title: Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.
abstract: 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.
authors.affiliation: Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK1.
authors.forename: Sharon
authors.initials: S
authors.lastname: Evans
authors.affiliation: Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK1.
authors.forename: David
authors.initials: D
authors.lastname: Cavanagh
authors.affiliation: Division of Molecular Biology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK1.
authors.forename: Paul
authors.initials: P
authors.lastname: Britton
mesh_terms: D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D003673:Defective Viruses / Q000235:genetics* / Q000502:physiology; D005587:Fowlpox virus / Q000235:genetics* / Q000502:physiology; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D006378:Helper Viruses / Q000235:genetics / Q000502:physiology; D001351:Infectious bronchitis virus / Q000235:genetics* / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000821:virology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D013728:Terminator Regions, Genetic / Q000235:genetics; D014709:Vero Cells; D019065:Virus Assembly
chemical_list: D004274:DNA, Recombinant; D004279:DNA, Viral; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral