As of November 10, 2022, the ongoing global monkeypox (recently renamed mpox) outbreak has resulted in 79,231 cases in 110 countries and 49 deaths, according to the Centers for Disease Control and Prevention. Approximately 10% of patients with mpox are hospitalized.1

Tecovirimat (ST-246), cidofovir, and brincidofovir (CMX001) are the antiviral agents currently used for the treatment of mpox.2 The currently circulating mpox viruses (MPXVs) have genomic alterations that were not observed previously and appear to affect virus biology, as indicated by the clinical and epidemiologic features seen with the viruses in the current outbreak, which are different from those seen in previous mpox outbreaks.2-5 These alterations may also affect virus sensitivity to antiviral drugs.

We obtained MPXV isolates from 12 patients, who had no known relationship to one another, during the current outbreak to assess virus sensitivity to tecovirimat, cidofovir, and brincidofovir in commonly used cell-line models and primary cultures of pathologically relevant cell types (human foreskin fibroblasts [HFF] and human foreskin keratinocytes [HFK]) (Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org). All isolates reacted with primers that detect clade II (West African clade) and had mutational profiles that closely resembled that of a reference genome from the current worldwide outbreak (ON563414.2) (Figs. S1 and S2 and Table S2), results that are consistent with previous findings.2,3,5 However, the isolates and ON563414.2 harbored mutations in the viral DNA polymerase (gp57, the target of cidofovir and brincidofovir) and in F13L (gp45, the target of tecovirimat) that were not present in a reference genome (MT903344.1) before the current outbreak. These mutations have arisen without known selective pressure from antiviral medications.

Primary human foreskin fibroblasts (HFF) and human foreskin keratinocytes (HFK) were infected with MPXV isolates obtained from 12 patients with MPXV infection (multiplicity of infection, 0.01), and immunofluorescence staining was performed at 24 hours, 48 hours, and 72 hours after infection (Panel A). Cell nuclei were stained with 4′,6-diamidine-2-phenylindole (DAPI). Dose–response curves and 50% inhibitory concentrations (IC50) were determined at 72 hours after infection (Panel B).

All isolates replicated in both HFF and HFK cells, as indicated by immunofluorescence staining for orthopoxvirus (Figure 1A and Fig. S3) and the detection of virus DNA in cell supernatants (Fig. S4). Pronounced cytopathogenic effects were seen in HFK cells but not in HFF cells.

Tecovirimat, cidofovir, and brincidofovir inhibited MPXV infection in a dose-dependent manner (Figure 1B). The 50% inhibitory concentration (IC50) of the drugs ranged from 4 to 20 nmol for tecovirimat, from 5 to 32 μmol for cidofovir, and from 9 to 152 nmol for brincidofovir. IC50 values determined in continuous cell lines differed substantially from those in primary cultures, in particular for cidofovir and brincidofovir (Fig. S5 and Table S3), findings that stress the importance of physiologically relevant models.

The IC50 values for tecovirimat, cidofovir, and brincidofovir (4000 nmol, 80 μmol, and 600 nmol, respectively) are within the range of therapeutic concentrations in plasma (Table S4). The highest plasma concentration of tecovirimat after one dose was 200 to 1000 times as high as the IC50 values for the agent. The plasma concentrations of brincidofovir were 3.9 to 67.0 times as high as the IC50 values, and the plasma concentrations of cidofovir were 2.5 to 16.0 times as high as the IC50 values. Our data indicate that the currently circulating MPXVs are likely to remain sensitive to the available antiviral drugs.

Denisa Bojkova, Ph.D.
Marco Bechtel, M.Sc.
Tamara Rothenburger, M.Sc.
Katja Steinhorst, B.Sc.
Nadja Zöller, Ph.D.
Stefan Kippenberger, Ph.D.
University Hospital, Frankfurt am Main, Germany

Julia Schneider, M.Sc.
Victor M. Corman, M.D.
Charité–Universitätsmedizin, Berlin, Germany

Hannah Uri, M.Sc.
Mark N. Wass, Ph.D.
University of Kent, Canterbury, United Kingdom

Gaby Knecht, M.D.
Infektiologikum, Frankfurt am Main, Germany

Pavel Khaykin, M.D.
MainFachArzt Frankfurt, Frankfurt am Main, Germany

Timo Wolf, M.D.
Sandra Ciesek, M.D.
Holger F. Rabenau, Ph.D.
University Hospital, Frankfurt am Main, Germany

Martin Michaelis, Ph.D.
University of Kent, Canterbury, United Kingdom
[email protected]

Jindrich Cinatl, Jr., Ph.D.
University Hospital, Frankfurt am Main, Germany
[email protected]

Supported by the Frankfurter Stiftung für krebskranke Kinder and the Biotechnology and Biological Sciences Research Council (South Coast Biosciences Doctoral Training Partnership).

Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.

This letter was published on December 28, 2022, at NEJM.org.

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4. Otu A, Ebenso B, Walley J, Barceló JM, Ochu CL. Global human monkeypox outbreak: atypical presentation demanding urgent public health action. Lancet Microbe 2022;3(8):e554-e555.

5. Wassenaar TM, Wanchai V, Ussery DW. Comparison of Monkeypox virus genomes from the 2017 Nigeria outbreak and the 2022 outbreak. J Appl Microbiol 2022;133:3690-3698.