Nitazoxanide Inhibits Human Norovirus Replication and Synergizes with Ribavirin by Activation of Cellular Antiviral Response [PublishAheadOfPrint]

Norovirus is the main cause of viral gastroenteritis worldwide. Although norovirus gastroenteritis is self-limiting in immunocompetent individuals, chronic infections with debilitating and life-threatening complications occur in immunocompromised patients. Nitazoxanide (NTZ) has been empirically used in the clinic and demonstrated effectiveness against norovirus gastroenteritis. In this study we aimed at uncovering the antiviral potential and mechanisms of NTZ and its active metabolite, tizoxanide (TIZ) using a human norovirus (HuNV) replicon. NTZ and TIZ, collectively referred to as thiazolides (TZD) potently inhibited replication of HuNV and a norovirus surrogate feline calicivirus. Mechanistic studies revealed that TZD activated cellular antiviral response and stimulated the expression of a subset of interferon-stimulated genes (ISGs), particularly IRF-1 not only in Huh7-based HuNV replicon but also in naïve Huh7, Caco-2 and novel human intestinal organoids. Overexpression of exogenous IRF-1 inhibited HuNV replication; whereas knockdown of IRF-1 largely attenuated the antiviral activity of TZD, suggesting that IRF-1 mediated TZD inhibition of HuNV. By using a JAK inhibitor CP-690550 and STAT1 knockout approach, we found that TZD induced antiviral response independent of the classical Janus Kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Furthermore, TZD and ribavirin synergized to inhibit HuNV replication and completely depleted the replicons from host cells after long-term treatment. In summary, our results demonstrated that TZD combated HuNV replication through activation of cellular antiviral response, in particular inducing a prominent antiviral effector IRF-1. NTZ monotherapy or combination with ribavirin represented promising options for treating norovirus gastroenteritis, especially in immunocompromised patients.

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