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Volume 41 Issue 3
June 2026
    Citation: Chen Yang, Runchu Zhao, Shuting Huo, Yaqing Zhang, Jingxi Feng, Mingli Gong, Lin Zhu, Conggang Zhang, Linqi Zhang, Jing Xue, Rong Zhang, Qihui Wang, Qiang Ding. Bruton's tyrosine kinase inhibitor BTKi-2 inhibits mpox virus and vaccinia virus infection .VIROLOGICA SINICA, 2026, 41(3) : 700-710.  http://dx.doi.org/10.1016/j.virs.2026.06.011
    shu

    Bruton's tyrosine kinase inhibitor BTKi-2 inhibits mpox virus and vaccinia virus infection

    • a. School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China;

    • b. CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;

    • c. Institute of Laboratory Animals Science, CAMS & PUMC, Beijing 100021, China;

    • d. School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China;

    • e. School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200433, China

    • Corresponding author: Qiang Ding, qding@tsinghua.edu.cn
    • Received Date: 10 December 2025
      Accepted Date: 16 June 2026
      Available online: 19 June 2026
    • The recent global outbreak of mpox virus (MPXV) infections underscores the urgent need for antiviral therapies against orthopoxviruses. In this study, using a high-content screening (HCS) platform based on a modified vaccinia virus Tiantan strain with GFP insertion (MVTT-GFP) under BSL-2 conditions, we screened 1513 kinase inhibitors for antiviral activity. Among these, Bruton's tyrosine kinase inhibitor BTKi-2 emerged as a potent candidate, exhibiting IC50 of 0.535 μM against vaccinia virus (VACV) and 0.260 μM against MPXV in vitro, while maintaining low cytotoxicity. In a murine model of VACV-induced pneumonia, BTKi-2 treatment reduced lung viral loads by 90% and a significantly improved survival compared to vehicle-treated controls. Notably, mechanistic studies indicate that BTKi-2's antiviral effects cannot be completely attributed to the inhibition of BTK or EGFR/ErbB2 signaling. These findings highlight BTKi-2 as a promising antiviral agent in vitro and in vivo, suggesting that BTKi-2 may offer a potential avenue for future therapeutic development against orthopoxvirus infections.
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      1. Adler, H., Gould, S., Hine, P., Snell, L.B., Wong, W., Houlihan, C.F., Osborne, J.C., Rampling, T., Beadsworth, M.B., Duncan, C.J., Dunning, J., Fletcher, T.E., Hunter, E.R., Jacobs, M., Khoo, S.H., Newsholme, W., Porter, D., Porter, R.J., Ratcliffe, L., Schmid, M.L., Semple, M.G., Tunbridge, A.J., Wingfield, T., Price, N.M., NHS England High Consequence Infectious Diseases (Airborne) Network, 2022. Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect Dis 22, 1153-1162.

      2. Beerli, C., Yakimovich, A., Kilcher, S., Reynoso, G.V., Flaschner, G., Muller, D.J., Hickman, H.D., Mercer, J., 2019. Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility. Nat Microbiol 4, 216-225.

      3. Buller, R.M.L., Buller, R.M., Chakrabarti, S., Moss, B., Fredrickson, T.N., Fredricksont, T., 1988. Cell proliferative response to vaccinia virus is mediated by VGF. Virology 164, 182-192.

      4. Chaudhri, G., Panchanathan, V., Bluethmann, H., Karupiah, G., 2006. Obligatory Requirement for Antibody in Recovery from a Primary Poxvirus Infection. Journal of Virology 80, 6339-6344.

      5. Chen, J., Hu, L., Shi, N., Tian, J., Zhang, Y., Tian, S., Cao, X., Ha, Z., Cai, Q., Lu, Y., Smith, G.L., Xie, Y., Lu, H., Zhang, P., Zhang, R., 2025. Development of a replication-defective MPXV platform for fundamental and therapeutic research. https://doi.org/10.1101/2025.06.21.660854.

      6. Daelemans, D., Pauwels, R., De Clercq, E., Pannecouque, C., 2011. A time-of-drug addition approach to target identification of antiviral compounds. Nat Protoc 6, 925-933.

      7. De Clercq, E., Jiang, Y., Li, G., 2023. Therapeutic strategies for human poxvirus infections: Monkeypox (mpox), smallpox, molluscipox, and orf. Travel Med Infect Dis 52, 102528.

