Infectious Bronchitis Virus as a Vector for the Expression of Heterologous Genes

Kirsten Bentley,Maria Armesto,Paul Britton,Volker Thiel

doi:10.1371/journal.pone.0067875

Abstract

The avian coronavirus infectious bronchitis virus (IBV) is the causative agent of the respiratory disease infectious bronchitis of domestic fowl, and is controlled by routine vaccination. To explore the potential use of IBV as a vaccine vector a reverse genetics system was utilised to generate infectious recombinant IBVs (rIBVs) expressing the reporter genes enhanced green fluorescent protein (eGFP) or humanised Renilla luciferase (hRluc). Infectious rIBVs were obtained following the replacement of Gene 5 or the intergenic region (IR) with eGFP or hRluc, or the replacement of ORFs 3a and 3b with hRluc. The replacement of Gene 5 with an IBV codon-optimised version of the hRluc gene also resulted in successful rescue of infectious rIBV. Reporter gene expression was confirmed by fluorescence microscopy, or luciferase activity assays, for all successfully rescued rIBVs following infection of primary chick kidney (CK) cells. The genetic stability of rIBVs was analysed by serial passage on CK cells. Recombinant IBV stability varied depending on the genome region being replaced, with the reporter genes maintained up to at least passage 8 (P8) following replacement of Gene 5, P7 for replacement of the IR and P5 for replacement of ORFs 3a and 3b. Codon-optimisation of the hRluc gene, when replacing Gene 5, resulted in an increase in genome stability, with hRluc expression stable up to P10 compared to P8 for standard hRluc. Repeated passaging of rIBVs expressing hRluc at an MOI of 0.01 demonstrated an increase in stability, with hRluc expression stable up to at least P12 following the replacement of Gene 5. This study has demonstrated that heterologous genes can be incorporated into, and expressed from a range of IBV genome locations and that replacement of accessory Gene 5 offers a promising target for realising the potential of IBV as a vaccine vector for other avian pathogens.

Highlights

Coronaviruses are positive-sense RNA viruses with large genomes ranging in size from approximately 26 to 31 kb, and are known to infect a wide range of mammalian and avian species, with species and tissue specific tropisms
Our results demonstrated that heterologous gene expression could be achieved in cell culture for all successfully rescued recombinant IBVs (rIBVs), genome stability varied depending on the genomic location of the reporter gene and the MOI at which rIBVs were passaged in vitro
The growth characteristics of the rIBVs were compared to the control viruses BeauRD5ab, BeauRD3ab [41] and BeauRDIR [42] in which the regions being replaced were deleted

Summary

Introduction

Coronaviruses are positive-sense RNA viruses with large genomes ranging in size from approximately 26 to 31 kb, and are known to infect a wide range of mammalian and avian species, with species and tissue specific tropisms. A process of discontinuous transcription during negative strand synthesis, regulated by short AU rich sequences known as transcription regulatory sequences (TRSs), leads to expression of the structural and accessory proteins as a nested set of subgenomic (sg) mRNAs (reviewed in [1,2,3]). The large size of coronavirus genomes, combined with the possibility of expressing heterologous genes via the generation of novel sg mRNAs, has meant that coronaviruses have long been attractive targets for use as viral-vector vaccines and gene delivery systems. A reverse genetics system for IBV, utilizing vaccinia virus, has been established and so made it possible to explore the use of rIBVs for vaccine development [20,21,22]

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Results

Conclusion