Efficient rescue of infectious bursal disease virus using a simplified RNA polymerase II-based reverse genetics strategy

Abstract

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.

VP1 and VP3 Are Required and Sufficient for Translation Initiation of Uncapped Infectious Bursal Disease Virus Genomic Double-Stranded RNA

“…In support of this explanation, we also showed that the in vitrotranscribed and artificially 5= capped IBDV RNA could successfully be translated into viral proteins and rescued into infectious IBDV by itself ( Fig. 5A) and that IBDV could also be efficiently rescued from the capped viral RNAs generated by the RNA polymerase II promoter (37). The defect in virus production and translation of IBDV uncapped RNA imposed by the lack of a 5= cap was rescued by the viral proteins VP1 and VP3 provided in trans, which is in line with previous observations (38).…”

Simultaneous alteration of residues 279 and 284 of the VP2 major capsid protein of a very virulent Infectious Bursal Disease Virus (vvIBDV) strain did not lead to attenuation in chickens

“…Plasmid constructs pVAXSA.Rib and pVAXSB.Rib [ 32 , 33 ] contain the full-length cDNA sequence of the cell culture-adapted and attenuated P2 strain segments A and B, respectively [ 34 ]. In both constructs, the 5′ end of each genomic segment was fused to the transcription start site of the immediate early CMV promoter, while a hepatitis delta virus (HDV) ribozyme sequence was added at the 3′ end.…”

“…In both constructs, the 5′ end of each genomic segment was fused to the transcription start site of the immediate early CMV promoter, while a hepatitis delta virus (HDV) ribozyme sequence was added at the 3′ end. This cloning strategy was designed so that the host RNA polymerase II will produce segments A and B transcripts with authentic 5′ and 3′ termini; an in vivo reverse strategy which has significantly improved the titer of rescued IBDV [ 32 ].…”

“…Next, pCR.PO7poly.wt was restricted with Bsp EI/ Eco RI and subcloned into Bsp EI/ Eco RI-digested pVAXSA.Rib. Since Eco RI digestion of pVAXSA.Rib eliminates the HDV ribozyme sequence, the latter was restored at the 3′ end of PO7 segment A, using the same previously described strategy [ 32 ]. The resulting plasmid construct, pVAXSA.PO7wt.Rib (Figure 1 ), contains a full-length chimeric segment A cDNA sequence where the 5′ end first 215 nucleotides originated from the attenuated P2 strain, while the remaining sequence (216 to 3261) derived from the Tunisian PO7 vvIBDV strain.…”

“…To rescue chimeric IBDV virus expressing the wild-type or the D279N/A284T mutated PO7 polyprotein, 5 μg each of plasmid constructs pVAXSA.PO7wt.Rib or pVAXSA.PO7mt.Rib were cotransfected with the same amount of pVAXSB.Rib. For comparative purposes, pVAXSA.Rib and pVAXSB.Rib plasmid constructs were cotransfected in parallel in order to rescue the cell culture-adapted and attenuated P2 strain, as previously described [ 32 ]. Co-transfections of CEF cells growing at 80% confluence in 100-mm culture dishes were performed using FuGENE-6, according to the manufacturer’s protocol (Roche Applied Science).…”