Avian coronavirus, also known as infectious bronchitis virus (IBV), belongs to the genus Gammacoronavirus and is the causative agent of infectious bronchitis, a highly contagious respiratory disease in the poultry industry. In virology studies, reverse genetics systems based on BACs are extremely valuable because they allow us to manipulate viral genes. In our study, we assembled the complete genome of the IBV strain Beaudette-FUB into an artificial bacterial chromosome (BAC), producing an infectious BAC clone. From this constructed IBV BAC clone, we successfully rescued infectious viruses with identical growth characteristics to the parental viruses. To establish genetically stable EGFP viruses, we then inserted the EGFP ORF into 11 putative cleavage sites of 3CLpro. Of these, we identified three insertion sites located at the outermost 3’ end of the replicase gene– between the coding sequences of Nsp13 (helicase), Nsp14 (RNA exonuclease), Nsp15 (RNA endonuclease), and Nsp16 (RNA methyltransferase) could tolerate heterologous genes in the IBV genome. Additionally, we found that fluorescent proteins expressed by the replicase gene can be efficiently cleaved by the 3CLpro and released from the replicase polyprotein. Furthermore, we also determined the genetic stability of these three EGFP-replicase viruses. Among them, the engineered Nsp13-EGFP-Nsp14 virus still exhibited high stability in DF-1 cells after 20 serial passages. The colocalization results showed that EGFP, together with dsRNA or RdRp, accumulated in the well-defined foci at the early stage of infection. When the infection progressed, EGFP proteins were produced and distributed throughout the cytoplasm. Our studies have shown that the replicase-EGFP viruses could be used to study viral replication and transcription, to screen antiviral drugs on a large scale, to develop multivalent vaccines, and even that the potential positions could be applied to other coronaviruses.
