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Journal of Virology, March 2004, p. 2711-2721, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2711-2721.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Presence of an Encephalomyocarditis Virus Internal Ribosome Entry Site Sequence in Avian Infectious Bronchitis Virus Defective RNAs Abolishes Rescue by Helper Virus

Brian Dove, David Cavanagh, and Paul Britton*

Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, United Kingdom

Received 11 August 2003/ Accepted 18 November 2003

Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system. The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette. However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes. The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system. IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation. CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase. However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV. Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.

* 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, March 2004, p. 2711-2721, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2711-2721.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • van den Born, E., Posthuma, C. C., Knoops, K., Snijder, E. J. (2007). An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene. J. Gen. Virol. 88: 1196-1205 [Abstract] [Full Text]  
  • Hodgson, T., Britton, P., Cavanagh, D. (2006). Neither the RNA nor the Proteins of Open Reading Frames 3a and 3b of the Coronavirus Infectious Bronchitis Virus Are Essential for Replication. J. Virol. 80: 296-305 [Abstract] [Full Text]  
  • Casais, R., Davies, M., Cavanagh, D., Britton, P. (2005). Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication. J. Virol. 79: 8065-8078 [Abstract] [Full Text]  


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