Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been reported globally with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. MPXV vaccines are limited and only two antivirals, tecovirimat and brincidofovir, approved by the United States (US) Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit Orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (Scarlet or GFP) and luciferase (Nluc) reporter genes to identify compounds with anti-Orthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed antiviral activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating the broad-spectrum antiviral activity against Orthopoxviruses and their potential to be used for the antiviral treatment of MPXV, or other Orthopoxvirus, infections.
Importance: Despite the eradication of smallpox, some Orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, there is presently limited access to those vaccines. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV, and other potentially zoonotic Orthopoxvirus infections. Here, we show that thirteen compounds, derived from two different libraries, previously found to inhibit several RNA viruses, exhibit also antiviral activity against VACV. Notably, eleven compounds also displayed antiviral activity against MPXV, demonstrating their potential to be incorporated into the therapeutic armamentarium to combat Orthopoxvirus infections.