The emergence of
drug-resistant influenza A virus (IAV) strains represents a serious threat to global human health and underscores the need for novel approaches to anti-
influenza chemotherapy. Combination
therapy with drugs affecting different IAV targets represents an attractive option for
influenza treatment. We have previously shown that the thiazolide anti-infective
nitazoxanide (
NTZ) inhibits H1N1 IAV replication by selectively blocking
viral hemagglutinin maturation. Herein we investigate the anti-
influenza activity of
NTZ against a wide range of human and avian IAVs (H1N1, H3N2, H5N9, H7N1), including
amantadine-resistant and
oseltamivir-resistant strains, in vitro. We also investigate whether
therapy with
NTZ in combination with the
neuraminidase inhibitors
oseltamivir and
zanamivir exerts synergistic, additive, or antagonistic
antiviral effects against influenza viruses.
NTZ was effective against all IAVs tested, with 50% inhibitory concentrations (IC50s) ranging from 0.9 to 3.2 μM, and selectivity indexes (SIs) ranging from >50 to >160, depending on the strain and the multiplicity of
infection (MOI). Combination
therapy studies were performed in cell culture-based assays using A/Puerto Rico/8/1934 (H1N1), A/WSN/1933 (H1N1), or avian A/chicken/Italy/9097/1997 (H5N9) IAVs; dose-effect analysis and synergism/antagonism quantification were performed using isobologram analysis according to the Chou-Talalay method. Combination index (CI) analysis indicated that
NTZ and
oseltamivir combination treatment was synergistic against A/Puerto Rico/8/1934 (H1N1) and A/WSN/1933 (H1N1) IAVs, with CI values ranging between 0.39 and 0.63, independently of the MOI used. Similar results were obtained when
NTZ was administered in combination with
zanamivir (CI=0.3 to 0.48).
NTZ-
oseltamivir combination treatment was synergistic also against the avian A/chicken/Italy/9097/1997 (H5N9) IAV (CI=0.18 to 0.31). Taken together, the results suggest that regimens that combine
neuraminidase inhibitors and
nitazoxanide exert synergistic anti-
influenza effects.