Efficient rescue of infectious bursal disease virus using a simplified RNA polymerase II-based reverse genetics strategy.
We describe a simplified RNA polymerase II-based reverse genetics approach that allows for the efficient rescue of high-titer infectious bursal disease virus (IBDV) from cloned cDNAs of genomic segments A and B. Unlike the previously reported RNA polymerase II-based methods, the developed strategy does not necessitate the introduction of a ribozyme sequence at both ends of the genomic cDNA sequences. This was achieved by fusing the 5' terminal sequence of the cDNA of each segment to the transcription start site of the immediate early cytomegalovirus promoter, while a ribozyme sequence was only introduced at the 3' end. Using this strategy, and without complementing with IBDV structural proteins, titers as high as 10(11) tissue culture infectious dose 50 were reproducibly obtained in chicken embryo fibroblast cells immediately upon co-transfection with cDNAs of both segments. We anticipate that this modification could improve reverse genetics for any other RNA virus and may be beneficial for vaccine development and dissection of the viral life cycle.
Ben Abdeljelil
N
N
Unit of Typing and Genetics of Mycobacteria, Institut Pasteur de Tunis, 13, Place Pasteur, B.P. 74, 1002 Tunis-Belvédère, Tunis, Tunisia.
Khabouchi
N
N
Mardassi
H
H
D020413:3' Untranslated Regions / Q000378:metabolism; D020121:5' Untranslated Regions / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D007243:Infectious bursal disease virus / Q000502:physiology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D014779:Virus Replication
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012319:RNA Polymerase II