IBV : potential as a vaccine vector and identification of a novel subgenomic mRNA

Abstract

Using an IBV reverse genetics system a series of recombinant viruses were generated to investigate the potential for utilizing IBV as a vaccine vector. Through the replacement of non-essential regions of the IBV genome, with eGFP or hRluc, factors influencing the stability of recombinant viruses expressing heterologous genes were determined. Expression of heterologous proteins was possible from a variety of virus constructs. The stability of recombinant viruses varied depending on the genome location of the heterologous gene, with replacement of Gene 5 proving to be most stable following passage in cell culture. Stability was strongly influenced by the MOI at which viruses were passaged, with low MOIs resulting in increased stability. The replacement of Gene 5 with a heterologous virus gene may be a suitable target for development of a bivalent vaccine capable of protecting against IBV and a second avian viral disease. Analysis of recombinant IBV mRNA expression profiles led to an investigation into an uncharacterized RNA species, and its link to the IBV intergenic region. A novel subgenomic mRNA was identified associated with the intergenic region that was shown to be transcribed via a non-canonical transcription regulatory sequence. In contradiction to the current model of coronavirus transcription this mRNA has a transcription regulatory sequence derived mainly from the leader, and not the body, transcription regulatory sequence. The non-canonical sequence was shown to be responsible for reduced transcription levels of the intergenic region mRNA. This project proposes the presence of an additional IBV subgenomic mRNA, transcribed via a non-canonical mechanism, and encoding a novel 5th accessory protein of IBV and closely related gammacoronaviruses