In vitro selection and characterization of tecovirimat-resistant mpox virus
Mpox virus (MPXV) is the causative agent of mpox, a disease characterized by skin lesions, and has remained a global threat since the 2022 outbreak. Tecovirimat is an antiviral agent that inhibits MPXV replication in vitro and in vivo by targeting the viral phospholipase VP37 protein and blocking viral envelope wrapping. Although tecovirimat has been administered to many mpox patients, the emergence of tecovirimat-resistant MPXVs carrying mutations in the VP37 protein have been reported, particularly in immunocompromised individuals. However, the impact of these respective mutations on drug susceptibility and viral fitness have not been fully characterized. Here, we report in vitro isolation and side-by-side characterization of MPXV harboring clinically observed mutations, I372N, T245I, Y258C, and N267D in the VP37 protein. We found that the VP37-N267D and -Y258C mutants showed markedly reduced susceptibility to tecovirimat compared with the VP37-I372N and -T245I (+I7L-A388S) mutants, while retaining susceptibility to an alternative anti-MPXV agent, brincidofovir. The VP37-I372N, T245I (+I7L-A388S), Y258C, and N267D mutants exhibited viral fitness comparable to the parental virus in A549 and Vero cells, as well as in human lung fibroblasts and keratinocytes. These findings provide insights into the phenotype of each VP37 mutation and emphasize the importance of developing antivirals with distinct mechanisms of action to effectively combat tecovirimat-resistant MPXV.