      8. Duraffour, S., Lorenzo, M.M., Zoller, G., Topalis, D., Grosenbach, D., Hruby, D.E., Andrei, G., Blasco, R., Meyer, H., Snoeck, R., 2015. ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping. Journal of Antimicrobial Chemotherapy 70, 1367-1380.

      9. Edghill-Smith, Y., Golding, H., Manischewitz, J., King, L.R., Scott, D., Bray, M., Nalca, A., Hooper, J.W., Whitehouse, C.A., Schmitz, J.E., Reimann, K.A., Franchini, G., 2005. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nature Medicine 11, 740-747.

      10. Estupinan, H.Y., Berglof, A., Zain, R., Smith, C.I.E., 2021. Comparative Analysis of BTK Inhibitors and Mechanisms Underlying Adverse Effects. Front. Cell Dev. Biol. 9. https://doi.org/10.3389/fcell.2021.630942.

      11. Fang, D., Liu, Y., Dou, D., Su, B., 2024. The unique immune evasion mechanisms of the mpox virus and their implication for developing new vaccines and immunotherapies. Virologica Sinica 39, 709-718.

      12. Fluckiger, A.-C., Li, Z., Kato, R.M., Wahl, M.I., Ochs, H.D., Longnecker, R., Kinet, J.-P., Witte, O.N., Scharenberg, A.M., Rawlings, D.J., 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. The EMBO Journal 17, 1973-1985.

      13. Foster, S.A., Parker, S., Lanier, R., 2017. The Role of Brincidofovir in Preparation for a Potential Smallpox Outbreak. Viruses 9, 320.

      14. Frischknecht, F., Moreau, V., Rottger, S., Gonfloni, S., Reckmann, I., Superti-Furga, G., Way, M., 1999. Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature 401, 926-929.

      15. Gilchuk, I., Gilchuk, P., Sapparapu, G., Lampley, R., Singh, V., Kose, N., Blum, D.L., Hughes, L.J., Satheshkumar, P.S., Townsend, M.B., Kondas, A.V., Reed, Z., Weiner, Z., Olson, V.A., Hammarlund, E., Raue, H.-P., Slifka, M.K., Slaughter, J.C., Graham, B.S., Edwards, K.M., Eisenberg, R.J., Cohen, G.H., Joyce, S., Crowe, J.E., 2016. Cross-Neutralizing and Protective Human Antibody Specificities to Poxvirus Infections. Cell 167, 684-694.e9.

      16. Hajjo, R., Sabbah, D.A., Abusara, O.H., Kharmah, R., Bardaweel, S., 2023. Targeting Human Proteins for Antiviral Drug Discovery and Repurposing Efforts: A Focus on Protein Kinases. Viruses 15, 568.

      17. Jefferies, C.A., Doyle, S., Brunner, C., Dunne, A., Brint, E., Wietek, C., Walch, E., Wirth, T., O'Neill, L.A.J., 2003. Bruton's Tyrosine Kinase Is a Toll/Interleukin-1 Receptor Domain-binding Protein That Participates in Nuclear Factor κB Activation by Toll-like Receptor 4 *. Journal of Biological Chemistry 278, 26258-26264.

      18. Kang, Y., Gao, P., Chen, X., Zhai, X., Wang, X., Han, X., Jia, B., Hou, B., Zhou, X., Song, J., Zhang, F., 2025. Lrrk2 promotes M1 macrophage polarization via regulating cytokine (IFN-γ) and TLR4 (LPS)-mediated responses. hLife 3, 98-111.

      19. Karim, M., Lo, C.-W., Einav, S., 2023. Preparing for the next viral threat with broad-spectrum antivirals. J Clin Invest 133.

      20. Keating, J.A., Striker, R., 2012. Phosphorylation events during viral infections provide potential therapeutic targets. Rev Med Virol 22, 166-181.

      21. Langhammer, S., Koban, R., Yue, C., Ellerbrok, H., 2011. Inhibition of poxvirus spreading by the anti-tumor drug Gefitinib (Iressa). Antiviral Res 89, 64-70.

      22. Lee, K.-G., Xu, S., Kang, Z.-H., Huo, J., Huang, M., Liu, D., Takeuchi, O., Akira, S., Lam, K.-P., 2012. Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response. Proc Natl Acad Sci U S A 109, 5791-5796.

