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Journal of Virology, December 2001, p. 12359-12369, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12359-12369.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

Rosa Casais,1 Volker Thiel,2 Stuart G. Siddell,2 David Cavanagh,1 and Paul Britton1,*

Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, United Kingdom,1 and Institute of Virology and Immunology, University of Würzburg, 97078 Würzburg, Germany2

Received 24 May 2001/Accepted 6 September 2001

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

* Corresponding author. Mailing address: Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, United Kingdom. Phone: 44-1635-578411. Fax: 44-1635-577263. E-mail: paul.britton{at}bbsrc.ac.uk.

Journal of Virology, December 2001, p. 12359-12369, Vol. 75, No. 24
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.24.12359-12369.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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