Figure 2. Phylogenetic inference relationships of the open reading frames encoding the hemagglutinin protein of selected strains of vaccinia, variola, cowpox, and monkeypox viruses and monkeypox virus isolates described in this study. Sequences used were cowpox virus Brighton (AF375089), variola virus Bangladesh (AF375129), vaccinia virus Lister (AY678276), monkeypox virus mpv 1997 (AF375096), mvp-squir (AF375112), mpv Zaire 77-0666 (Z99052), mpv-cncr (AF375102), mpv 74-226 (AF375099), mpv-082 (AF375095), mpv-utc (AF375113), and mpv-3945 (AF375098). ClustalW, version 1.83 (10), was used to generate amino acid multiple sequence alignments (pairwise gap opening = 35 and gap extension = 0.75; multiple alignment gap opening = 15 and gap extension = 0.30; Gonnet series). Each alignment was processed using RevTrans (11). Bayesian posterior probability inference of phylogeny used MrBayes, version 3.084. MrBayes settings for the best-fit model (GTR+I+G) were selected by hierarchies for the likelihood ratio test in MrModeltest 2.0 (12). Bayesian analysis was performed with MrBayes; the maximum likelihood model used 6 substitution types (nst = 6). Rate variation across sites was modeled by using a gamma distribution, with a proportion of sites being invariant (rates = invgamma). The Markov chain Monte Carlo search was run for 1 million generations; trees were sampled every 100 generations (the first 4,000 trees were discarded as burn-in). NL, the Netherlands; DRC, Democratic Republic of Congo.