      23. Lin, Y., Hu, Z., Fu, Y.-X., Peng, H., 2024. Mucosal vaccine development for respiratory viral infections. hLife 2, 50-63.

      24. Matia, A., Lorenzo, M.M., Romero-Estremera, Y.C., Sanchez-Puig, J.M., Zaballos, A., Blasco, R., 2022. Identification of β2 microglobulin, the product of B2M gene, as a Host Factor for Vaccinia Virus Infection by Genome-Wide CRISPR genetic screens. PLOS Pathogens 18, e1010800.

      25. Mercer, J., Knebel, S., Schmidt, F.I., Crouse, J., Burkard, C., Helenius, A., 2010. Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry. Proc Natl Acad Sci U S A 107, 9346-9351.

      26. Minhaj, F.S., 2022. Orthopoxvirus Testing Challenges for Persons in Populations at Low Risk or Without Known Epidemiologic Link to Monkeypox - United States, 2022. MMWR Morb Mortal Wkly Rep 71.

      27. Mitja, O., Ogoina, D., Titanji, B.K., Galvan, C., Muyembe, J.-J., Marks, M., Orkin, C.M., 2023. Monkeypox. The Lancet 401, 60-74.

      28. Ndembi, N., Folayan, M.O., Komakech, A., Mercy, K., Tessema, S., Mbala-Kingebeni, P., Ngandu, C., Ngongo, N., Kaseya, J., Abdool Karim, S.S., 2025. Evolving Epidemiology of Mpox in Africa in 2024. N Engl J Med 392, 666-676.

      29. Newsome, T.P., Weisswange, I., Frischknecht, F., Way, M., 2006. Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus. Cellular Microbiology 8, 233-241.

      30. Niu, K., Wang, X., Jiang, Q., Liu, K., Xie, S., Song, B., Wu, W., Li, X., Peng, C., 2025. Ibrutinib inhibits the replication of multiple poxviruses by targeting the Bruton tyrosine kinase. J Virol 99, e00517-25.

      31. Peng, C., Zhou, Y., Cao, S., Pant, A., Campos Guerrero, M.L., McDonald, P., Roy, A., Yang, Z., 2020. Identification of Vaccinia Virus Inhibitors and Cellular Functions Necessary for Efficient Viral Replication by Screening Bioactives and FDA-Approved Drugs. Vaccines 8, 401.

      32. Perez-Garcia, J.-A., Dans-Vilan, L., Montero-Morales, L., Culebras, E., Fernandez-Castelao, S., Miguel-Benito, A., Puerta-Lopez, T., Delgado-Iribarren Garcia-Campero, A., 2026. First case of Mpox clade Ib transmitted from human-to-human outside the African continent and not linked to travel. Int J Infect Dis 163, 108224. https://doi.org/10.1016/j.ijid.2025.108224.

      33. Reeves, P.M., Bommarius, B., Lebeis, S., McNulty, S., Christensen, J., Swimm, A., Chahroudi, A., Chavan, R., Feinberg, M.B., Veach, D., Bornmann, W., Sherman, M., Kalman, D., 2005. Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases. Nat Med 11, 731-739.

      34. Reeves, P.M., Smith, S.K., Olson, V.A., Thorne, S.H., Bornmann, W., Damon, I.K., Kalman, D., 2011. Variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and SRC family tyrosine kinases. J Virol 85, 21-31.

      35. Ren, W., Lan, J., Ju, X., Gong, M., Long, Q., Zhu, Z., Yu, Y., Wu, J., Zhong, J., Zhang, R., Fan, S., Zhong, G., Huang, A., Wang, X., Ding, Q., 2021. Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption. https://doi.org/10.1371/journal.ppat.1010053.

      36. Ritter, J.M., Martines, R.B., Bhatnagar, J., Rao, A.K., Villalba, J.A., Silva-Flannery, L., Lee, E., Bullock, H.A., Hutson, C.L., Cederroth, T., Harris, C.K., Hord, K., Xu, Y., Brown, C.A., Guccione, J.P., Miller, M., Paddock, C.D., Reagan-Steiner, S., Mpox Pathology Working Group, 2024. Pathology and Monkeypox virus Localization in Tissues From Immunocompromised Patients With Severe or Fatal Mpox. J Infect Dis 229, S219-S228.

