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Vaccination uses live attenuated vaccines (LAVs) that are generated via serial passage of a virulent field isolate through embryonated hens\u2019 eggs, typically 80\u2013100 times. The molecular basis of attenuation is unknown and varies with each attenuation procedure. To investigate specifically targeted attenuation, we utilized reverse genetics to target the macrodomain 1 (Mac1) domain within non-structural protein 3 of the virulent M41 strain. Macrodomains are found in a variety of viruses, including coronaviruses, and have been associated with the modulation of the host\u2019s innate response. Two recombinant IBVs (rIBVs) were generated with specific single point mutations, either Asn42Ala (N42A) or Gly49Ser (G49S), within the Mac1 domain generating rIBVs M41K-N42A and M41K-G49S, respectively. Replication\n                      <jats:italic>in vitro<\/jats:italic>\n                      was unaffected, and the mutations were stably maintained during passaging\n                      <jats:italic>in vitro<\/jats:italic>\n                      and\n                      <jats:italic>in ovo<\/jats:italic>\n                      . While M41K-N42A exhibited an attenuated phenotype\n                      <jats:italic>in vivo<\/jats:italic>\n                      , M41K-G49S was only partially attenuated. The attenuated\n                      <jats:italic>in vivo<\/jats:italic>\n                      phenotypes observed do not appear to be linked to a reduction in viral replication and additionally M41K-N42A highlighted the N42A mutation as a method of rational attenuation. Vaccination of chickens with either rIBV M41K-N42A or a rIBV containing the Mac1 N42A mutation and our previously identified attenuating Nsp10 and 14 mutations, Pro85Leu and Val393Leu respectively, offered complete protection from homologous challenge. The presence of multiple attenuating mutations did not appear to negatively impact vaccine efficacy.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Infection of chickens with the\n                      <jats:italic>Gammacoronavirus<\/jats:italic>\n                      infectious bronchitis virus (IBV) causes an acute respiratory disease, resulting in reduced weight gain and reductions in egg laying making it a global concern for poultry industries and food security. Vaccination against IBV uses live attenuated viruses (LAVs), generated by multiple passages of a virulent virus through embryonated hens\u2019 eggs. The molecular basis of attenuation is unknown and unpredictable requiring a fine balance between loss of virulence and vaccine efficacy. In this study, we targeted the macrodomain of IBV for rational attenuation demonstrating a single point mutation can result in loss of pathogenicity. An IBV vaccine candidate was subsequently generated containing three specific attenuating mutations, to reduce the risk of reversion, which completely protected chickens. The targets in this study are conserved among IBV strains and the coronavirus family offering a potential method of rational attenuation that can be universally applied for vaccine development.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.01663-24","type":"journal-article","created":{"date-parts":[[2025,2,27]],"date-time":"2025-02-27T08:47:39Z","timestamp":1740646059000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":9,"title":["Recombinant infectious bronchitis virus containing mutations in non-structural proteins 10, 14, 15, and 16 and within the macrodomain provides complete protection against homologous challenge"],"prefix":"10.1128","volume":"99","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9583-630X","authenticated-orcid":true,"given":"Sarah","family":"Keep","sequence":"first","affiliation":[{"name":"The Pirbright Institute","place":["Pirbright, United 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