{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T23:21:23Z","timestamp":1771888883662,"version":"3.50.1"},"reference-count":38,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2019,7,18]],"date-time":"2019-07-18T00:00:00Z","timestamp":1563408000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["CIISA-UID\/CVT\/00276\/2019"],"award-info":[{"award-number":["CIISA-UID\/CVT\/00276\/2019"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["SFRH\/BD\/89426\/2012"],"award-info":[{"award-number":["SFRH\/BD\/89426\/2012"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["SFRH\/BD\/104261\/2014"],"award-info":[{"award-number":["SFRH\/BD\/104261\/2014"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Seventh Framework Programme","award":["FP7\/2007-2013, 311931, ASFORCE"],"award-info":[{"award-number":["FP7\/2007-2013, 311931, ASFORCE"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>African swine fever (ASF) is a fatal viral disease of domestic swine and wild boar, considered one of the main threats for global pig husbandry. Despite enormous efforts, to date, neither the classical vaccine formulations nor the use of protein subunits proved to be efficient to prevent this disease. Under this scenario, new strategies have been proposed including the development of disabled infectious single cycle (DISC) or replication-defective mutants as potential immunizing agents against the ASF virus (ASFV). In this study, we describe the methodology to generate an ASFV-DISC mutant by homologous recombination, lacking the A104R gene, which was replaced by the selection marker (GUS gene). The recombinant viruses were identified when the infected cells acquired a blue color in the presence of X-Gluc (100 \u00b5g\/mL), which is the substrate for the GUS gene. Since these viral particles result from loss-of-function mutations, being unable to replicate, helper-cell lines expressing the viral pA104R protein were produced. Vero and COS-1 cell lines were transfected by different methods, both physical and chemical, in order to stably express the ASFV-pA104R. Best results were obtained by using Lipofectamine 2000 and Nucleofection methodology of Vero with the pIRESneo vector and by using Flp-FRT site-directed recombination technology system in Flp-In CV-1 cells (transformed COS-1 cells with a single integration site in a transcriptional active region). In order to ensure an efficient and stable integration of the viral ORF on the host cellular genome, the maintenance of the insert was verified by PCR and its expression by immunofluorescence and immunoblot analysis. Although the isolation of the recombinant virus was not achieved, the confirmation of ASFV-\u0394A104R sequence, and the detection of the recombinant mutant through three passages, suggest that this approach is feasible and could be a potential strategy to generate safe and efficient DISC vaccine candidates.<\/jats:p>","DOI":"10.3390\/vaccines7030068","type":"journal-article","created":{"date-parts":[[2019,7,19]],"date-time":"2019-07-19T03:14:41Z","timestamp":1563506081000},"page":"68","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Towards the Generation of an ASFV-pA104R DISC Mutant and a Complementary Cell Line\u2014A Potential Methodology for the Production of a Vaccine Candidate"],"prefix":"10.3390","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1218-5052","authenticated-orcid":false,"given":"Ferdinando B.","family":"Freitas","sequence":"first","affiliation":[{"name":"CIISA\u2014Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"}]},{"given":"Margarida","family":"Sim\u00f5es","sequence":"additional","affiliation":[{"name":"CIISA\u2014Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"},{"name":"Laborat\u00f3rio de Virologia, Instituto Nacional de Investiga\u00e7\u00e3o Agr\u00e1ria e Veterin\u00e1ria (INIAV), Quinta do Marqu\u00eas, 2780-157 Oeiras, Portugal"}]},{"given":"Gon\u00e7alo","family":"Frouco","sequence":"additional","affiliation":[{"name":"CIISA\u2014Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"}]},{"given":"Carlos","family":"Martins","sequence":"additional","affiliation":[{"name":"CIISA\u2014Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5765-576X","authenticated-orcid":false,"given":"Fernando","family":"Ferreira","sequence":"additional","affiliation":[{"name":"CIISA\u2014Centro de Investiga\u00e7\u00e3o Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterin\u00e1ria, Universidade de Lisboa, Avenida da Universidade T\u00e9cnica, 1300-477 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2019,7,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.jcpa.2014.09.003","article-title":"An update on the epidemiology and pathology of African swine fever","volume":"152","author":"Mur","year":"2015","journal-title":"J. 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