      37. Roschewski, M., Lionakis, M.S., Sharman, J.P., Roswarski, J., Goy, A., Monticelli, M.A., Roshon, M., Wrzesinski, S.H., Desai, J.V., Zarakas, M.A., Collen, J., Rose, K.M., Hamdy, A., Izumi, R., Wright, G.W., Chung, K.K., Baselga, J., Staudt, L.M., Wilson, W.H., 2020. Inhibition of Bruton tyrosine kinase in patients with severe COVID-19. Science Immunology 5, eabd0110.

      38. Sanjana, N.E., Shalem, O., Zhang, F., 2014. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783-784.

      39. Shaw, M.L., 2020. Second-Generation BTK Inhibitors Hit the Treatment Bullseye With Fewer Off-Target Effects. August 2020 26, SP226-SP227.

      40. Smith, T.G., Gigante, C.M., Wynn, N.T., Matheny, A., Davidson, W., Yang, Y., Condori, R.E., O'Connell, K., Kovar, L., Williams, T.L., Yu, Y.C., Petersen, B.W., Baird, N., Lowe, D., Li, Y., Satheshkumar, P.S., Hutson, C.L., 2023. Tecovirimat Resistance in Mpox Patients, United States, 2022-2023. Emerging Infectious Diseases. https://doi.org/10.3201/eid2912.231146.

      41. Tasso, B., Spallarossa, A., Russo, E., Brullo, C., 2021. The Development of BTK Inhibitors: A Five-Year Update. Molecules 26, 7411.

      42. Taylor, L., 2025. WHO Declares Mpox Emergency over but Says Threat Remains. BMJ (Clinical research ed.), 390, r1908.

      43. The PALM007 Writing Group, 2025. Tecovirimat for Clade I MPXV Infection in the Democratic Republic of Congo. New England Journal of Medicine 392, 1484-1496.

      44. Titanji, B.K., Hazra, A., Zucker, J., 2024. Mpox Clinical Presentation, Diagnostic Approaches, and Treatment Strategies: A Review. JAMA 332, 1652-1662.

      45. Tiwari, A., Kalonji, T., Miller, T., Van Den Bossche, T., Krolicka, A., Muhindo-Mavoko, H., Mitashi, P., Tahita, M.C., Lood, R., Pitkanen, T., Maketa, V., 2025. Emergence and Global Spread of Mpox Clade Ib: Challenges and the Role of Wastewater and Environmental Surveillance. J Infect Dis 231, e825-e829.

      46. Treon, S.P., Castillo, J.J., Skarbnik, A.P., Soumerai, J.D., Ghobrial, I.M., Guerrera, M.L., Meid, K., Yang, G., 2020. The BTK inhibitor ibrutinib may protect against pulmonary injury in COVID-19-infected patients. Blood 135, 1912-1915.

      47. Vakaniaki, E.H., Kacita, C., Kinganda-Lusamaki, E., O'Toole, A., Wawina-Bokalanga, T., Mukadi-Bamuleka, D., Amuri-Aziza, A., Malyamungu-Bubala, N., Mweshi-Kumbana, F., Mutimbwa-Mambo, L., Belesi-Siangoli, F., Mujula, Y., Parker, E., Muswamba-Kayembe, P.-C., Nundu, S.S., Lushima, R.S., Makangara-Cigolo, J.-C., Mulopo-Mukanya, N., Pukuta-Simbu, E., Akil-Bandali, P., Kavunga, H., Abdramane, O., Brosius, I., Bangwen, E., Vercauteren, K., Sam-Agudu, N.A., Mills, E.J., Tshiani-Mbaya, O., Hoff, N.A., Rimoin, A.W., Hensley, L.E., Kindrachuk, J., Baxter, C., de Oliveira, T., Ayouba, A., Peeters, M., Delaporte, E., Ahuka-Mundeke, S., Mohr, E.L., Sullivan, N.J., Muyembe-Tamfum, J.-J., Nachega, J.B., Rambaut, A., Liesenborghs, L., Mbala-Kingebeni, P., 2024. Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo. Nat Med 30, 2791-2795.

      48. Wang, L., Zhang, T., Wang, N., Liu, X., Kang, D., 2026. Recent Progress in the Discovery of Anti-Mpox Agents. Medicinal research reviews 46. https://doi.org/10.1002/med.70004.

      49. Wu, S., Deng, J., Du, M., Liu, M., Liu, J., 2025. Global transmission characteristics of mpox outbreaks: a systematic review and meta-analysis. EClinicalMedicine 89, 103526.

