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

Infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically important disease of chickens. Vaccination uses live attenuated vaccines (LAVs) that are generated via serial passage of a virulent field isolate through embryonated hens' eggs, typically 80-100 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's 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 in vitro was unaffected, and the mutations were stably maintained during passaging in vitro and in ovo. While M41K-N42A exhibited an attenuated phenotype in vivo, M41K-G49S was only partially attenuated. The attenuated in vivo 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.