Tecovirimat is active against various MPXV strains, while cidofovir, brincidofovir, trifluridine, and gemcitabine have no detectable MPXV-specific antiviral activity.

In treating patients with mpox, current treatment options are limited, with tecovirimat (TEC) being one of the few available. TEC has been approved by the European Medicines Agency (EMA) for treating patients with mpox and is in clinical use in Europe and Japan. However, following exposure to TEC, TEC-resistant variants such as A290V-containing variant (MPXV) emerge quickly. In such cases involving MPXV, alternative agents such as brincidofovir (BCV) have been used, although their efficacy remains controversial and their anti-MPXV activity is yet to be clearly defined. In the present work, we evaluated the anti-MPXV features of five agents (TEC; cidofovir, CDV; BCV; trifluridine, TFT; and gemcitabine, dFdC) reportedly active against various MPXV strains including MPXV, MPXV, MPXV and MPXV, employing cell-based quantitative assays using multiple target cell types such as VeroE6 cells as well as morphometric assays focusing on their cytostatic and cytotoxic natures. The EC values of TEC against MPXV MPXV, and MPXV were 0.001, 0.005, and 0.004 µM, respectively, without tangible cytotoxicity, while that against MPXV was ∼130-fold greater with 0.13 µM. The EC values of CDV, BCV, TFT, and dFdC against MPXV were 18, 1.8, 3.8, and 0.02 µM, respectively; however, the apparent anti-MPXV activity of these four agents was highly associated with their cytotoxicity as they were examined with qualitative and quantitative cell-based-morphometric assays. The data strongly show that none the four agents examined exhibited significant anti-MPXV activity and indicate that effective anti-MPXV agents active against wild-type and drug-resistant variants are urgently needed.