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16 Pág., Poxviruses have large DNA genomes, and they are able to infect multiple vertebrate and invertebrate animals, including humans. Despite the eradication of smallpox, poxvirus infections still remain a significant public health concern. Vaccinia virus (VV) is the prototypic member in the poxviridae family and it has been used extensively for different prophylactic applications, including the generation of vaccines against multiple infectious diseases and/or for oncolytic treatment. Many attempts have been pursued to develop novel attenuated forms of VV with improved safety profiles for their implementation as vaccines and/or vaccines vectors. We and others have previously demonstrated how RNA viruses encoding codon-deoptimized viral genes are attenuated, immunogenic and able to protect, upon a single administration, against challenge with parental viruses. In this study, we employed the same experimental approach based on the use of misrepresented codons for the generation of a recombinant (r)VV encoding a codon-deoptimized A24R gene, which is a key component of the viral RNA polymerase. Similar to our previous studies with RNA viruses, the A24R codon-deoptimized rVV (v-A24cd) was highly attenuated in vivo but able to protect, after a single intranasal dose administration, against an otherwise lethal challenge with parental VV. These results indicate that poxviruses can be effectively attenuated by synonymous codon deoptimization and open the possibility of using this methodology alone or in combination with other experimental approaches for the development of attenuated vaccines for the treatment of poxvirus infection, or to generate improved VV-based vectors. Moreover, this approach could be applied to other DNA viruses. IMPORTANCE The family poxviridae includes multiple viruses of medical and veterinary relevance, being vaccinia virus (VV) the prototypic member in the family. VV was used during the smallpox vaccination campaign to eradicate variola virus (VARV), which is considered a credible bioterrorism threat. Because of novel innovations in genetic engineering and vaccine technology, VV has gained popularity as a viral vector for the development of vaccines against several infectious diseases. Several approaches have been used to generate attenuated VV for its implementation as vaccine and/or vaccine vector. Here, we generated a rVV containing a codon-deoptimized A24R gene (v-A24cd), which encodes a key component of the viral RNA polymerase. v-A24cd was stable in culture cells and highly attenuated in vivo but able to protect against a subsequent lethal challenge with parental VV. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated VV and/or vaccine vectors., This work was supported by grants E-RTA2014-00006 and E-RTA2017-0066 from Ministerio de Economía y Competitividad and Ministerio de Ciencia, Innovación y Universidades as part of the Plan Estatal de Investigación Científica, Desarrollo e Innovación Tecnológica, and grant COV20-00901 from Instituto de Salud Carlos III (ISCIII). This research was funded by a “Ramon y Cajal” Incorporation grant (RYC-2017) from the Spanish Ministry of Science, Innovation and Universities to A.N. Research in L.M.-S. was partially funded by the Department of Defense (DoD) W81XWH1910496 and the National Institute of Health (NIH) R21 AI1135284 grants, and startup funding from the Department of Microbiology and Immunology at University of Rochester.