Ibrutinib inhibits the replication of multiple poxviruses by targeting the Bruton tyrosine kinase

The recent surge in global monkeypox (mpox) outbreaks highlights the critical need for developing antiviral agents targeting orthopoxvirus infections. Ibrutinib, a selective Bruton tyrosine kinase (BTK) inhibitor initially approved by the FDA in 2013 for treating chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), has emerged as a potential antiviral candidate based on a high-throughput screening for its efficacy against vaccinia virus (VACV). This study evaluated the antiviral capability of ibrutinib against VACV, mpox virus (MPXV), and lumpy skin disease virus (LSDV), demonstrating its strong antiviral activity against multiple poxviruses. <i>In vitro</i> assays confirmed that ibrutinib significantly inhibited the replication of VACV, MPXV, and LSDV across multiple cell lines. <i>In vivo</i> studies using VACV-infected BALB/c mice revealed that ibrutinib treatment extended survival, mitigated weight loss and lesion formation, and reduced viral loads in infected mice. Mechanistic investigations indicated that ibrutinib inhibited viral early and late protein synthesis as well as DNA replication. Furthermore, BTK manipulation influenced viral replication, underscoring ibrutinib's antiviral action through a cellular target. Notably, BTK phosphorylation and nuclear translocation were observed during poxvirus infection, suggesting its role in viral replication. Overall, our findings suggest that ibrutinib is a promising candidate for treating poxvirus infections and that its cellular target, BTK, contributes to poxvirus replication.IMPORTANCEPoxviruses are important zoonotic pathogens affecting human and animal health. The recent outbreak of monkeypox virus (MPXV) highlights the persistent threat posed by poxviruses to public health. In addition, lumpy skin disease virus (LSDV) causes economic impact by affecting the cattle industry. Currently, there are no effective drugs to combat LSD, which poses a major challenge to livestock health and productivity. Therefore, there is an urgent need to develop antiviral drugs against multiple poxviruses. Through high-throughput screening of antiviral drugs targeting vaccinia virus (VACV), we identified ibrutinib as a candidate antiviral drug. In our study, ibrutinib effectively inhibited the replication of MPXV, VACV, and LSDV <i>in vitro</i> and <i>in vivo</i>. Knockdown of Bruton tyrosine kinase (BTK), the cellular target of ibrutinib, significantly inhibited virus replication, while overexpression of BTK enhanced virus replication, which displays its role as a pro-viral effector. Overall, ibrutinib is a promising anti-poxvirus agent that can combat various poxviruses by targeting BTK.