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High-throughput DNA sequencing enables the study of experimental evolution in near real time. Until now, mutants with deletions of nonessential host range genes were used in experimental evolution of vaccinia virus (VACV). Here, we guided the selection of adaptive mutations that enhanced the fitness of a hybrid virus in which an essential gene had been replaced with an ortholog from another poxvirus genus. Poxviruses encode a complete system for transcription, including RNA polymerase and stage-specific transcription factors. The abilities of orthologous intermediate transcription factors from other poxviruses to substitute for those of VACV, as determined by transfection assays, corresponded with the degree of amino acid identity. VACV in which the A8 or A23 intermediate transcription factor subunit gene was replaced by the myxoma (MYX) virus ortholog exhibited decreased replication. During three parallel serial passages of the hybrid virus with the MYXA8 gene, plaque sizes and virus yields increased. DNA sequencing of virus populations at passage 10 revealed high frequencies of five different single nucleotide mutations in the two largest RNA polymerase subunits, RPO147 and RPO132, and two different Kozak consensus sequence mutations predicted to increase translation of the MYXA8 mRNA. Surprisingly, there were no mutations within either intermediate transcription factor subunit. Based on homology with Saccharomyces cerevisiae RNA polymerase, the VACV mutations were predicted to be buried within the internal structure of the enzyme. By directly introducing single nucleotide substitutions into the genome of the original hybrid virus, we demonstrated that both RNA polymerase and translation-enhancing mutations increased virus replication independently. IMPORTANCE Previous studies demonstrated the experimental evolution of vaccinia virus (VACV) following deletion of a host range gene important for evasion of host immune defenses. We have extended experimental evolution to essential genes that cannot be deleted but could be replaced by a divergent orthologous gene from another poxvirus. Replacement of a VACV transcription factor gene with one from a distantly related poxvirus led to decreased fitness as evidenced by diminished replication. Serially passaging the hybrid virus at a low multiplicity of infection provided conditions for selection of adaptive mutations that improved replication. Notably, these included five independent mutations of the largest and second largest RNA polymerase subunits. This approach should be generally applicable for investigating adaptation to swapping of orthologous genes encoding additional essential proteins of poxviruses as well as other viruses., (Copyright © 2018 American Society for Microbiology.)