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Intracellular particles are fully infectious, and therefore virus mutants lacking essential functions in the exit pathway are unable to form plaques but can multiply intracellularly. We isolated virus mutants in which two of the genes required for virus spread (F13L and A27L) were deleted independently or concurrently. The phenotypes of the mutant viruses were consistent with the need of A27L and F13L for intercellular virus transmission, the effect of the \u0394A27L mutation being more severe than that of \u0394F13L. Despite their defect in spread, \u0394A27L mutant viruses could be expanded by infecting cell cultures at high multiplicity of infection, followed by the release of virions from infected cells by physical means. We developed a novel system for the isolation of recombinant Vaccinia virus in which selection is efficiently achieved by recovering plaque formation capacity after re-introduction of A27L into a \u0394A27L virus. This system allowed the insertion of foreign DNA into the viral genome without the use of additional genetic markers. Furthermore, starting with a double mutant (\u0394A27L-\u0394F13L) virus, A27L selection was used in conjunction with F13L selection to mediate simultaneous dual insertions in the viral genome. This selection system facilitates combined expression of multiple foreign proteins from a single recombinant virus.<\/jats:p>","DOI":"10.1038\/s41598-019-52053-4","type":"journal-article","created":{"date-parts":[[2019,10,30]],"date-time":"2019-10-30T20:29:05Z","timestamp":1572467345000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Genes A27L and F13L as Genetic Markers for the Isolation of Recombinant Vaccinia Virus"],"prefix":"10.1038","volume":"9","author":[{"given":"Mar\u00eda M.","family":"Lorenzo","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juana M.","family":"S\u00e1nchez-Puig","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rafael","family":"Blasco","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2019,10,30]]},"reference":[{"key":"52053_CR1","doi-asserted-by":"publisher","first-page":"112","DOI":"10.1016\/j.omtm.2017.09.007","volume":"7","author":"ZS Guo","year":"2017","unstructured":"Guo, Z. 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