A(H5Nx) viruses, two A(H5N1), one A(H5N6), and one A(H5N8), recently isolated from human cases. Niclosamide inhibited all four viruses in plaque reduction assays with MDCK cells, with low inhibitory concentration 50% (IC50) values (0.68-1.40 μM) and minimal cytotoxicity at effective concentrations. These values were more potent than the IC50 values observed for the RdRp inhibitor favipiravir. Niclosamide treatment plus either baloxavir marboxil or favipiravir resulted in additive or near-additive interactions, as indicated by synergy scores of ±10. Importantly, niclosamide retained antiviral activity against HPAI A(H5Nx) viruses bearing resistance-associated amino acid substitutions (i.e., PA-I38T, baloxavir resistance and PB1-K229R, favipiravir resistance), consistent with its host-directed mechanism of action. Although there are barriers to be overcome such as a narrow therapeutic window, largely attributable to its poor bioavailability and some cytotoxicity, our findings suggest niclosamide has potential as a host-targeting therapeutic option against emerging zoonotic influenza viruses, particularly in settings involving antiviral-resistant escape mutants.