Generation of a recombinant GFP-tagged infectious bronchitis virus (IBV)

Infectious Bronchitis Virus (IBV) is a highly contagious gammacoronavirus which infects poultry. Using reverse genetics, enhanced green fluorescence protein (eGFP) has been inserted into a pathogenic strain of IBV, M41, in place of a newly identified open reading frame (ORF), ORF7. ORF7 has been identified in several IBV strains including M41 and apathogenic strain Beau-R. ORF7 is located immediately downstream of the N gene, preceding the 3’ UTR. This region has been chosen because we are interested in investigating whether a protein can be transcribed from the associated transcription regulatory sequence (TRS-B). M41 has a deletion at the 3’ end of the genome and does not encode all of ORF7, suggesting it is not essential for the viral replication and is not required for a pathogenic phenotype. The TRS-B, TAACA for ORF7 in Beau-R, is non-canonical and is located after the stop codon for the N gene. In M41 only part of the TRS-B is present, TAA, so nucleotides CA were added alongside the eGFP sequence. A number of viruses were successfully rescued. Growth kinetics in primary Chicken Kidney cells (CK) were comparable to the parent virus M41-K. The stability of the eGFP sequence has been assessed in CK cells and in embryonated eggs. The newly constructed TRS-B however was not utilised suggesting that it is not solely the TRS-B that controls the transcription of ORF 7.