      50. Yang, C., Yu, Y., Peng, Q., Song, J., Sun, B., Shi, Y., Ding, Q., 2024. Drupacine as a potent SARS-CoV-2 replication inhibitor in vitro. Biosafety and Health. https://doi.org/10.1016/j.bsheal.2024.09.001.

      51. Yang, G., Pevear, D.C., Davies, M.H., Collett, M.S., Bailey, T., Rippen, S., Barone, L., Burns, C., Rhodes, G., Tohan, S., Huggins, J.W., Baker, R.O., Buller, R.L.M., Touchette, E., Waller, K., Schriewer, J., Neyts, J., DeClercq, E., Jones, K., Hruby, D., Jordan, R., 2005. An Orally Bioavailable Antipoxvirus Compound (ST-246) Inhibits Extracellular Virus Formation and Protects Mice from Lethal Orthopoxvirus Challenge. Journal of Virology 79, 13139-13149.

      52. Young, L., Sung, J., Stacey, G., Masters, J.R., 2010. Detection of Mycoplasma in cell cultures. Nat Protoc 5, 929-934.

      53. Zhang, J.-H., Chung, T.D.Y., Oldenburg, K.R., 1999. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. SLAS Discovery 4, 67-73.

      54. Zhao, R., Wu, L., Sun, J., Liu, D., Han, P., Gao, Y., Zhang, Y., Xu, Y., Qu, X., Wang, H., Chai, Y., Chen, Z., Gao, G.F., Wang, Q., 2024. Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections. Nat Commun 15, 4660.

      55. Zhu, W., Fang, Q., Zhuang, K., Wang, H., Yu, W., Zhou, J., Liu, L., Tien, P., Zhang, L., Chen, Z., 2007. The attenuation of vaccinia Tian Tan strain by the removal of the viral M1L-K2L genes. Journal of Virological Methods 144, 17-26.

      56. Zhu, Z., Han, Y., Gong, M., Sun, B., Zhang, R., Ding, Q., 2024. Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry. Journal of Virology 98, e01957-23.

      57. Zucker, J., Fischer, W.A., Zheng, L., McCarthy, C., Saha, P.T., Javan, A.C., Greninger, A., Hamill, M.M., Leslie, K., Brooks, K.M., Berardi, J., Smith, D., Hosey, L., Aldrovandi, G., Ferbas, K., Day, C., Ignacio, R.A.B., Bolan, R., Glesby, M.J., Landovitz, R.J., Luetkemeyer, A.F., Madero, J.S., Gandhi, R.T., Nachman, S., Eron, J., Currier, J.S., Wilkin, T., 2026. Tecovirimat for the Treatment of Mpox. New England Journal of Medicine 394, 884-895.

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      Bruton's tyrosine kinase inhibitor BTKi-2 inhibits mpox virus and vaccinia virus infection

        Corresponding author: Qiang Ding, qding@tsinghua.edu.cn
      • a. School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China;
      • b. CAS Key Laboratory of Pathogen Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
      • c. Institute of Laboratory Animals Science, CAMS & PUMC, Beijing 100021, China;
      • d. School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China;
      • e. School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200433, China
      • Received Date: 10 December 2025
      • Accepted Date: 16 June 2026

      Abstract: The recent global outbreak of mpox virus (MPXV) infections underscores the urgent need for antiviral therapies against orthopoxviruses. In this study, using a high-content screening (HCS) platform based on a modified vaccinia virus Tiantan strain with GFP insertion (MVTT-GFP) under BSL-2 conditions, we screened 1513 kinase inhibitors for antiviral activity. Among these, Bruton's tyrosine kinase inhibitor BTKi-2 emerged as a potent candidate, exhibiting IC50 of 0.535 μM against vaccinia virus (VACV) and 0.260 μM against MPXV in vitro, while maintaining low cytotoxicity. In a murine model of VACV-induced pneumonia, BTKi-2 treatment reduced lung viral loads by 90% and a significantly improved survival compared to vehicle-treated controls. Notably, mechanistic studies indicate that BTKi-2's antiviral effects cannot be completely attributed to the inhibition of BTK or EGFR/ErbB2 signaling. These findings highlight BTKi-2 as a promising antiviral agent in vitro and in vivo, suggesting that BTKi-2 may offer a potential avenue for future therapeutic development against orthopoxvirus infections.

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