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Animal: RNA Viruses |
Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK1
Author for correspondence: Paul Britton. Fax +44 1635 577263. e-mail Paul.Britton{at}bbsrc.ac.uk
Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.
This article has been cited by other articles:
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  T. Hodgson, P. Britton, and D. Cavanagh 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., January 1, 2006; 80(1): 296 - 305. [Abstract] [Full Text] [PDF]
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R. Casais, M. Davies, D. Cavanagh, and P. Britton Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication J. Virol., July 1, 2005; 79(13): 8065 - 8078. [Abstract] [Full Text] [PDF]
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B. Dove, D. Cavanagh, and P. Britton Presence of an Encephalomyocarditis Virus Internal Ribosome Entry Site Sequence in Avian Infectious Bronchitis Virus Defective RNAs Abolishes Rescue by Helper Virus J. Virol., March 15, 2004; 78(6): 2711 - 2721. [Abstract] [Full Text] [PDF]
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K. Hackney, D. Cavanagh, P. Kaiser, and P. Britton In Vitro and In Ovo Expression of Chicken Gamma Interferon by a Defective RNA of Avian Coronavirus Infectious Bronchitis Virus J. Virol., May 15, 2003; 77(10): 5694 - 5702. [Abstract] [Full Text] [PDF]
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R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus J. Virol., December 15, 2001; 75(24): 12359 - 12369. [Abstract] [Full Text] [PDF]
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