Phylogeny of Orthopoxviruses (OPVs). (A) New-World (highlighted grey) and Old-World OPVs (dotted box) were clearly separated by the unrooted phylogenetic tree. (B) Enlarged unrooted phylogenetic tree of Old-World OPVs defined the species ectromelia virus (ECTV), monkeypox virus (MPXV), vaccinia virus (VACV), and variola virus (VARV) as monophyletic clades. Cowpox virus (CPXV) strains were polyphyletic and grouped into four different clades (CPXV-like 1, CPXV-like 2, VACV-like, VARV-like). Single-branch CPXV isolates are indicated by a black asterisk. Isolates sequenced in this study appear in bold face. Scales represent substitutions per position. All species and clade segregating branches are supported by bootstrap values of at least 80% (see Supplementary Figure S1).